Thermo Fisher Scientific Chromeleon 6.60 User Manual

CHROMELEON
®
Chromatography Management System
Tutorial
and
User Manual
Version: 6.60
March 2004
© Dionex 2004
Doc.: CM_Manual_660.doc
License Agreement
i
CHROMELEON Software
Individual License Agreement
This legal document is an agreement between you, the end user, and
Dionex Corporation. The CHROMELEON Chromatography Management
System is provided to you only on the condition that you agree to become
bound by the terms of this agreement, which includes the software license
and the software disclaimer warranty. If you do not agree to the terms of
this agreement, promptly return the disk package(s) and the accompanying
items (including printed materials and binders or other containers) to
Dionex for a full refund.
INDIVIDUAL CHROMELEON LICENSE
1. GRANT OF LICENSE In exchange for the payment of the LICENSE
FEE, Dionex grants the LICENSEE a non-exclusive right to use and display
the CHROMELEON software (the "SOFTWARE") provided with this LICENSE
in accordance with the terms and restrictions below. The LICENSEE is the
end-user organization and its subsidiaries (excluding sub-contractors)
billed by Dionex for this LICENSE. If the organization billed is not the enduser, the LICENSEE is the first end-user organization.
2. OWNERSHIP OF THE SOFTWARE
The LICENSEE owns the
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subsequently recorded, but Dionex retains title and ownership of all
SOFTWARE recorded on such media, regardless of the form in which it is
recorded. This license is not a sale of the SOFTWARE or any copy.
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materials are copyrighted, and may contain proprietary and confidential
information of Dionex.
The LICENSEE may make one (1) copy of the original SOFTWARE
diskettes provided with this LICENSE for backup purposes only. Any such
copy automatically becomes the property of Dionex, and the LICENSEE
must include the copyright notice and any confidentiality or proprietary
legend with any such copy. The LICENSEE may copy the SOFTWARE
from the original or backup diskettes to the computers used in accordance
with the use restrictions below.
ii
License Agreement
All other copying of the SOFTWARE and the accompanying materials is
expressly forbidden. The LICENSEE may be held legally responsible for
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the LICENSEE to abide by the terms of this license.
4. USE RESTRICTIONS Under this LICENSE, the LICENSEE may use
the SOFTWARE on one (1) single-user computer, or one (1) terminal of a
multi-user computer. The LICENSEE must purchase a CHROMELEON Data
Processing License for each additional computer on which the
SOFTWARE provided under this LICENSE is used.
The SOFTWARE may be stored on the computer's local mass storage
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The LICENSEE may not modify, adapt, translate, reverse engineer,
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This LICENSE is effective until terminated. It will
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LICENSEE may terminate this LICENSE by destroying all copies of the
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7. UPDATE POLICY Dionex may (but is not obliged to) release updated
versions of the SOFTWARE. One copy of any such versions will be
provided to the LICENSEE at no additional charge for each registered
SYSTEM that has a valid CHROMELEON Subscription Agreement
("SUBSCRIPTION") in effect at the time of the release. Any such software
update that is provided under a SUBSCRIPTION may be used to collect
data only on the one SYSTEM to which the SUBSCRIPTION applies. All
terms of this LICENSE shall apply to any updates provided to the
LICENSEE.
License Agreement
iii
8. MISCELLANEOUS This LICENSE, including the disclaimer of warranty
and limited warranty, is governed by the laws of the State of California,
excluding its choice of law rules.
DISCLAIMER OF WARRANTY AND LIMITED WARRANTY
The SOFTWARE and the accompanying materials (including instructions
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entire risk as to the results and performance of the SOFTWARE is
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Dionex warrants to the original LICENSEE that the disks on which the
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delivery of this individual LICENSE as evidenced by a copy of the receipt.
The entire liability of Dionex and the exclusive remedy to the LICENSEE
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or (b) replacement of any defective disks that are returned to Dionex with a
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or misapplication, Dionex shall have no responsibility to replace the disks
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the remainder of the original warranty period or thirty (30) days, whichever
is longer.
THE ABOVE ARE THE ONLY WARRANTIES OF ANY KIND AND
DIONEX HEREBY DISCLAIMS ALL OTHER WARRANTIES, EITHER
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NEITHER DIONEX NOR ANYONE ELSE WHO HAS BEEN INVOLVED IN
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iv
License Agreement
ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. IN THE EVENT
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DIONEX TO THE LICENSEE OR ANY THIRD PARTY SHALL NOT
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Some states do not allow exclusion or limitation of the duration of the
implied warranties or exclusion or limitation of liability for incidental or
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Trademarks and Product Names
v
Trademarks and Product Names
Below please find a list containing various trademarks and product names.
All trademarks (™) and registered trademarks (®) belong to Dionex
Corporation unless indicated otherwise. All other trademarks or registered
trademarks are the property of their respective owners.
A
AAA-Direct™ Amino Acid Analysis System
AAA-Certified™
AAES™
Acclaim® columns
Acrodisk® (Gelman Sciences, Inc.)
Acurate™ Flow Splitter
Advanced Computer Interface (ACI)
Advanced Gradient Pump (AGP)
AD20 UV/Vis Absorbance Detector
AES® Atlas Electrolytic Suppressor
Alliance® (Waters Corp.)
AminoPac® Columns
AminoTrap™ Columns
AMMS®-ICE, AMMS-ICE II Anion MicroMembrane Suppressor
AnchorChips™ (Bruker BioSciences)
Anion Atlas® Electrolytic Suppressor
Anion Self-Regenerating Suppressor®
Anotop™ (Whatman)
AppliCard™
AQA™ Mass Spectrometer (Thermo Electron Corp.)
vi
Trademarks and Product Names
Aroclor™ (Monsanto Corp.)
AS40, AS50 Automated Sampler
AS3500 Autosampler
ASE® 100, 200, or 300 Accelerated Solvent Extractor or Solvent Controller
ASI-100™ or ASI-100T™ Automated Sample Injector
ASI-100P™ or ASI-100PT™ Automated Sample Injector
ASRN™ Anion Self-Regenerating Neutralizer
ASRS®, ASRS® II, or ASRS® ULTRA Anion Self-Regenerating Suppressor
Atlas® Suppressor
Auto OnGuard™ (but OnGuard®)
AutoASE® software
autoflex™ (Bruker BioSciences)
Autoion®
AutoNeutralization™ system or technology
AutoQ™
AutoRegen® System
AutoSelect™
AutoSuppression® device
AXIMA-QIT™ (Shimadzu Biotech)
B
BAKER INSTRA-ANALYZED® Acids (J. T. Baker)
Biodialyser™ (AmiKa, Inc.)
BioLC® System
BioPlus™ Columns
BioSelect™ (The Separations Group)
BorateTrap™ Column
Trademarks and Product Names
C
CA Carbamate Analyzer
CAES® Cation Atlas® Electrolytic Suppressor
CarboPac™ MA1, PA1, PA10, PA-100 columns
Cation Atlas™ Electrolytic Suppressor
Cation MicroMembrane Suppressor®
Cation Self-Regenerating Suppressor®
CD Builder™ (AppletWare Inc.)
Chemraz® (Greene, Tweed & Co.)
ChromatoCritters™
CH-2 Column Heater
CHX650 Column Temperature Controller
CHROMELEON® Chromatography Management Software
CMD™ Carbohydrate Membrane Desalter
CMMS® Cation MicroMembrane Suppressor
Continuously Regenerating Anion Trap Column (CR-ATC)
Continuously Regenerating Cation Trap Column (CR-CTC)
CSRN™ Cation Self-Regenerating Neutralizer
CSRS®, CSRS® II, CSRS® ULTRA Cation Self-Regenerating Suppressor
CD20 Conductivity Detector
CMMS® II Cation MicroMembrane Suppressor®
Cryptand columns
D
DataDetective™ (AppletWare Inc.)
dBASE® (Borland International, Inc.)
Dequest® (Monsanto Corporation)
Dionex®
vii
viii
Trademarks and Product Names
DNAPac® column (for NucleoPac)
DNAPhor™ SB1.5 kB Sieving Buffer Kit (for NucleoPhor)
Dowex® (Dow Chemical Company)
DX-80 Ion Analyzer
DX-120 Ion Chromatograph
DX-320 Ion Chromatograph
DX-500 or DX-600 Ion Chromatography System or HPLC System
DX-800 Process Analyzer
DX-LAN™ Instrument Interface
E
ED40, ED50 Electrochemical Detector
EG40, EG50 Eluent Generator
Electrochrom
EluGen®
EO1 Eluent/Solvent Organizer
F
FAMOS™ Fully Automated Micro Autosampler
FastLoc™ (Thermo Electron Corp.)
Finnigan AQA™, Finnigan MSQ™
Flarefit®
FluoBoost Micro Fluorescence Flow Cell
Foxy® (Isco, Inc.)
FPLC® (Pharmacia LKB)
Freon® (E.I. du Pont de Nemours & Co.)
Fusica columns
Trademarks and Product Names
G
GM-2, GM-3, or GM-4 Eluent Gradient Mixers
GP40, GP50 Gradient Pump
GS50 Gradient Pump
H
HPICE® (mostly replaced by IonPac® ICE)
Hydromatrix™ (Varian Associates, Inc.)
I
ICE (ion-exclusion columns, e.g., ICE-AS6)
ICS-90, ICS-1000, ICS-1500, ICS-2000, ICS-2500
InkJet® (Hewlett-Packard)
IonPac® columns
IonPhor™ Electrolyte Buffers
IonSep® reagents
IP20 Isocratic Pump
Irganox® (Ciba)
J
Just Add Water
K
Kalrez® (E.I. du Pont de Nemours & Co.)
KEL-F® (3M Corporation)
ix
x
Trademarks and Product Names
L
LANtastic® (Artisoft, Inc.)
LaserJet® (Hewlett-Packard Corporation)
LC5 Injection Module
LC10 Chromatography Organizer
LC20 Chromatography Enclosure
LC25, LC30 Chromatography Oven
M
Mascot® (Matrix Science Ltd.)
MassLynx™ (Micromass)
µ-Guard™ columns
µ-Dumper
µ-Fluidics
MetPac™ Reagents
MFC-1 (Metal-Free Column)
MICRO® (International Products Corp.)
Micro Precolumn
MicroBead™ resin
MicroInjection valve
MicroMembrane™ Suppressor
Millennium® (Waters Corp.)
Milli-Q® (Millipore)
MMS™ MicroMembrane™ Suppressor
Monolithic capillary column
Mono Q® (Pharmacia LKB)
Trademarks and Product Names
MonoStandard®
M Path™ (Thermo Electron Corp.)
MPIC® (Mobile Phase IC)
MS-DOS® (Microsoft Corporation)
MSQ™ Mass Spectrometer (Thermo Electron Corp.)
N
Nano precolumn
N-EVAP® (Organomation Associates, Inc.)
NovaPak® (Waters Corp.)
NucleoPac (now DNAPac®)
NucleoPhor (now DNAPhor™)
O
OligoStandards™
OmniFLEX™ (Bruker BioSciences)
OmniPac® Columns
OnGuard®, OnGuard® II Sample Prep Station (but Auto OnGuard™)
Optima™ (Fisher Scientific)
ORBO™ (Supelco, Inc.)
P
P680 HPLC Pump
PaintJet® (Hewlett-Packard)
PC10 Postcolumn Pneumatic Controller
PC10 Reagent Delivery Module
PD40 Diode Array Detector
PDA-100 Photodiode Array Detector
xi
xii
Trademarks and Product Names
PeakNet® Chromatography Workstation
Pentium® (Intel)
PepMap™
Pico-Buffer®
PicoView™ (New Objective, Inc.)
PolyVial™
PowerPoint® (Microsoft)
Probot™ Microfraction Collector
Process 450 (Datensystem für 8200er-Serie)
ProPac® Columns
ProteinChips® (Ciphergen)
Q
QSTAR® (Applied Biosystems)
R
Reacti-Therm™ (Pierce Chemical Company)
Reagent-Free™
S
SC-CSRS®
SELDI ProteinChips® (Ciphergen)
Self-Contained Cation Self-Regenerating Suppressor
SelectaPore™ Columns (The Separations Group)
Self-Regenerating Suppressor®
SEQUEST® (University of Washington, Seattle, USA)
Series 600 SFC and SFC/GC Systems
SFE-723 Supercritical Fluid Extractor
SmartFlow™
Trademarks and Product Names
SP10 AutoNeutralization™ System
SpeedVac™ (Savant Corp.)
SRC SRS Controller)
SRN™ Self-Regenerating Neutralizer
SRS® Self-Regenerating Suppressor
Summit® HPLC System
SUPELCOSIL™, Supelguard™ (Supelco, Inc.)
Superba columns
Superose® (Pharmacia)
Supor® (Pall Corporation)
SupraPur® (EM Industries, Inc.)
Switchos™ Microcolumn Switching Device
T
TAC-1 (Trace Anion Concentrator)
TCC-1 (Trace Cation Concentrator)
TCC-100 (Thermostatted Column Compartment)
Teflon®, Tefzel® (E.I. du Pont de Nemours & Co.)
ThermoFlare™
Thermos Column Oven
Triton® X-100 (Rohm & Haas)
TurboVap® (Zymark Corporation)
Tween® 20 (Atlas Chemical Co.)
U
UCI-100 or UCI-50 Universal Chromatography Interface
UI20 Universal Interface
ultraflex (Bruker BioSciences)
xiii
xiv
Trademarks and Product Names
UltiChrom™ software
UltiMate™
Ultrex® (J.T. Baker)
UV-Booster
UVD 170S/170U UV/Vis Detector
UVD 340S/340U Photodiode Array Detector
U-Z View™ Capillary Flow Cell
V
Vespel® (E.I. du Pont de Nemours & Co.)
VHP™ (The Separations Group, Inc.)
Vortex-Genie® (Scientific Industries, Inc)
Voyager™ (Applied Biosystems)
Vydac® columns (The Separations Group, Inc.)
W
Windows® 2000/XP (Microsoft Corporation)
Windows NT® (Microsoft Corporation)
Wonderware InTouch® (Wonderware Corporation)
Z
Zitex® (Norton Chemplast)
Zorbax® (E.I. du Pont de Nemours & Co.)
Abbreviations
xv
Abbreviations
Below please find a list of the most frequently used abbreviations in
Chromeleon, the online Help, and the manual:
¾AIA
Analytical Instrument Association
BCD
¾Binary-Coded Decimal Code
BOOTP Bootstrap Protocol (Internet protocol)
CAN
Controller Area Network
CD
Compact Disk
CDS
Chromatography Data System
CE
Capillary Electrophoresis
CFR
Code of Federal Regulations
(often synonymous with 21 CFR Part 11)
CM
Chromeleon® Chromatography Management System
CMB
¾Backup container/file (file extension)
CMS
Chromatography Management System
CS
Cluster Server
DHCP
Dynamic Host Communication Protocol
DNS
Domain Name Server
DX
Dionex
GC
Gas Chromatography
¾GLP
Good Laboratory Practice
GMP
Good Manufacturing Practice
GPIB
General Purpose Interface Bus
HPLC
High Performance Liquid Chromatography
IC
Ion Chromatography
IPC
Chromeleon Instruments Server or Instruments PC
¾IQ
Installation Qualification
Abbreviations
xvi
ISTD
Internal Standard
LAN
Local Area Network
LIB
UV Spectra Library (file extension)
LIC
Chromeleon ¾License Server
¾LIMS
Laboratory Information Management System
LOD
Limit of Detection
MAC
Media Access Control address
(unique address of each network interface card)
MS
¾Mass Spectrometer
NFP
¾Network Failure Protection
¾ODBC Microsoft Open Database Connectivity
¾OQ
Operational Qualification
PAN
¾Control Panel for chromatographic system control
(file extension)
¾PDA
Photodiode Array Detector
¾PGM
Control program for chromatographic system control
(file extension)
PN
PeakNet® Chromatography Management System
¾PPA
Peak Purity Analysis
¾PPI
Peak Purity Index
¾PQ
Performance Qualification
¾QNT
Quantification method for signal interpretation and evaluation
(file extension)
QRF
¾Query file (file extension)
RDF
¾Report Definition File; includes the layout definition
for the report and export (file extension)
¾RPC
Remote Procedure Call
RSD
Relative Standard Deviation
¾SDK
Software Development Kit for Chromeleon
Abbreviations
SEQ
¾Sequence; that is, the sequence of samples plus the
corresponding files (file extension)
¾SIM
Selected Ion Monitoring (MS channel for specific masses)
¾SOR
Signed Off Results (File) = signed sequence
¾SQL
Structured Query Language (computer language)
SST
¾System Suitability Test
TCP/IP
Transmission Control Protocol/Internet Protocol
(class of Internet protocols)
¾TIC
Total Ion Current
(MS channel for the entire mass range or a part thereof)
¾TTL
Transistor-Transistor Logic
UDP
User Datagram Protocol (IP communication protocol)
UDC
¾User-Defined Column
USB
Universal Serial Bus
VCD
¾Virtual Channel Driver (driver for virtual devices)
WSP
Workspace; that is, the definition of a screen layout
(file extension)
xvii
For a list of the ¾Institutes and Institutions for Industry Standards, refer to
the Glossary.
xviii
Abbreviations
Tutorial
Doc.: CM_Tutorial_660.doc
Table of Contents
T-I
Table of Contents
Chromeleon - Tutorial................................................. T-1
Table of Contents........................................................ T-2
Sequence - Flow Chart ............................................... T-4
I. Starting Chromeleon ............................................... T-5
II. The Browser ............................................................ T-9
Browser Appearance and Functions .................................................T-9
Opening Files .................................................................................... T-11
III. Online Help ........................................................... T-13
1. Context-sensitive Online Help..................................................... T-13
2. Systematic Online Help ................................................................ T-14
IV. Control .................................................................. T-17
1a) The Control Panel ....................................................................... T-17
1b) Connecting a Control Panel to a Timebase ............................. T-19
1c) Using the Control Panel............................................................. T-22
2. The Control File (PGM File).......................................................... T-26
3. The Program Wizard ..................................................................... T-27
4. Editing the Control File ................................................................ T-38
T-II
Table of Contents
V. The Analysis.......................................................... T-39
1. The Sample List (Sequence)........................................................ T-39
2. Starting the Analysis .................................................................... T-47
VI. Quantification Method (QNT File) ...................... T-51
1. Detecting Peaks (Detection Tab)................................................. T-53
2. Graphical Input of Detection Parameters................................... T-58
3. Automatically Generating the Peak Table .................................. T-60
4. Identifying Peaks (Peak Table Tab)............................................. T-62
5. Quantifying Substances (Amount Table/Peak Table Tabs)..... T-63
6. Defining the Calibration Mode and Calibration Type ................ T-67
VII. Data Analysis ...................................................... T-71
1. The Integration Window ............................................................... T-74
2. Modifying the Chromatogram...................................................... T-76
3. Report Tables ................................................................................ T-77
4. Modifying a Report Table ............................................................. T-79
5. Saving Changes ............................................................................ T-80
VIII. Printing Results ................................................. T-81
1. Creating a Print Template (Printer Layout) ............................... T-81
2. Printing........................................................................................... T-83
IX. Special Functions ................................................ T-87
1. Installing Datasources.................................................................. T-87
2. Finding Samples (Query) ............................................................. T-90
3. Backup and Restore ..................................................................... T-91
4. Signing Sequences Electronically .............................................. T-93
5. Modifying a Control Panel............................................................ T-98
6. Internal and External Standards................................................ T-100
Table of Contents
T-III
7. Spectra Library Screening ......................................................... T-101
8. Defining the Workspace ............................................................. T-103
Addendum: Keyboard Shortcuts/Short Glossary T-105
Keyboard Shortcuts........................................................................ T-105
Datasource and Database.............................................................. T-108
Raw Data.......................................................................................... T-109
Sequence ......................................................................................... T-109
(Chromatography) Server .............................................................. T-110
Timebase.......................................................................................... T-111
Index.............................................................................. T-i
T-IV
Table of Contents
Chromeleon - Tutorial
T-1
Chromeleon - Tutorial
Welcome to Chromeleon, the innovative chromatography management
system!
Use this tutorial to become familiar with Chromeleon. Learn how to
• Start the program
• Perform an analysis
• Reprocess your data
For an overview of the topics, refer to the ÎTable of Contents.
Tip:
The arrow (Î) references topics in the Tutorial.
There are no references between the Tutorial and the User and/or
Administrator Manual.
T-2
Table of Contents
Table of Contents
I.
ÎStarting Chromeleon: If your computer is
running under Windows NT; Windows 2000, or
Windows XP, you can start Chromeleon by
clicking Start > Programs > Chromeleon on the
task bar.
II.
Managing Data: Chromeleon allows you to
manage data in folders and directories similar
to Microsoft Windows. The tool that helps you
to handle your chromatography data is the
ÎBrowser.
III.
Online Help: The Tutorial can only deal with
selected aspects of Chromeleon. For more
information, refer to ÎOnline Help and/or to the
User Manual.
IV.
Controlling your HPLC system: Make sure
that your chromatography or CE instruments
are correctly connected to the PC via a serial
port. If the instruments are connected correctly,
you can operate the pump, autosampler,
detector, etc. from a ÎControl Panel on your
PC. In addition, you can create a ÎControl File
(PGM File)
to
control
your
system
automatically.
V.
Analyzing Samples: Can you control your
instruments from the PC? If you can, you can
start the chromatographic analysis. To use all
functions provided by Chromeleon, we
recommend that you create a ÎSample List
(Sequence) first:
Use the ÎSequence Wizard to include the
samples to process in the sample list. The
Wizard allows you to define the number of
samples, the order of sample processing, the
injection volumes, the sample type (analysis or
standard), and the location where Chromeleon
finds information about how to perform the
analysis.
Table of Contents
V.
(cont'd)
T-3
To make this information available to
Chromeleon, create the corresponding files
(PGM File, QNT File, see below) and enter a
program and a method name.
When you have created the sample list, you
can start the ÎAnalysis. The resulting data is
automatically saved.
VI.
Quantifying: The ÎQuantification Method
(QNT Method) allows you to minimize the
reprocessing effort for single chromatograms.
The QNT Method defines the detection
parameters, the peak identification, and the
calibration of the substances in several
samples.
VII.
Data Analysis: You can display the result of a
chromatographic analysis on the screen.
ÎData Analysis allows you to manually change
integration limits directly in the single
chromatogram or to re-evaluate samples based
on new parameters ("offline.")
VIII.
Printing Results: You can Îprint your
analysis data immediately after data acquisition
or at any time later. For perfect adjustment to
your personal requirements, create your own
report templates in the ÎPrinter Layout, define
your own result variables, or embed your
company logo.
IX.
Managing Data (Special Functions): In
addition to the basic functions, the Chromeleon
provides various special functions. For
example, you can search samples according to
defined criteria (query), backup and restore
data, electronically sign sequences, and search
spectra. For more information, refer to
ÎSpecial Functions.
For an overview of the most important steps in Chromeleon, see
ÎSequence - Flow Chart
T-4
Sequence - Flow Chart
Sequence - Flow Chart
I. Starting Chromeleon
T-5
I. Starting Chromeleon
Objectives of this chapter:
• To make you familiar with the basics of a client/server software
• To start the client and server programs
Chromeleon is a client/server program, operable under Windows 2000 and
Windows XP. The two independent program parts, the client and the
server, allow you to operate the program from different PCs on a network:
Server: The server PC is directly connected to the different modules of the
chromatography systems. The server communicates with the individual
modules of a ÎTimebase and controls them actively. You usually perform
only two actions in the server program: you start and stop the server. In the
client program, you specify which control actions the server shall perform.
Client: The client program allows you to work offline, i.e., to perform certain
tasks independent from the server, for example, creating sequences,
processing data, searching single spectra in a spectra library, etc. The
client can be installed locally. In this case, the client and server programs
can be installed on the same PC that is connected to the chromatography
system. This is typical for a single-site installation (one timebase only).
In addition, remote client PCs can be connected to the chromatography
server on the company network. You can then perform client functions on a
PC that is not connected directly to the chromatography system but to the
server PC on the network.
T-6
I. Starting Chromeleon
A characteristic of client/server systems is that certain processes are
performed on the client, while others run on the server. The cooperation
between the client and the server is crucial to the full functionality of the
program.
Since the client and the ÎServer are independent programs, you have to
start them separately.
Starting the Client Program
• Click Start > Programs > Chromeleon to start the Chromeleon client.
• If User Management was created and enabled during installation, a
logon dialog box appears. In this case, enter your user ID and personal
password.
Starting the Server Program
If you do not only want to view data but control an entire chromatography
system and perform your own analyses, start the Server Monitor program.
The server controls all connected instruments and allows direct instrument
operation and control via the PC.
• Select Server Monitor to start the Server Monitor program. The
Chromeleon icon appears on the Windows task bar next to the Windows
system clock.
I. Starting Chromeleon
T-7
Tip:
During installation, Chromeleon usually creates a link in the Autostart group
and the corresponding icon is displayed. You can also configure the
program in such a way that it starts whenever the computer is started.
• Move the mouse cursor to the icon. You will see the quick info message:
CHROMELEON Server is not running.
• Open the context menu (right-click) and select Start Server (or doubleclick the icon and click Start). The icon can have different colors. Gray
coloring indicates that the server is running idle.
• Both, the client program and the server program are active now. The
client control panel allows direct access to all instruments that are
connected to the server.
Note:
The instruments of a system are combined in a common ÎTimebase.
Instruments and timebases are installed and configured in the Server
Configuration program. Since your Dionex Service Representative usually
performs this task during the initial installation of Chromeleon, the Tutorial
does not provide more information about this.
T-8
I. Starting Chromeleon
II. The Browser
T-9
II. The Browser
The Browser window loads automatically whenever Chromeleon is started.
Opening the Browser
• Click the Browser icon
on top.
to open the Browser window or to display it
Browser Appearance and Functions
The Browser, which is the "control room" of Chromeleon, serves for data
management, storage, and retrieval. The Browser's appearance and
functions are similar to the Windows Explorer.
Caution:
Although the Browser is very similar to the Windows Explorer, you should
not confuse these two windows! Do not use the Windows Explorer for
operations within the Chromeleon datasources! Usually, your administrator
will prohibit these operations!
The left window section shows a tree structure with different levels. The
right window section shows detailed information about the currently
selected item.
T-10
II. The Browser
The ÎDatasources, for example, ComputerName_local
level of the structure and serve to store data and results.
, are the top
• Click the + or - character next to a datasource to expand or collapse the
corresponding directory structure (yellow folders).
The lowest levels contain the ÎSequences (blue folders:
) created by
the user. During installation, one directory is created. The directory name
corresponds to the name of the timebase. The timebase contains a
sequence that stores manually acquired data. Thus, it is called manual.
• Select a directory to display its subdirectories in the right window
section.
• Select a sequence to view files and samples contained in the
sequence.
When you click a sequence, the upper part of the right windows displays
the files contained in the sequence. The different icons indicate the file
types:
Symbol
File Extension
File Type
*.pgm
Control file or PGM File
*.qnt
Quantification method
*.rdf
Report template or report definition
*.pan
Control panel
The lower part of the right window displays the sample list showing the
individual samples of the sequence. The different icons indicate up to
seven different sample types:
Symbol
Name
Sample Type
Unknown
Unknown sample
Blank Run
Blank
Validation
Validation sample
Standard
Standard sample
Matrix
Matrix blank sample
Spiked
Spiked sample
Unspiked
Unspiked sample
II. The Browser
T-11
Opening Files
Double-click to open a file name in the Browser. Chromeleon loads the
appropriate chromatographic environment according to the file type:
• Double-click a control file (PGM File) to open the PGM Editor.
• Double-click a quantification method (QNT File) to open the QNT editor.
• Double-click a PAN file to open a control panel.
• Double-click a report template (report definition file = RDF) to open the
Printer Layout.
Opening Samples
When opening samples, consider the following (independent of the file
type):
• If raw data exist for several channels of a processed sample (Status =
finished, multiple, or interrupted), double-clicking the sample opens one
channel only (for example, a UV channel at a wavelength of 254 nm).
• Right-click a sample (here: sample1) and select Open from the context
menu. A submenu is opened on which Chromeleon lists all channels
that exist for this sample:
T-12
II. The Browser
If you record four different channels of a sample with a diode array detector
when you process the sample (as was in this example), a separate
chromatogram will be available for each channel (here: 3DFIELD and
UV_VIS-1 to UV_VIS-4).
• Select the channel or the chromatogram to display.
The highlighted channel (here: UV_VIS-1) is used as the "preferred
channel." The preferred channel automatically opens when you doubleclick a sample. Select Preferences on the File menu, and then specify
which channel Chromeleon shall use as the preferred channel.
Moving and Copying Items
Different ways are available to move or copy a subdirectory, sequence, or
file:
• Left mouse button: Use this button to move or copy the element
according to the settings made for the Left mouse button Drag/Drop
behavior on the Browser tab page of the Preferences (on the File
menu).
• Right mouse button: Use this button to move or copy the element as
desired.
• Keyboard shortcut: Use keyboard shortcuts to perform the desired
action, for example, <Crtl> <C>, to copy the element or <Crtl> <V> to
paste the element.
III. Online Help
T-13
III. Online Help
There may be issues and questions that are not discussed in this Tutorial.
In this case, refer to the User Manual and the online Help for more
information. You can access online Help anywhere in the program:
• ÎContext-sensitive Help answers your questions that may occur at the
current cursor position.
• ÎSystematic Help allows you to
• Select an interesting topic on the Contents tab page
• Search the index for any required information
• Search for specific words and phrases in help topics by
searching the entire text of the help system.
1. Context-sensitive Online Help
Access to the online Help depends on your current cursor position. Usually,
a window opens from which you can access systematic online Help. In
other cases, a popup window opens providing a short context-sensitive
description and links for access to related topics.
Help via the Context Menu
Right-click to open the context menu:
Select How to ... to display detailed information about actions that can be
performed at the current cursor position. Select What’s this? for a
description of the selected item, command, etc. In some cases, What’s
this? topics provide information that is more general. Usually, links are
available for specific information.
T-14
III. Online Help
Help via the Help Button and Icon
First, click the
icon on the toolbar to display a question mark
appended to the cursor, and then click the item of interest for specific
information.
In some cases, a Help button is available instead of the context menu or
the question mark. Click Help to display information about the current
screen.
In some windows in which you can enter parameters and variables, such as
in the QNT editor, click Explain Param. or Explain Variable for information
about your selection.
Help via the F1 Key
In addition, you can always press the F1 key. This help information
corresponds to the context-sensitive help information that is available via
the question mark cursor or the Help button.
2. Systematic Online Help
Select Index on the Help menu for an overview of the topics that are
available in the online Help. The help window provides the following tabs:
• Contents
• Index
• Find
Contents Tab
The Contents tab page contains the table of contents of the online Help.
Double-click a book symbol to display the topic titles. Double-click an open
book symbol to close the book again.
Topic titles are marked by a page symbol with a blue question mark.
Double-click to open the topic.
III. Online Help
T-15
Index Tab
The Index tab page enables you to find a specific term by searching the
index entries. Type the first few letters of the word you are looking for in the
first field until the desired term appears in the second field. Click the index
entry you want, and then click Display to open the desired topic. If several
topics are found, the Topics Found window opens. Select the desired
entry and then click Display.
Find Tab
The Find tab page enables you to search for specific words and phrases in
help topics by searching the entire text of the help system. (The query is
not limited to the index entries.) First, Chromeleon generates a list that
contains every word from the online Help file. You can then search the
resulting list for a specific term. The procedure is similar to searching for a
specific term on the Index tab page.
T-16
III. Online Help
The query finds all topics that contain the entered word(s). That is why the
Topics Found list is far more extensive than the one found when searching
the index. To narrow the search, you can select matching words in the
second field:
IV. Control
T-17
IV. Control
Objectives of this chapter:
• To describe the purpose and functions of a control panel
• To connect the devices to Chromeleon, using a control panel
• To display the current chromatogram
1a) The Control Panel
A control panel (often just called panel) allows you to control an entire
chromatography system from your PC. The system may consist of one or
several analytical instruments that are installed under the same timebase.
The control panel comprises the different elements required to control your
system and to display the current chromatogram.
The above image shows a control panel for controlling the basic functions
of a pump, an autosampler, and a two-channel UV detector. On the right,
you see the detector signal.
Chromeleon includes more than a hundred default control panels that cover
most applications. Although it is possible to change the existing panels, you
rarely have to do so. For more information, see ÎModifying a Control
Panel in the Special Functions section.
T-18
IV. Control
Opening a Default Control Panel
From the Browser
• Open the Dionex Templates > Panels directory and select the control
panel that corresponds to your system.
• Double-click to open the panel.
From the File or context Menu
• Select Open on the File or context menu.
IV. Control
T-19
• In the Open dialog box, select Control Panel in the Object of type
field.
• In the Look in field, select the local ÎDatasource of the system. This
datasource is automatically saved on your computer during the
Chromeleon installation. The datasource is named <NAME_LOCAL>,
wherein <NAME> is the computer name. If the Look In field contains
further datasources, you can also search here for an appropriate control
panel.
• Open the Dionex Templates > Panels directory and select one of the
default control panels (*.pan).
• Double-click to open the panel.
As soon as a control panel has been opened, Chromeleon tries to connect
to the ÎTimebase that is linked to this control panel.
If the connection cannot be established, the control elements are grayed
and an error message is displayed. In this case, connect the control panel
to the timebase as described in the next topic (see below).
1b) Connecting a Control Panel to a
Timebase
When a control panel is open, you can connect to any Chromeleon
timebase at any time. However, verify that the server on which the
timebase is installed has been started as described in ÎStarting
Chromeleon.
• Select Connect to Timebase on the Control menu to create and/or to
change the timebase assignment.
• In the dialog box that opens, determine the ÎServer and the
ÎTimebase to which you want to connect the control panel.
T-20
IV. Control
• If the server is started locally on your computer, click the + character to
open the My Computer submenu. If the server is running, the names of
all timebases that are installed on the respective server will be
displayed. If the server is not running, a corresponding message will
appear. In this case, start the server first and then select the desired
timebase, for example, HPLC. The Computer field will automatically
show your PC, that is, the name that was specified during the Windows
installation. The Protocol field shows the entry My Computer.
• If your PC is connected to other computers via a network or modem,
you can also access a server that was not started on your local
computer, but on another PC. To do so, the select the network protocol
under Protocol. (Access to other computers and timebases is usually
via the Internet TCP/IP protocol.) Open the Network Neighborhood to
browse for the required timebase.
• Chromeleon remembers the servers accessed on other computers.
These servers are listed under Favorites.
As soon as you complete the dialog, Chromeleon tries to access the
selected timebase. If communication fails, this may be due to several
reasons.
IV. Control
T-21
Possible Problems when Connecting to a Timebase
Cannot connect to timebase "..."
⇒ The error message indicates that the corresponding server is not
running, that the selected timebase does not exist on this server, or that a
wrong communication protocol has been selected.
• Start the server as described in Starting Chromeleon or select the
required timebase or protocol.
Tip:
Chromeleon can communicate via various network protocols such as IPX,
TCP/IP, or NetBEUI. Communication between two stations is possible only
if the same (!) network protocol is installed and selected. Generally, it is
sufficient to install the corresponding Microsoft ("IPX/SPX-compatible
protocol"; "NetBEUI"; "TCP/IP") or Novell ("Novell IPX ODI Protocol")
network protocols via Settings > Control Panel > Network >
Configuration. The protocol that is actually used depends on the current
network installation. Please contact your network administrator.
This timebase contains no device/object named "..."
⇒ The control panel tries to access an instrument (or a function of this
instrument) that is not part of the current installation environment. The
control panel and installation environment do not match. Load the
appropriate control panel or change the existing configuration of the
timebase in the Server Configuration program. For more information, refer
to the online Help and/or the User Manual.
Device is not remote.
⇒ The control panel tries to access an instrument that is currently not
ready for operation. Check whether all instruments listed in the
configuration are actually connected to the server PC and that they are
turned on. It may be necessary to restart the instrument to ensure proper
functionality.
T-22
IV. Control
The connection is established correctly (no error message), but the
controls are inactive.
⇒ The Monitor Only mode is enabled. It is not possible to actively operate
the control panel. The Monitor Only mode is always enabled when the
selected timebase is already controlled by a different workstation. Disable
the Monitor Only mode on the Control menu. You can now actively
control the timebase. (This is indicated by the controls on the control
panel.) Simultaneously, the previously controlling workstation loses the
control rights and is in Monitor Only mode.
Tip:
Have a look at the Audit Trail entries. They often provide precious
information that helps you to correct the error.
1c) Using the Control Panel
As soon as a control panel is correctly connected to a timebase, you can
operate the individual instruments of this timebase using the controls
provided on the panel.
Depending on the control panel that is currently defined as default, various
active and passive controls and display elements (Lamp, ... Signal Plot)
are available for controlling instruments and displaying system functions.
You can change the appearance of the controls (size, shape, and color).
IV. Control
T-23
For example, the above control panel allows you to control an HPLC
system that consists of a pump, an autosampler, a UV detector, and a
fluorescence detector. In this example, all modules except the fluorescence
detector are connected. Different buttons are available for issuing control
commands such as Inject+Acq.On (for injection and data acquisition),
autozero, etc. In addition, the current protocol data (Audit Trail) are
displayed. After data acquisition has been started, the current
chromatogram is displayed in the signal plot.
The control panel is usually configured during installation. We recommend
storing the control panels in the directory of the corresponding timebase.
(You cannot store them in the Dionex Templates directory because this
directory is read-only).
Online-Toolbar: The basic commands such as the pump flow control, the
inject command, or the start of the data acquisition are available on a
separate toolbar. The toolbar is only active when a control panel is open.
T-24
IV. Control
The functions of the single buttons are as follows (from left to right):
• Flow...: Define the flow and gradient composition and start the flow
• Inject...
• Acquisition on/off: Start and stop data acquisition
• Stop Flow: Stop the flow, interrupt data acquisition, and hold the sample
• Hold: Freeze the solvent composition and hold data acquisition and the
sample
• Continue: Continue the flow, gradient, and sample
• Edit Batch: Edit the order in which the samples are processed
• Start/Stop Batch
However, how does the system know that moving a slider should change
the pump flow, and not the oven temperature? In addition to changing the
size, color, and shape of each control, you can also change its functionality.
These assignments are preset for the controls provided on the standard
control panels. Although you can adapt them to your requirements, this is
seldom required. For more information, see ÎModifying a Control Panel in
the Special Functions section.
Manual Sample Processing/Starting the Analysis
If the panel is connected to the corresponding timebase, you can use the
Online toolbar to start sample analysis:
• Open the control panel and connect to the desired timebase.
• Select Flow... on the Control menu (or click the
icon on the Online
toolbar). Enter the flow rate and the solvent composition the pump shall
deliver. The pump immediately adjusts to the selected settings.
icon on the
• Select Inject... on the Control menu (or click the
Online toolbar). Specify the position from which to draw the solvent and
the quantity (in µl) you want to draw and inject. This command, too, is
executed immediately. Injection is performed directly after the command
is issued.
• Select Acquisition On on the Control menu (or click the
icon on
the Online toolbar). Define the signals to record and click OK to start
data acquisition. Chromeleon records the data supplied by the detector.
IV. Control
T-25
You can also execute these commands directly from the control panel if
your panel supports the corresponding option.
• To finish data acquisition and to complete the analysis, select
Acquisition Off on the Control menu or click the Acquisition on/off
icon again on the Online toolbar.
If your control panel is connected to a timebase on the local server, the
recorded data is usually saved to the Manual sequence in the <Timebase
Name> directory of the local datasource. This sequence is automatically
generated during installation; it contains one sample only.
Note:
With each new manual sample processing, the existing data is overwritten
by the data of the newly analyzed sample (default setting). However, using
the Save to sequence command, you may also save the data to any other
sequence after data acquisition. Use this type of sample processing only for
test runs etc.!
You will usually want to analyze several samples automatically one after
another. To do so, you have to define both the order in which the different
samples shall be processed and the commands that are required at the
respective time. In Chromeleon, the processing information is contained in
a ÎSample List (Sequence). The information about the command order is
part of a ÎControl File (PGM File).
T-26
IV. Control
2. The Control File (PGM File)
Objectives of this chapter:
• To describe the definition and purpose of the program
• To introduce the Program Wizard that guides you through program
creation
• To describe the functions for program reprocessing
The different commands instruct the single instruments of a
chromatography system to perform specific tasks. Enter the commands via
the controls on the control panel or by clicking the corresponding buttons
(for more information, see the Control Panel section).
For automatic and synchronized operation, the single commands must be
listed in a file. When starting the file, the listed commands are executed
consecutively, observing the relative time differences. This type of file is
called control file or PGM File.
Control File Properties
For a very simple control file, the PGM Editor could look like this, for
example:
IV. Control
T-27
In the Command view, the various commands of the PGM File are listed
on the right below the Title line. The commands are assigned to the
corresponding retention time. For information about how to create a
program, refer to the following sections.
3. The Program Wizard
Creating a Program
We recommend that you use the Chromeleon Program Wizard to create a
basic program structure and thus, avoid syntax errors when you enter the
commands.
• Verify that the server is running. If the server is not running, start the
server as described in ÎStarting Chromeleon.
• Select New on the File menu.
• Select Program File from the list and click OK to start the Program
Wizard.
Depending on the installed instruments, the Program Wizard provides
various steps. Below please find an example describing the different steps
required to create a program for a typical HPLC timebase that contains the
following devices:
• P680 Pump
• ASI-100 Autosampler (without cooling option) or AS50 Autosampler
• TCC-100 Thermostatted Column Compartment
• UVD340U Photodiode Array Detector
For more information about each Wizard page, press the F1 key or click the
Help button. Click Back or Next to toggle between the pages.
T-28
IV. Control
Step 1: Timebase and Server
Determine the server and the timebase to be used:
• If the server was started locally, click the + character to display the items
under My Computer, and then select the desired timebase. Select My
Computer in the Protocol field. If you want to access a server that was
not started on your computer but on a remote PC, click the + character
to display the items under Network Neighborhood, and then select the
appropriate timebase. In this case, you also have to select a network
protocol in the Protocol field. For more information, press the F1 key.
Clicking Next takes you to the next wizard page. Click Finish to
generate a program with the settings made on the different wizard
pages.
IV. Control
T-29
Step 2: ColumnOven Options (here: Dionex TCC-100)
Use this Wizard page to set the nominal temperature and the upper and
lower temperature limits:
Step 3: Pump Options (here: Dionex P680)
Use this Wizard page to define how to operate the pump:
• From the Type drop-down list, select Isocratic to deliver a constant
solvent composition. If more than one solvent is used, the Start field
determines the solvent composition in percent (for example: B = 45%,
C = 0%, D = 5%). %A is calculated by the system from the portions of B,
C, and D. Enter the flow rate in the Start field under Total Flow.
T-30
IV. Control
• If you select Ramp instead of Isocratic, use the Start and End input
fields to set the start and end time of the gradient ramp. You can also
select a Multi-Step Gradient. In this case, an additional Wizard page is
opened when you click Next.
Step 3a: Flow Gradient Options (here: Dionex P680)
If you selected Multi-Step Gradient from the Type drop-down list, use this
Wizard page to specify the desired gradient by entering the:
• Retention time
• Flow
• Solvent composition
Tip:
Before you enter a multi-step gradient, determine the flow rate and the start
composition for the solvent on the previous Wizard page. This facilitates the
entry.
IV. Control
T-31
To append lines to the table, place the cursor in the last line of the table
and press the arrow down key on the keyboard. The window will show the
graphical representation of your gradient. A blue line indicates the flow,
while the area represents the gradient composition. The following example
shows a gradient at a flow rate of 1 ml/min, with a constant 5%D during the
entire gradient. The portion of solvent B changes according to the entries
made in the %B column.
Note:
If you have a Dionex GP40, GP50, or GS50 pump installed, you can
program curved or linear gradients. The default is Curve 5, that is, a linear
gradient. Curves 1 to 4 result in a convex gradient. Use curves 6 to 9 for a
concave gradient.
T-32
IV. Control
Step 4: Sampler Options (here: Dionex ASI-100)
Use this page of the Wizard to determine the autosampler options:
• Determine the syringe and sample options:
• Dispense Speed: Enter the speed with which the injection shall be
performed.
• Draw Speed: Enter the speed with which the syringe is filled.
• Sample Height: Enter the height at which the sample is drawn,
measured from the vial bottom to the needle tip.
• Syringe Delay: Enter the time that the needle will remain in the vial
after loading.
• Determine the speed for the needle movements:
• Up/Down Speed: Enter the speed for moving the needle up and
down.
• Radial Speed: Enter the speed for moving the needle radially.
• Select the Synchronize injection with pump check box to synchronize
injection with the pump cycle.
IV. Control
T-33
Or else: Steps 4 + 4a: Sampler Options
(here: Dionex AS50)
If you have an AS50 autosampler installed, use this Wizard page to define
the autosampler options that do not change during the program:
• If you have the respective option installed, define the Column
Temperature and the Tray Temperature. Select the Wait for stable
temperature check box to wait before the injection until the temperature
is stable.
• In the Cycle Time field, enter the time between two injections.
• In the Syringe Speed field, define the speed for drawing the sample.
• In the Sample Needle Height field, define how deep the needle will dip
into the vial, that is, the position from which the sample is drawn.
• In the Cut Volume field, enter the sample volume that is finally
dispensed into the waste.
• In the Flush Volume field, specify the volume for flushing the injection
port.
T-34
IV. Control
Also, define the different AS50 sample preparation steps:
Note:
In the example, the different functions have been entered one after the
other. A "real" application would look different, of course.
• First, select the desired function in the Function column. Seven
different functions are available.
• Determine the desired parameters. (For example, for the Dispense
function these are the Source, the Volume, and the Destination
parameters.)
• Click Insert to enter the sample preparation step. To delete an existing
sample preparation step, select the respective step and click Delete.
IV. Control
T-35
Step 5: Acquisition Options
(here: especially Dionex UVD 340U)
Determine the signals to be recorded during sample processing.
• Select the signals. Under Acquisition Time, specify how long the
individual sample shall be analyzed.
For a photodiode array detector, for example, an entire 3D field and the
UV_VIS_1 and UV_VIS_2 channels are recorded for 10 minutes (From
0.000 min to 10.000 min). 0.000 means that data acquisition starts
immediately after injection (t = 0.000 min).
Step 6: Signal Parameters
(here: especially Dionex UVD 340U)
Define the signal parameters for each signal selected in step 4.
• Depending on the signal type, you must set various parameters (for
example, Excitation Wavelength for the fluorescence detector). In the
case of a UV detector, specify the wavelength, the bandwidth, and the
step used for recording (for example: Wavelength = 225nm,
Bandwidth = 1nm, Step = Auto, Average = On (selected)).
T-36
IV. Control
• In case of ECD channels, specify the Data Collection Rate (= number
of collected data points per second) and the SRS Current (= suppressor
current).
• In addition, enter the temperature compensation factor and the cell
temperature as desired.
IV. Control
T-37
Step 7: Completing and Saving the Program
• Click Finish to complete the Program Wizard. In addition to your input,
the complete program displayed in the PGM Editor also includes
commands that are automatically added by Chromeleon, such as the
Inject and End commands. These commands are required to execute
the file. When these commands are missing, the program can be
executed in exceptions only. The complete program could look as
follows:
• You can edit a program later, as necessary. If you enter invalid
commands, Chromeleon recognizes them and marks the respective line
in red color (Glow instead of Flow in the above example). For more
information about the error, refer to the status bar.
• Select Save as on the File menu to save the PGM File under a
descriptive name.
T-38
IV. Control
4. Editing the Control File
You can edit each line directly via the keyboard. Make sure that the control
file is connected to a timebase. If it is not, Chromeleon cannot perform the
Ready Check and input via the F8 key (see below) is not supported.
• To open the PGM File, select Open on the File menu or double-click the
corresponding file name directly in the Browser.
• Verify that your server is running. If the server is not running, start the
server as described in ÎStarting Chromeleon.
• Select Connect to Timebase on the Control menu.
Note:
If the timebase to which the program is connected is not available, select
Connect to Timebase on the Control menu to connect the program to a
different timebase.
You can now edit the program. Instead of overwriting the command syntax
directly in the PGM File, we recommend to proceed as follows:
• Move the mouse cursor in the line to edit and press <F8 key>.
Chromeleon opens the Commands dialog box for the currently selected
command. You can now edit the input by selecting defined values,
without having to worry about the correct command syntax.
• Save the edited PGM File using the Save as command.
V. The Analysis
T-39
V. The Analysis
1. The Sample List (Sequence)
Objectives of this chapter:
• To describe the definition and purpose of a sequence
• To introduce the Sequence Wizard that guides you through sequence
creation
• To describe the functions for sequence reprocessing
• To automate the analysis by using a sample batch
To allow a chromatographic system to process several samples one after
another without interruption, you have to define the order and the program
to be used for sample processing. Chromeleon stores this information in a
sequence.
Blue folders mark sequences in the left Browser pane (see "Isocratic" in the
following picture). When you click a sequence, the right pane displays all
files and samples contained in the sequence. The upper right Browser pane
is reserved for files that are required for sample processing. The lower
section lists all samples of the selected sequence. This section is referred
to as Sample List.
T-40
V. The Analysis
Sample List Contents
The sample list forms the basis for sample processing. It lists the samples
intended for processing (one line corresponds to one sample) and indicates
how processing is or was performed (contents of individual columns).
No.
Name
Type
Position
Inj. Vol.
Program
Method
Status
Inj. Date/Time
Weight
Dil. Factor
ISTD Amount
Number of the sample in the sequence.
Sample name. The symbol indicates the sample type.
Sample type.
Unknown: unknown sample
(symbol in front of the sample name: )
Standard: calibration sample ( )
Validate:
sample for validating the calibration ( )
Blank:
blank value (usually without injection) ( )
Matrix:
blank value of the sample matrix ( )
Spiked:
spiked sample ( )
Unspiked: unspiked sample ( )
Sample position in the autosampler.
Injection volume in µl
ÎControl File (PGM File)
ÎQuantification Method File (QNT Method)
Sample status
Single:
The sample has not yet been processed.
Multiple:
The sample can be analyzed several times
(the old sample will be overwritten).
Finished:
The sample has already been processed.
Running:
The sample is currently being processed
(green background).
Interrupted: The sample was interrupted during
processing.
Injection date and time
Sample weight
Dilution factor
Amount (quantity or concentration) of the used internal
standard
Samples are always processed in the order of their appearance in the
sample list.
V. The Analysis
T-41
1.a) Creating a New Sequence
The easiest way to create a new sequence is to use the Sequence Wizard.
As an alternative, you may as well save an existing sequence under a new
name and then modify the sequence as desired.
Start the Sequence Wizard to create the basic structure of a sample list.
Starting the Sequence Wizard
• Select New on the File menu.
• From the list, select Sequence (using Wizard).
• Click OK to start the Sequence Wizard.
A sequence is created in five steps: Each step is performed on a separate
page. For each page, detailed information is available via the F1 key or the
Help button. Click Back or Next to toggle between the pages.
Step 1
Specify the timebase on which the sequence to be created shall run. Select
a timebase in the right window section. The corresponding entries for
Computer and Protocol are automatically made.
T-42
V. The Analysis
Step 2
Create all unknown samples to be processed.
V. The Analysis
T-43
• Select Use Template.
• Enter a sample name and complete the name using the arrow key to
add, for example, #n. This is to number the samples consecutively, for
example, sample#n. If you want to inject several times from the same
vial, it may make sense to use the position number (#p) and the
injection number (#r) as well (see the example above).
• Enter the number of injections (Injections per Vial).
• Specify the position of the first sample (Start position).
• Specify the sample volume to inject (Injection Volume).
• Click Apply to display the result of your input in the sample list
(Sequence Preview). If the result requires editing, for example,
because the injection volume should vary or individual vials are not
positioned consecutively, you can change the information manually in
the Browser after closing the Sequence Wizard. The Rack Preview
displays the positions of the samples in the rack.
Step 3
Create all standard samples to process.
T-44
V. The Analysis
• Proceed as described in step 2. In addition, specify any number of
standards (Variation:) that shall be analyzed after a specified number of
samples (after each).
Step 4
Determine how to process, evaluate, and print the sequence.
• Enter the name and the directory of the ÎPGM File and the ÎQNT File
to be used in the Program and Quantification Method fields. Or else,
click Browse... and navigate to the desired file. If you have not
generated a new PGM File or QNT File before, you can leave these
fields empty for now.
• For ion chromatography systems, you can select an application
template instead. This template provides the appropriate PGM and QNT
files. First, specify the suppressor type you are using:
V. The Analysis
T-45
• For HPLC, IC, and GC sequences, select the DEFAULT.RDF report
template in the Preferred Report field. The template is provided in the
REPORT directory of the local database. The Preferred Channel field
determines the channel for which data are displayed.
Note:
You can leave the Quantification Method, Preferred Report, and
Preferred Channel fields empty here. Any input is optional only.
T-46
V. The Analysis
Step 5
Specify the name for the sequence to create and where to save it.
• Enter any name in the Sequence Name field and select, for example,
the local data <USERNAME_LOCAL> from the Datasource field.
Generate a separate sequence directory by specifying a directory name
in the Directory field (you can also create a new directory).
Tip:
Do not use special characters (such as an umlaut) in sequence and
directory names as this may cause problems in Novell networks!
• On the last Wizard page, click Done to complete your input.
V. The Analysis
T-47
1. b) Editing an Existing Sequence
After you have clicked Done, Chromeleon generates a sequence based on
the entered information. The sequence is displayed in the Browser. As
mentioned before, the sequence is a basic structure that needs to be
adjusted to special requirements.
For example, if you wish to inject different volumes, you can edit the
sequence later accordingly. Or else, if you have not specified the PGM
and/or QNT file in step 4 of the Sequence Wizard, you can select this file
later. Edit the sequence afterward in the Browser.
• In the sample list, change individual fields directly via the keyboard or
open an edit box by pressing the F8 key.
• You may add additional samples. (Select Append Sample or Insert
Sample on the context menu. Or else, place the cursor in the last line,
and then press the arrow down key.)
• Of course, you can also copy and paste or delete samples via the
corresponding commands on the context menu.
Note:
If you did not specify a control file when you created a sequence (Step 4 of
the Sequence Wizard), the Program column cells on the sample list are
empty! In this case, copy the required PGM File to the sequence directory.
For each sample, select the PGM File to be used for processing.
2. Starting the Analysis
Analyzing one sample can be performed manually. For several samples,
automatic sample processing is recommended. This requires a ÎPGM
File, a ÎSequence, and a ÎQuantification Method (if possible).
Automatic Sample Processing
You can start automatic sample processing as soon as the
chromatographic conditions, the samples to process, and their order have
been defined during sequence creation.
Automatic sample processing is also called Online Batch or Batch
Processing.
• Open a Control Panel. Select Edit on the Batch menu. Or else, click the
following icon on the Online toolbar:
T-48
V. The Analysis
• On Batch List tab page, click Add to open the Browse dialog box.
Under Object of Type, select Sequence from the drop-down list box.
Select the desired sequence in your datasource and click Open.
This action enters the path and the name of the sequence in the Batch
dialog box. If a sequence contains a sufficient number of samples,
sample processing is possible around the clock. Instead of listing all
samples in one sequence, you can create several sequences. In this
case, enter all required sequence names in the Batch List.
• Repeat adding sequences until all required sequences are listed in the
display field.
The order of the listed sequences determines the order of sample
processing: When starting a batch, samples 1 to n of the first sequence are
analyzed, followed by samples 1 to n of the second sequence, etc.
• Select a sequence and change the processing order by clicking the
Move Up and Move Down buttons.
• Click Ready Check to check whether automatic sample processing
(batch) is possible; i.e., Chromeleon checks whether all devices to be
used are ready for use (turned on, connected, lamp switched on, etc.) In
addition, this action verifies that all data are available and the memory
capacity is sufficient.
• Click Start to start the analysis.
V. The Analysis
T-49
As soon as the online batch starts, all samples of the sequence(s) that
have the status Single and Multiple (depending on the setting those with
the status Interrupted as well) are analyzed in the listed order. During a
running batch, the currently processed sequence is labeled with a green
triangle in the batch list.
T-50
V. The Analysis
VI. Quantification Method (QNT File)
T-51
VI. Quantification Method
(QNT File)
VI. Quantification Method (QNT File)
Objectives of this chapter:
•
To define a method for peak detection and recognition, quantification,
and evaluation
•
To use this method for many samples and sequence and thus, save
time
Introduction
After a chromatogram has been recorded, you need to integrate and assign
the peaks first before you can quantify them. It would be a time-consuming
task to do this in the report for each single sample. That is why all these
steps are combined and saved in the QNT Method. Nevertheless, it is
possible to edit a single sample in the report later. However, the better the
QNT Method evaluates your samples, the less effort will be necessary for
reprocessing the samples.
Strictly speaking, the QNT File is required after the analysis only. However,
we recommend creating the file earlier. This allows you to follow and
evaluate the course of the analysis.
Creating a Quantification Method
• Select New on the File menu.
• Select Method File as file type and click OK to create a new method.
T-52
VI. Quantification Method (QNT File)
The QNT File Window
The QNT File window contains various worksheets (General, Detection,
Peak Table, Amount Table, Peak Tracking, MS Tracking, Calibration,
Spectra Library Screening, SST, and MS). Open the required worksheet
by clicking the respective tab on the lower window bar.
To create a simple evaluation method, it is sufficient to enter the Peak
Table settings.
Creating a Peak Table
The peak table allows you to recognize peaks (= ÎDetecting Peaks),
assigning names to the peaks in a chromatogram (= ÎIdentifying
Substances), and converting the determined peak areas into substance
amounts (= ÎQuantifying Substances and ÎDefining the Calibration Mode
and Calibration Type). The required information is usually entered before
the analysis.
Saving the QNT File
Use the Save as command to save the quantification method under a
separate name.
VI. Quantification Method (QNT File)
T-53
Editing the QNT File
When the QNT File is edited, all changes will be implemented immediately
in all involved components (only if the Auto Recalibrate check box has
been selected on the General tab page).
If you have opened a channel of a specific sample (see: ÎThe Browser Appearance and Functions) and you see the corresponding chromatogram,
this representation is updated immediately after changing the QNT File.
The same applies to the representation of numerical values as they appear
in any report.
Caution:
If you have not specified a quantification method when creating a sequence
(Step 4 of the ÎSequence Wizard), the Method column cells in the sample
list will be empty! Enter the name of the desired QNT File for each of the
samples. To evaluate all samples of a sequence with the same QNT File,
select the QNT File for the first sample, and then copy the entry for all
entries using the F9 key.
1. Detecting Peaks (Detection Tab)
Before peaks can be identified and quantified, they have to be detected.
Based on default values for the peak recognition algorithm, Chromeleon is
able to detect even the smallest peaks. Normally, however, you will not be
interested in these small peaks so that you may want to include certain
filters to exclude them from being displayed. This and other detection tasks
are defined on the Detection tab page.
Double-click the default parameter (Minimum Area = 0.001 {Signal)*min) to
open the Edit detection parameter dialog box:
T-54
VI. Quantification Method (QNT File)
Frequently used Detection Parameter
Minimum Area
Usually, the Minimum Area default parameter will be sufficient as filter.
Minimum Area defines the area threshold, below which peaks are not
identified during peak detection or integration.
In HPLC-UV, 1.000 mAU*min usually is an appropriate value. Set the
parameter to the desired value and click OK to accept the changes. This
action usually suppresses the small peaks that are of no interest:
VI. Quantification Method (QNT File)
T-55
To add more detection parameters, select the Lines command, and then
select Append Lines. You can also use the Minimum Height, Minimum
Width, Maximum Peak Height, and Maximum Width detection
parameters as additional filters.
Tip:
Changing the Minimum Area, Minimum Height, or Minimum Width
parameters influences the baseline; changing the Maximum Area Reject
or Maximum Height Reject parameter does not. If you change the
Maximum Area Reject or Maximum Height Reject parameters, it may
happen that the corresponding peaks are no longer displayed.
Inhibit Integration
Use the Inhibit Integration detection parameter to disable peak
integration. If the parameter is set to On, peak detection is disabled. This
may be useful for disabling the integration of injection peaks. Negative
injection peaks may cause an undesired baseline; for example the Water
Dip peak in ion chromatography. Enable the Inhibit Integration detection
parameter to avoid this effect:
T-56
VI. Quantification Method (QNT File)
If the value is set to On before the first peak to be inhibited, peak detection
is disabled until the parameter is disabled (Off), i.e., no peaks are
recognized. The chromatogram is drawn on the screen but it is not
integrated in this area.
Inhibit Integration can be used to inhibit the injection peak by enabling the
parameter at the start time of the chromatogram and by disabling it shortly
after the void time. (In the above example, the retention time of the
negative peak is 2.513 min.)
If the first peak follows shortly after the negative water peak and if the
retention times of the separate chromatograms show considerable
fluctuations it may be difficult to set the end time for Inhibit Integration
exactly. If the end time is set too early or too late, the first peak may not be
integrated as desired.
In this case, use the Void Volume Treatment parameter instead. If the
peak start is still set too early, use the Fronting Sensitivity parameter to
remedy the situation.
VI. Quantification Method (QNT File)
T-57
Note:
The Void Volume Treatment parameter only inhibits integration of the negative peak
with the lowest signal value.
Detect Negative Peaks
The default setting is that negative peaks are not detected. Negative peaks,
for example, the negative water peak in ion chromatography, may cause an
undesired baseline. Enable the Detect Negative Peaks detection
parameter to avoid this:
T-58
VI. Quantification Method (QNT File)
To detect a negative peak this switch must be activated before the peak
start. To correct the baseline without labeling the peaks or including peaks
in the peak list, select Don't label.
Note:
In the result report, the area of negative peaks is indicated as a positive
value.
More Detection Parameters
Use the Rider Threshold and Maximum Rider Ratio detection
parameters to define the peaks that shall be classified as riders. Use Rider
Skimming to define how to divide a rider peak from the main peak.
Besides, you can use the Valley to Valley parameter to enforce valley-tovalley integration, and the Fronting Sensitivity Factor or Tailing
Sensitivity Factor parameters to influence the determination of the peak
start and peak end.
2. Graphical Input of Detection Parameters
Click the detection parameter tool to display the position of the detection
parameters in the chromatogram.
This is especially useful if you want to change the position of detection
parameters or graphically insert additional parameters. Use the Detection
Parameter Tool to change the position of detection parameters. Open the
tool by selecting the respective command on the context menu or by
icon. In the chromatogram, the parameters are marked by
clicking the
a dotted line at which the abbreviation for the parameter and the respective
parameter value is indicated. For example, the figure below shows the
values for the Rider Threshold (in short: RidThd), Rider Skimming (RidSki),
and Maximum Rider Ratio (RidRat) at 1.000 min. Position the mouse on a
detection parameter in the chromatogram to activate the detection
parameter tool so that you can move this parameter via the left mouse
button.
VI. Quantification Method (QNT File)
T-59
To insert a detection parameter at the position of the mouse cursor, select
Detection Parameters on the context menu:
For example, this setting enforces valley-to-valley integration as from the
selected time.
You can also enter detection parameters graphically by holding down the
right mouse button and selecting a chromatogram area. The context menu
provides the following options:
• Set Averaged Baseline Start and Set Average Baseline End
• Set Background Subtraction Range
• Set Minimum Area
• Set Minimum Height
• Set Minimum Width
• Set Peak Slice & Sensitivity
• Set Inhibit Integration Range
T-60
VI. Quantification Method (QNT File)
Tip:
You can undo the graphical input of detection parameters. Click one of the
QNT editor tables and select Undo on the Edit menu. (In the
chromatogram itself, you can only undo changes that were made in
currently open chromatograms.) In the Detection table of the QNT Editor,
you may just as well delete the parameters that are no longer required.
3. Automatically Generating the Peak Table
You can create the peak table manually by entering the single peak names
and retention times. However, generating the peak table automatically by
using the Autogenerate Peak Table command considerably facilitates this
task. (For more information, refer to ÎAutomatically generating the Peak
Table.)
Select Autogenerate Peak Table on the context menu. Chromeleon
automatically generates the peak table and enters the retention times of all
peaks detected in the currently open chromatogram as set times in the
QNT Method. You only have to complete the table by entering component
names and altering the default window and other values as necessary.
Peaks, which are not of interest, can be deleted from the table. These will
consequently be excluded from the report if the Including all not detected
peaks of the peak table parameter in the Integration Report Properties
has been disabled. (To change the setting of this parameter, select Table
Properties on the Edit or context menu.)
VI. Quantification Method (QNT File)
T-61
During automatic table generation, the variables described below are set as
follows:
• The Peak Names are constructed from the name of the QNT Method,
for example, in the Anion Qnt method, detected peaks will be assigned
the names Anion Qnt -1, Anion Qnt -2, Anion Qnt -3, etc.
• The Window values are entered as absolute values (indicated by the
suffix A).
• No Group.
• The Amount values and the Response Factors are set to 1.0.
• The Peak Type is Auto (i.e., based on the peak, it is determined by
Chromeleon).
• The entry in the Comment column is Autogenerated.
When you generate peak tables via the Autogenerate Peak Table from
the Edit menu, all peaks in the chromatogram are usually enumerated
(type Enumerate peaks of current chromatogram). However, you can also
use the results of the spectra library screening (see ÎSpectra Library
Screening) for peak naming (type: Use spectra library screening results).
T-62
VI. Quantification Method (QNT File)
4. Identifying Peaks (Peak Table Tab)
The Peak Table is used to assign peak names to all peaks of interest in a
chromatogram. Peaks are typically identified by the retention time.
However, it is also possible to identify peaks by the corresponding spectra.
• Select the Peak Table tab to open the peak table.
• Enter the names of all peaks to be identified in the Peak Name column.
Assign the expected retention time to each peak in the Ret.Time
column (= nominal retention time (Tret)). (You can add additional lines
using the Lines .../ Append Line commands on the context menu).
If a peak is detected at the specified time, the name is assigned
automatically (fig. a). An additional tolerance range for peak detection is
defined in the Window column (fig. b). The width corresponds to the double
Window value. To determine the retention time area, this value is added or
subtracted from the retention time.. If a peak is detected in this range, it is
identified even if the actual retention time does not correspond exactly to
the entered nominal retention time (fig. c). If several peaks are detected
within this range, the Chromeleon identifies the greatest, the first, or the
nearest peak to the retention time (fig. d), depending on the extension of
the Window parameter.
VI. Quantification Method (QNT File)
set
peak name
actual
window
T-63
Tret (f)
Tret (n)
Tret (g)
Tret
a)
b)
c)
d)
• In the Window column, enter, for example, 0.25 AG, for each peak to
identify the largest peak within a 30-second window. Time input is in
industry minutes. For example, the value 1.00 corresponds to 60 s. The
value 0.25 thus corresponds to 15 s, which means a window range of
±15 s. The setting 0.25AF identifies the first peak; the setting 0.25AN
identifies the nearest peak to the set retention time.
5. Quantifying Substances
(Amount Table and Peak Table Tabs)
You can use standard samples (sample type: Standard) to quantify
unknown samples. To do so, you have to enter the known substance
amount of these standard samples in the amount table together with the
standard method. Enter this information in the Amount and Standard
columns.
Amount Column
• Search the amount table for the name of the standard substance(s). If
the required names are not listed, enter the names and the retention
times as described ÎIdentifying Peaks.
• Move along the line to the Amount column.
• Enter the amount values of the substances (Substance A, Substance B,
etc.).
T-64
VI. Quantification Method (QNT File)
Enter either a concentration value such as µg/µl or an absolute value such
as µg. (Enter the used dimension in the Amount Interpretation section on
the General tab.)
Multi-Point Calibration
Multi-point calibration can be achieved by
• Using different standards with different concentrations
• Injecting different volumes from a single sample vial
Different standards with different concentrations
If standards are available in various concentrations, i.e., in several vials,
enter the concentrations of all substances to be calibrated in a separate
Amount column. Do this for each vial from which injection is performed.
• To add additional Amount columns, select Columns.../Edit Amount
Columns... on the context menu:
• Standard samples, validation samples, and spiked samples are
available for this.
VI. Quantification Method (QNT File)
T-65
• Click New to create a new amount column. Enter any name of your
choice.
• Select Unassigned from the left list box and then move the desired
sample to the new column. (For example, Sample 2 in the above
image.)
• Enter the concentration value of the second vial in the new Amount
column.
Injecting different volumes from a single sample vial
If the range to calibrate does not contain several powers of ten, you can
inject different volumes from the same vial. Chromeleon automatically
considers the different volumes. In this case, you only need one Amount
column, as the concentration is constant.
For this type of multi-point calibration, the decisive column of the sample list
in the Browser is the Inj. Vol. column. The column could look as follows:
If you enter the Amount as amount value (and not as concentration value),
make sure that the amount corresponding to the reference injection volume
is entered in the Amount column. The reference injection volume does not
have to equal the actual injection volume, i.e., the injection volume
specified in the sample list of the Browser. Enter the reference injection
volume on the General tab page of the QNT Editor:
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VI. Quantification Method (QNT File)
"Standard" Column
The standard method determines how a calibration is performed. A general
distinction is made between calibrations based on external or internal
standards.
An external standard refers to a calibration that is performed based on one
or several standard samples (normal case). An internal standard is the
known amount of a standard substance that is added to the unknown
sample. (The Tutorial provides only a short description for this special case.
For more information, see ÎInternal and External Standard in the Special
Functions section.)
• Make sure that the correct standard method for each peak to calibrate is
entered in the Standard column (= External = default value).
For information about further calibration options (Internal or
Internal/External), see the online Help or the User Manual (for example, in
the Calibration section).
Further Columns
All other peak table columns determine how the determined area values
are converted into the resulting substance amount values. Generally, the
standard settings can be used. No changes are required.
Verify that the following default settings in your QNT Method are correct for
your application:
• Calibration Type = Lin
• Integr. Type = Area
• Response Factor = 1,000
VI. Quantification Method (QNT File)
T-67
6. Defining the Calibration Mode and
Calibration Type
The ÎQuantifying Substances topic already described the basic elements
of the calibration with Chromeleon. However, Chromeleon provides many
more calibration functions. The complete description of these functions
would go beyond the scope of this Tutorial. Nevertheless, the most
important functions are described below.
Calibration Mode
The calibration mode allows you to define which standard samples shall be
used for calibrating which unknown samples. Define the calibration mode
on the General tab page. The following options are available:
Select the Total mode to use all standard samples for calibrating all
unknown samples. Use the Fixed mode to use certain standards (also from
previous sequences) for calibrating all unknown samples. Select the
Standard Addition mode to analyze unknown spiked or unspiked samples.
For more information about the Group, Additional and Bracketed modes,
see below:
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VI. Quantification Method (QNT File)
Calibration Type
The calibration type defines and weighs the calibration function to be used
as necessary. Enter the calibration type on the Peak Table tab page of the
QNT Editor:
• Press the F8 key in the Cal.Type column to open the Calibration Type
for <Peakname> dialog box.
• In the Calibration Function section, select the desired calibration
function for the current peak. This is usually the Linear or Linear with
Offset function (if the calibration function does not pass the origin).
However, quadratic and exponential functions and a polygon (Point-toPoint) through the calibration points are available as well.
• In the Weights section, define the weights for the single calibration
points. If you selected No Weights, higher amounts/signal values are
weighted more. Select 1/Amount or 1/Response for proportionate
weighting of small and high amounts/signal values. Select 1/Amount^2
and 1/Response^2 to weight smaller values more than high values.
VI. Quantification Method (QNT File)
T-69
• In the Further Options section, more options are available. Select
Average all response values for each calibration level before curve
fitting to average the calibration points of each calibration level before
calculating the calibration curve. Select Include point (0.0) for curve
fitting to include the origin as calibration point into the calibration if you
selected a calibration function with offset. However, this does not mean
that the calibration curve runs through the origin.
• Special rules apply to calibrations that use the Standard Addition
function. For more information, refer to the corresponding sections in the
Chromeleon online Help.
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VI. Quantification Method (QNT File)
VII. Data Analysis
T-71
VII. Data Analysis
Objectives of this chapter:
• To describe the chromatogram, calibration curve, and spectra views
• To display the sample results in report tables
• To evaluate and edit single chromatograms
Introduction
Chromeleon allows you to display sample data under various aspects. You
can:
• Display a single chromatogram.
• Compare several chromatograms.
• Check the peak purity.
• Display calibration curves.
• Search single spectra in a spectra library.
Each action is performed in a separate window or pane. Each pane is
intended for a specific task and has its own window arrangement and menu
structure.
Data Representation
To display data, select the corresponding data first:
• In the Browser, click the sequence of interest.
• Open a sample in the sequence by double-clicking the sample name.
• The sample opens in the Integration window. The Integration window
usually displays the chromatogram and the report table.
Sometimes, only the chromatogram is opened. This depends on the
report definition file (RDF):
T-72
VII. Data Analysis
Use the icons on the Method toolbar to quickly change from one view to
another:
The first five icons open a separate window each:
• Integration (opens the screen report and displays the results of the
analysis)
• PPA (opens a window that displays the data of a diode array detector)
• QNT-Editor (opens the view for editing the quantification method)
• Printer Layout (opens the view for preparing the printout)
• Signed Results (opens the signed results for the current sample)
VII. Data Analysis
T-73
The following icons open an additional part window:
• Show Report (displays the report table)
• Show Trend (displays the trend plot--changes of variables are
graphically displayed from sample to sample)
• Show Chromatogram/Split Zoom (displays the chromatogram in two
panes: complete view and enlarged view)
• Show Spectra (displays the UV spectrum)
• Show Calibration Curve (displays the calibration curve)
• Show Mass Spectra (displays the mass spectrum)
Click the four icons on the very right to:
• Display the Previous Chromatogram or the Next Chromatogram
• Display the Previous Channel or the Next Channel
The Integration window is the most important window for data
representation. For more information, see ÎThe Integration Window
below. For information about the ÎQNT Editor and the ÎPrinter Layout
windows, see the associated sections in this Tutorial.
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VII. Data Analysis
1. The Integration Window
• To open a sample chromatogram, double-click the sample name in the
Browser.
In the easiest case, you will only see the chromatogram of the sample. For
example:
• Select Decorations on the context menu to change the appearance of
the chromatogram. You can change the captions, axis ratios, and
coloring as well as the actual chromatogram representation (display of
peak heights, additional chromatograms, or grid).
• Double-click an individual peak to display its properties:
VII. Data Analysis
T-75
• Select a peak and click on the corresponding icons to display the
Spectra Plot and the Report.
If the analysis was performed with a diode array detector, the spectrum of
the selected peak, for example, anthracene, is displayed on the top right;
the lower section displays part of the integration report (four lines, the line
with the currently selected peak is highlighted by a different background
color). You can change the Spectra Plot window via the Decorations
command as described above. Select Decorations to display the available
options. This command also minimizes the spectrum in the chromatogram.
The displayed default report contains information about various
parameters. Each worksheet has a number of default variables that are an
integral part of the specific report type. For example, the integration report
contains the Ret.Time, Area and Amount columns; the calibration report
includes the Offset (c0), Slope (c1), and Curve (c2) columns, etc.
Double-click a peak name in a report window to change the selection of the
current peak. Instead of anthracene, pyrene is highlighted by a different
color. The spectra representation is updated as well.
T-76
VII. Data Analysis
2. Modifying the Chromatogram
In some cases, it may be useful to change the sample integration that was
performed manually, for example, by moving peak delimiters. To do this,
use the icons on the Integration toolbar:
Use the Automatic Tool to perform the most important actions. The shape
of the mouse cursor indicates the performed action.
Move left/right peak delimiter
Change baseline point (left/right/center)
Move baseline point (left/right/center)
Move perpendicular line
Move baseline segment
Change retention window
Move detection parameter
Insert peak
Display spectra
Zoom out an area
Action not possible
The action that can be performed at the current position is indicated on the
status bar.
Example:
The mouse cursor changes its shape when it is near the blue peak
delimiters.
indicates the peak start, while
marks the peak end.
• Left-click to change the integration limits of a peak by moving the peak
delimiters.
It is not possible to "run over" another peak delimiter. After moving a peak
delimiter, Chromeleon draws a new baseline between the peak start and
the peak end. All peak properties such as area, width, substance amount
etc. are recalculated. The integration report is immediately updated.
• Select Save Manipulations on the context or Edit menu to save the
reprocessed results.
Similar to moving peak delimiters, you can perform other actions. For
example, you can insert or delete peaks or change the baseline.
VII. Data Analysis
T-77
3. Report Tables
The default report (default.rdf) contains five tabs. The Integration tab
mentioned above describes the general characteristics of the respective
peaks in the current sample such as the Ret. Time, Area, and Amount
columns or the concentration of the respective substance.
The Calibration tab displays the calibration data of the current sample:
The Calibration tab page includes by default:
• The calibration type and the number of calibration points in the
calibration curve
• The relative standard deviation (of the points from the calibration curve)
• The correlation coefficient (independent of the calibration curve)
• The calibration curve parameters (Offset, Slope, and Curve)
The Peak Analysis tab provides a summary of the characteristics of the
single peaks such as the peak width, peak height, peak type, resolution,
asymmetry, and the number of theoretical plates.
In contrast to the first three tabs, the summary includes all samples in the
sequence:
T-78
VII. Data Analysis
The Summary always refers to the current peak. To display the summary
for any other peak, click the corresponding peak in the chromatogram.
The last report tab (Audit Trail) displays the corresponding sample
protocol:
It lists all commands sent to the corresponding timebase during the
analysis including time and retention time.
VII. Data Analysis
T-79
4. Modifying a Report Table
The individual report sheets are designed such that they contain the most
important peak and sample characteristics for most applications.
Nevertheless, it may be possible that you do not need an existing column
or that you want to add a new one.
• Double-click the column to be changed. The Properties Report
Column dialog box is opened:
The report columns are listed under Categories. Under Variables, all
result variables calculated and evaluated by Chromeleon are listed. Select
a variable to display its properties (Formula..., Header..., Dimension...,
Format...).
Header.. shows the text of the column header. Dimension.. shows the
dimension name. Format.. defines the number of decimal places. Select
the Selected Channel option to display the values of the current channel.
Or else, select Fixed Channel to display the data of a specific channel
independently of the representation in the integration window.
T-80
VII. Data Analysis
• Click OK to replace the selected column with the newly defined column.
• Select a column, and then select Delete Column(s) to remove the
column.
Note:
These report options influence the on-screen representation only. In the
Printer Layout, define the appearance of the printout!
5. Saving Changes
Select Save Report Definition.. to save all changes within a report as a
separate report definition file (RDF). The arrangement and the appearance
of the individual report window sections are saved plus the variables to be
displayed.
With each new start of Chromeleon, the report opens just as it was saved
last. If you have created your own Report Definition Files (RDF), you can
select a different appearance via Load Report Definition.
Keep in mind that each sequence is saved with a preferred profile. Upon
opening a sample of the corresponding sequence, it is not the appearance
of the window saved last that is used but the appearance of a specific
report definition file (RDF). Proceed as follows to define a sequence for a
preferred report:
• Select a sequence in the Browser and then select Properties on the
context menu.
• Under Preferred RDF File, enter the name of the report definition to
load.
VIII. Printing Results
T-81
VIII. Printing Results
1. Creating a Print Template
(Printer Layout)
The Printer Layout window displays custom print templates. To open a
print template for a specific sample:
• Double-click a sample name in the Browser. Chromeleon displays the
chromatogram in the Integration plot window.
• Select Printer Layout on the View menu to change from the
Integration plot to the Printer Layout.
Tip:
The Method toolbar allows you to quickly change from one view to another
(for more information, see ÎData Analysis). Place the mouse cursor on an
icon to read its label in the quick info field.
The image shows part of a report based on the default report template
(DEFAULT.RDF):
T-82
VIII. Printing Results
The Integration, Calibration (Curr. Peak), Calibration (Batch), Peak
Analysis, SST, Summary, and Audit Trail worksheets are part of the
DEFAULT.RDF report template.
• Select one of the tabs on the window bottom to open the corresponding
sheet of the report.
The appearance and the structure of the individual worksheets are very
similar to Microsoft Excel spreadsheets. Each sheet consists of a large
number of columns (256) and lines (16000) and thus is much larger than a
single printed page. A worksheet can consist of many horizontally or
vertically arranged printed pages. Chromatograms or tables that exceed
one printed page are automatically printed on two or more pages. Define
the order in which the pages are printed in the Page Setup (on the File
menu).
Editing an Existing DEFAULT.RDF Template
It is less effort to change an existing report template than to create a
completely new one. Different templates, such as the DEFAULT.RDF and
the DEFLTDAD.RDF templates, are available on the Chromeleon software
CD. Copy any template you wish to use as the basis for a new template to
a different and writable directory.
• Select Save Report Definition on the context or Workspace menu and
save the template to a different and writable directory.
You can now edit and save the copied template. For information about how
to proceed and the available options, see below. For more information
about the individual steps, refer to How to ...: Actions in the Window
Printer Layout section in the User Manual or the online Help.
• Select Layout Mode on the Edit menu.
• Select the worksheet you wish to edit or add an additional (empty)
worksheet by selecting Insert Sheet on the Edit menu.
• Select Delete Sheet on the Edit menu to delete an unnecessary
worksheet.
• Double-click a tab page to change its name, for example, Integration
Special.
VIII. Printing Results
T-83
• Select Insert Row(s)/Column(s) or Delete Row(s)/Column(s) to add
or remove single rows or columns on the worksheet.
• Select a field, a column, or an area, and select then Clear ... on the
context menu to remove unnecessary information from the worksheet.
Having removed all unnecessary parts, you can fill cells, columns, or areas
of the worksheet with new contents. You can either use the Windows
clipboard (copy & paste) or the Insert ... command from the context menu.
Follow the steps below:
• Select Insert ... to further specify the desired command, for example:
• Insert ... Chromatogram inserts a chromatogram.
• Insert ... Calibration Plot inserts a calibration curve.
• Insert ... Spectra Plot inserts a UV spectrum, etc.
• Use the small cross to mark the area where to insert the desired
element. The element is automatically inserted at the desired position.
• If you want to insert a report table or a single report variable, mark an
empty field first. Then, use the corresponding command to insert the
table or variable at the selected position.
• Report columns can be inserted only in existing tables.
• Select Save Report Definition on the context menu or the Workspace
menu and save your changes.
2. Printing
Printing in Automatic Batch Operation
You can start printing the results during automatic sample processing
(Online Batch). Make the corresponding settings in the Batch dialog box.
• Select Reporting on the Batch menu in the Browser or on a control
panel.
• Enable the Print/Export Report check box to print or export the sample
processing results.
T-84
VIII. Printing Results
• Select Print each sample immediately to start printing immediately
after the sample has been analyzed. Select Print when the entire
batch has finished to print all sample results after the entire sequence
is completed.
• To specify the report type and the extent of the printed output, click the
Report Setup button. The Batch Report dialog box is opened:
VIII. Printing Results
T-85
Select a report template in the Use Report Definition field. The default
report Chromeleon templates, i.e., DEFAULT.RDF and DEFLTDAD.RDF,
are available in the Dionex Templates > Reports directory.
• From the With Selected Channel field, select the channel to print, for
example, UV_VIS_1. If the field remains empty, the preferred channel is
printed (see ÎThe Browser: Appearance and Function).
• Select the Printout option to print the sheets.
• The Printer field shows the name of the previously selected default
printer. Select Setup to select another printer.
You have now specified the channel, the printer, and the report definition
for the printed output. The Printer Layout allows you to determine the
appearance of the report and to specify the information to be printed.
As described in detail in the ÎCreating a Print Template (Printer Layout)
topic, a report template contains several special sheets for different print
data. The sheets included in the selected report template are listed in the
Selected sheets to be printed field. The Integration, Calibration
(Curr.Peak), Calibration (Batch), Peak Analysis, Summary, and Audit
Trail sheets are included in the default report template.
• Under Selected sheets to be printed, select the worksheets of the
report template you wish to print.
• Determine for each worksheet whether all samples or only samples of a
specific type shall be printed.
• Select Print for every sample to print the results for each sample.
• Select Print under certain conditions to print the results only when a
certain condition is met (for example, only for a specific sample type).
For example, if you want to print the Calibration (Batch) worksheet for the
last standard sample only, select the Print under certain conditions
option and then click Conditions. In the dialog box that opens, enable the
Last Sample in a List of Standards check box.
T-86
VIII. Printing Results
• Click OK to return to the Batch Report dialog box and define the other
worksheets.
• Click Start as described in ÎStarting the Analysis to start sample
processing.
Printing Samples
Of course, sample-processing results can also be printed independently of
the online batch. This applies to all samples for which raw data are
available. Chromeleon assigns Finished as sample status in the sample
list. Proceed as follows to print the results of any samples.
• Open the Browser and select Print Setup on the File menu. Select a
printer for printing the results and exit the dialog box by clicking OK.
• Select the name of a sequence to print the results of all included
samples.
• If you only want to print specific samples of a sequence, select them
one by one with the mouse. Simultaneously press the CTRL key and
left-click to select several samples.
• Select Batch Report... on the File or context menu and determine how
to print the report (see Printing in Automatic Batch Operation).
IX. Special Functions
T-87
IX. Special Functions
In the previous chapters, the Tutorial described the basic Chromeleon
functions. Most users for routine operation use these functions. In addition,
Chromeleon provides many special functions that can save time for special
operations. However, these functions will certainly not be relevant for all
users.
Below please find a short introduction to the most important special
functions and the actions they perform:
In the Browser:
ÎInstalling Datasources
ÎFinding Samples (Query)
ÎBackup and Restore
ÎSigning Sequences Electronically
In the Control Panel:
ÎModifying a Control Panel
In the QNT Editor:
ÎInternal and External Standards
ÎSpectra Library Screening
In addition:
ÎDefining the Workspace
1. Installing Datasources
Datasources represent the top level in the Browser. Each user can access
at least one datasource that is the default datasource created during
installation (<PC-NAME_LOCAL>).
Creating additional datasources or accessing datasources on other
computer in the network is often useful. Therefore, these options are
supported as well. The section below describes how to create links to other
datasources (For more information about how to create a new datasource,
T-88
IX. Special Functions
refer to How to ...: Actions in the Browser/Setting up a Datasource in
the User Manual and online Help).
All steps required for setting up datasources start with the Datasources
command on the File menu.
• Open the Browser and select Datasources.
The Datasource Manager shows all datasources that the client can
currently access.
Connecting an Existing Datasource ("Connect")
If you know the directory in which the other datasource is located, you can
connect to this datasource.
• Click the Connect button of the Datasource Manager to establish the
connection.
• Select the format of the underlying database.
IX. Special Functions
T-89
Usually, you will select Chromeleon datasources (Native Chromeleon
Data Source). Other supported formats include default Microsoft Access
databases (mdb container), SQL server, and Oracle databases.
• Select the appropriate datasource type and click OK.
• In the following dialog box, navigate to the network folder that houses
the datasource.
• Select the folder and click Open.
Note:
If the folder and the datasource are located on a remote computer, the user
of that computer must share the corresponding folder! If (s)he does, you
can connect to the selected datasource. The name and the type icon of the
datasource will appear in the Browser.
T-90
IX. Special Functions
2. Finding Samples (Query)
You will often have to find samples according to defined problems. Instead
of endlessly poring over old folders, you can use the query function of the
Browser to do so easily, fast, and purposefully. Select New on the File
menu to start the Query Wizard:
On the first query page, define whether to perform the query in the current
datasource or in a fixed one. Also, define whether to search for sequence
properties, sample properties, or any other properties of samples.
On the next pages, define in the Data Field the variable whose properties
you want to find. Select an operator and define the value. The combo box
to the right of the respective line allows you to enter a logical connective
with another query property. Only after having entered AND or OR, you can
enter another property:
IX. Special Functions
T-91
The following query searches for all samples with
• Sample type = Matrix Blank and
• Whose comments start with Charge 123456 or
• An injection date between 2/2/2000 and 2/3/2000.
On the next page, enter the following settings in order to restrict the query
to samples
• Containing an Anthracene peak or
• Containing more than 10 calibration points
3. Backup and Restore
In the course of time, large amounts of data will result, especially when
PDA detectors are used, but also when a great number of sequences is
recorded. To ensure that you can continue to store data on your hard disc,
you have to delete some of your data from time to time. However, do not
destroy these data permanently. They still have to be available for future
use. Of course, you can save the respective sequences and datasource on
your network as well.
T-92
IX. Special Functions
However, Chromeleon provides the possibility to backup sequences,
directories, or entire datasources and save these backup files (which can
also be compressed), for example, on CD. Proceed as follows:
• Select the object to save.
• Select Export/Backup on the File menu and then, select Backup.
• Indicate where to save the backup file and define all further backup
options:
• Start the backup to create the backup file. The extension of the backup
file is cmb.
• You can then, for example, burn the backup file to a CD or attach it to
an Email.
IX. Special Functions
T-93
Data that were saved in this way can be used again via the Restore
function. Select Import/Restore on the File menu, and then select
Restore. Find the respective backup file and indicate where to restore it.
The Backup and Restore functions are an easy way to save large amounts
of data in such a way that they can be quickly accessed later. Structure and
cross-references remain intact. In addition, these functions facilitate
exchanging data via E-mail.
4. Signing Sequences Electronically
Electronic Signature allows you to sign the results that have been
generated from your ÎRaw Data. This function is important aspect for
quality assurance and GLP. When User Mode is enabled, you can sign and
protect ÎSequence reports that have been accepted as correct. In this
way, it is possible to review and reproduce the results at any time later.
Electronic Signature includes three steps:
• Submit
• Review
• Approve
Typically, the user who created the report signs and submits it. Afterward,
for example, the laboratory manager reviews the report and signs it as
well. Finally, the quality assurance manager approves the results.
Note:
Enable User Mode. If User Mode is disabled, electronic signature will not
be possible. Besides, electronic signature is available only for user
databases that have been created with a CmUser program version 6.10 or
higher. Update your database if an error message notifies you that
electronic signature will not be possible.
To sign a sequence proceed as follows:
• Click the sequence and select Submit Results (first signature step) on
the context menu.
• Select the Report Definition file (RDF) to be used for displaying the
sequence, select the channel for which you want to show the results,
and select the report sheets to be signed:
T-94
IX. Special Functions
• The Check Signed Results dialog box appears. This dialog box allows
you to check the report to be signed. Use the arrow keys to toggle
between different sequence samples.
For each sample, the tab pages for the selected report sheets are
displayed. Click OK when you have finished checking the report.
IX. Special Functions
T-95
T-96
IX. Special Functions
• The Submit Signature dialog box is opened. To sign the SOR file,
enter your User ID and the signature password:
• The extension of the newly created file is .SOR. The SOR file is stored
in the signed sequence.
• In this way, the sequence and its samples are protected against
changes.
• Select Electronic Signature on the File menu and then select Verify to
have Chromeleon check the signed sequence report once again, i.e.,
the sequence, the corresponding files (pgm, qnt, rdf, sor), and the
individual samples.
To view the signature status of a sequence, select the sequence and select
Properties on the context menu. The Signature tab page indicates the
status of the selected sequence:
IX. Special Functions
T-97
You can only remove a signature if you are authorized by the
corresponding privileges. Removing the signature also deletes the
corresponding SOR File.
T-98
IX. Special Functions
5. Modifying a Control Panel
Chromeleon includes more than a hundred default control panels for
different devices and functions. If you do not find a control panel for your
requirements, we recommend modifying the panel that is closest to your
requirements. You can add further controls or change the functionality and
appearance of existing ones.
Note:
If you cannot perform the following steps, you do not have the user
privileges required for these actions. In this case, contact your system
administrator.
Enabling the Layout Mode
• Enable Layout Mode on the context menu. You can modify the control
panel only in the Layout Mode.
Adding a control
• Move the cursor on the layout toolbar.
The Quick Info provides a brief description for the control on which the
cursor is currently positioned.
• Select the desired control by clicking. The corresponding object is now
attached to the cursor.
• Place the cursor on the location where to add the new object and leftclick.
Functionality and appearance of the control
• Right-click the control for which you want to change the functionality
and/or appearance.
• Select Properties to open the Properties dialog box.
IX. Special Functions
T-99
Depending on the selected control, the Properties dialog box includes
various tabs. For a slider, the Style tab looks as follows:
• Select the General, Color, or Style tab to change the appearance such
as the color, the shape, or the caption of the control.
• Select the Link tab to determine the function and the instrument to be
operated via the control.
T-100
IX. Special Functions
• From the Object list, select the instrument itself. From the Object
Property list, select the instrument's function. Instrument and function
selection depend on the components installed with the timebase.
With the settings shown above, you can use a slider to control the pump
flow rate.
6. Internal and External Standards
Substances in unknown samples are usually quantified using external
standard samples. However, it is also possible to add a known amount of a
standard substance (= internal standard, ISTD) to an unknown sample and
then use this standard for calibration. Use the Standard column on the
Peak Table tab page to specify whether you want to calibrate a substance
using an external or internal standard.
• Press the F8 key to open the Standard Method for <Peakname>
dialog box.
• Select External to use external standard samples for calibration.
• Select Internal to use internal standard samples for calibration.
• If you use several sample preparation steps during which substance
may be lost, correct this using the Internal/External method: Add to
your unknown sample a known amount of a substance that originally
was not part of the sample (= ISTD). Be sure that the chemical behavior
IX. Special Functions
T-101
of the substance is similar to the behavior of the substances to quantify.
During sample preparation, a corresponding amount of this standard
substance will be lost as is for the substances to quantify. In this case,
you can use the known internal standard in the Internal/External
method to correct the external calibration. First, define a substance as
internal standard (Use this peak as Internal Standard) and then use
this substance as Associated ISTD Peak.
• If you did not add exactly the same amount of internal standard to all
samples, select the Use sample amount as reference option. In this
case, you have to enter the amount of internal standard that was added
to the sample in the sample list of the Browser. This entry is then used
for the calculation (instead of the entry made in the Amount column of
the QNT editor).
For an example of the possible settings in the Standard column, see the
image below:
Dimethylfluoranthene and Chrysene are calibrated, using external
standards. Naphthalene and Fluoranthene are used as internal standards
with Naphthalene being used for internal calibrations and Fluoranthene for
internal/external calibrations. Phenanthrene and Anthracene are calibrated,
using Naphthalene as internal standard, whereas the internal/external
method is used with Fluoranthene as internal standard to quantify Pyrene.
7. Spectra Library Screening
The Spectra Library Screening tab page of the QNT editor allows
searching spectra libraries for reference spectra for the different peaks of
the current chromatogram. As the chromatographic conditions considerably
influence the spectra, we recommend filling your spectra library with
spectra from chromatograms that were recorded under the same conditions
as the current chromatogram.
T-102
IX. Special Functions
Note:
To create a new library, select New on the File menu in the Browser and
then copy the desired spectra in the spectra plot (Ctrl + C) and paste them
into the library (Strg + V).
On the Spectra Library Screening tab page, select the spectra library in
which to search for the reference spectra. In addition, enter the comparison
criteria for the spectra search. Normally, the default values of the Match
Criterion (Least Squares) and Hit Threshold (950) will be sufficient.
However, you can also specify many additional search criteria.
Click Apply to start the spectra search. The retention spectrum of the
actual peaks is compared to the reference spectrum that was found. To the
top right of the spectra plot, the corresponding match factor is displayed.
IX. Special Functions
T-103
The match factor indicates the correspondence of the two spectra. A value
of 1000 indicates that the spectra are identical. If the match factor were 0,
they would be completely different. If no corresponding spectrum is found
based on the selected criteria, a message stating No spectra library hits
found! appears.
Note:
You can use the spectra library screening results for automatic peak table
creation (see ÎAutomatically Generating the Peak Table).
8. Defining the Workspace
Chromeleon allows you to save the window arrangement of any work
situation, i.e., the combination of different windows, in a workspace.
This facilitates opening single files or windows and allows you to work in
whatever work environment you prefer.
The information about the windows involved is stored in a WSP file. If you
want to start working using a specific workspace, open the corresponding
WSP file or generate it by storing the screen contents. The following
commands are available on the Workspace menu:
• Select Open Workspace to open an existing workspace.
• Select Save Workspace to save the current workspace.
• Select Save Workspace as to save the current workspace with a new
name.
• Select Autosave Workspace to always save the most recent
workspace as the default workspace.
There is no restriction on the number of windows that can be saved with
each workspace
A useful workspace arrangement might combine, for example, the report, a
control panel, and the Browser (refer to the picture).
T-104
IX. Special Functions
Tip:
When Chromeleon is started, the most recently used workspace is loaded.
Addendum: Keyboard Shortcuts + Short Glossary
T-105
Addendum: Keyboard Shortcuts +
Short Glossary
Keyboard Shortcuts
Shortcuts are provided for many operations, especially in online control:
General
Action
Where
Right-click
Ctrl + Tab
All Chromeleon Opens the context menu.
windows
Changes to the next
window.
Browser
Aborts the drag or move
action.
Opens the contextsensitive Help;
Enables the Edit mode.
Browser +
Renames files (after the
Find command: Find
QNT Editor
Next).
Chromatogram Brings you to the next
sample.
Brings you to the previous
sample.
Updates the window.
Brings you to the next
partial window.
Browser
Optimizes the column
(F7 key only) + width.
QNT Editor
Optimizes the line height.
Opens the dialog box.
Shows the hotkey
underlines.
Browser
Displays the
chromatogram.
Browser +
Opens the corresponding
Control Panels Properties.
Esc
F1
F2
F3
F4
Shift + F4
F5
F6
F7
Shift + F7
F8
F10/Alt
Shift + F5
Alt + Enter
Description
T-106
Addendum: Keyboard Shortcuts + Short Glossary
Action
General
(cont'd)
Ctrl + Tab
Ctrl
Ctrl + N
Ctrl + O
Ctrl + S
Ctrl + R
Ctrl + P
Edit
Ctrl + Z
Ctrl + Y
Ctrl + X
Ctrl + C
Ctrl + V
Ctrl + F
F3
Ctrl + H
F9
Ins
Del
Where
Description
Toggles between open
windows.
Zooming (changes the
cursors to the zoom
cursor).
Signal plot in
the Report and
in the QNT
Editor
Report + Printer Creates a new file.
Layout
Opens the file.
Saves the file.
Opens the batch report.
Prints your selection.
Undoes the previous
action.
Repeats the previous
action.
Cuts the selected object.
Copies the selected object.
Pastes the selected
objects
Finds a string of
characters.
Finds the next string of
characters.
Replaces the entry in the
field.
Fills the column or the
selected cell(s) with the
first value of the selection.
Inserts the selected object.
Deletes the selected
object.
Addendum: Keyboard Shortcuts + Short Glossary
Action
Where
T-107
Description
Sample List
Ctrl + I
Ctrl + D
Ctrl + A
Inserts a sample.
Deletes a sample.
Adds a sample.
PPA
Alt + F4
Copies the spectrum to the
clipboard.
Control
Ctrl + F
Ctrl + I
Ctrl + Break
Break
Ctrl + A
Ctrl + B
Changes the pump's
settings
Initiates injection.
Stops the pump.
Enables Hold mode.
Starts data acquisition.
Edits the batch.
Signal plot
Double-click... - Overview
window
- Time axis
- Signal axis
- Plot range
(- or else:…)
Shift
- when zooming
Unzoom.
Auto Plot Speed.
Autoscale.
Signals...
Axis/decoration.
Retains scale ratio
between signal and value
axis (The shape of
chromatograms is
maintained).
Gauge/
Slider
Ctrl key
Press when
dragging
Toggles the Snap To
Scale option.
Script
Button
Click
Button hold
down
Indicates whether the
program is still running.
Stops the program upon
confirmation.
T-108
Addendum: Keyboard Shortcuts + Short Glossary
Action
Edit Field
Tab/Enter
Esc
Layout
Mode
Alt + Click
Where
Sends the new value.
Aborts the input.
In the selected
panel
Alt + Drag
Esc
Arrow keys
Shift +
Arrow keys
Shift +
Select
Ctrl + Select
Ctrl + Drag
Doubleclicking
Description
Draws a selection frame
on the control panel.
Temporarily toggles the
Snap To Grid option.
Deselects all.
Drags the selection pixel
by pixel.
Increases/reduces the
selection pixel by pixel.
Extends the selection.
In the Online
Signal Plot of
the panel
Highlights the selected
control (the highlighted
control is used in Align..).
Copies controls.
Opens the Properties...
Datasource and Database
A database that is mounted to the Chromeleon client is referred to as a
datasource. To mount a datasource, select Mount Datasource on the File
menu in the Browser and then specify the format and location of the
database.
During the initial installation of Chromeleon, a local default datasource is
created on each client PC. In network operation, the datasource name is
composed of the computer name and the extension LOCAL (<PC
name>_local). On a local station that is not available on a network, the
name stated under Control Panel > Network > Identification in the
operating system is used. If no identification is entered, the datasource is
named DEFAULT_LOCAL.
Addendum: Keyboard Shortcuts + Short Glossary
T-109
Chromeleon data is usually stored in an Access database, that is, in an
MDB (Microsoft Data Base) container. The default datasource is based on
an Access database, too. The ODBC capability of Chromeleon allows you
to use various other formats (SQL, Oracle, etc.) as well. Both, Sample data
and Sequence data are saved in a datasource, independently of the
chosen format. You can save a database on a local hard disk or any other
mass storage device.
Select New Directory on the File menu in the Browser to create individual
subdirectories under a datasource. You can then use these directories to
save ÎSequences and the corresponding data and programs.
If the datasource is located on a centralized network PC, all clients with the
appropriate access rights, which have been assigned by the system
administrator, can access the database. If the datasource is stored on a
local hard disk, the corresponding client grants database access via the
Windows File Sharing option. Chromeleon also allows you to lock
datasources, directories, or sequences.
Raw Data
Raw data refers to all analog and digital data points that are digitally stored
on a PC. That is why raw data exists for signals or channels only selected
by the user before data acquisition.
The scope and accuracy of the stored raw data depends on the selected
Sampling Rate, the Step, and the resolution of the detector signal.
Sequence
A sequence combines samples that belong together due to their origin or
processing. The names of all samples belonging to one sequence are
entered in the sample list (also called sequence table). Thus, a sequence is
a container for various samples.
Theoretically, the number of samples is not limited, but use more than 100
sample entries in exceptions only. Create additional sequences to reduce
the number of samples included in one sequence and thus to accelerate
access to the individual samples. In this way, it is also easier for you to
keep track of the processed samples.
T-110
Addendum: Keyboard Shortcuts + Short Glossary
The sequence table also defines how to process a sample. It includes
information about the sample itself (name, injection volume, position,
sample weight, dilution factor, etc.) and references chromatographic
methods that specify the program (PGM File) to be performed for the
analysis and the evaluation parameters to be used (QNT Method).
The entire data collected in connection with creating and processing a
sequence is saved in the associated sequence. This also includes the raw
data and protocol data recorded during the analysis.
Similar to ÎDatasources, sequences can be "locked." In this status, data
and results are read-only. You cannot modify or extend them.
(Chromatography) Server
PCs connected to the components of a system via interfaces can be used
as chromatography servers.
A chromatography server is automatically installed on the PC during
installation. The chromatography server controls the data exchange
between the chromatographic system and the PC. Upon starting, each
server is capable of serving up to six controlled or 16 non-controlled
systems (ÎTimebases). The server receives the commands that have
been entered on the control panel of a client PC and executes them at the
specified time, for example, by transmitting them to the corresponding
device driver of the HPLC system. The server also assumes this function in
the opposite direction. Thus, the raw data of each system is stored at the
location specified by the client and the entire system-relevant data is
forwarded to the client.
Configure the server and install the timebase(s) in the Server Configuration
program.
If the Chromeleon server and client software are located on the same PC,
the installation is called a local installation or referred to as workstation. If
they are not, the installation is called network installation.
Tip:
In case of manual data acquisition, ÎRaw Data is always stored in the
manual sequence of the timebase directory (if not otherwise defined). This
directory is available only in the local datasource of a local installation. In
the case of a batch, the user can decide where to store the data.
Addendum: Keyboard Shortcuts + Short Glossary
T-111
Timebase
All components that are combined in a chromatography system to enable
the chromatographic separation and related in a time context with each
other are assigned to the same timebase.
A timebase can be a very complex system. For example, it can consist of
two pumps, one autosampler, one column oven, two detectors switched in
series, and one fraction collector. However, an isolated gas chromatograph
can also represent a timebase.
Any other system that is completely independent from the first one
represents a new timebase. Administration of different timebases is on one
or several ÎChromatography Servers.
Create your timebase(s) in the Server Configuration program.
T-112
Addendum: Keyboard Shortcuts + Short Glossary
Index
T-i
Index
A
Amount Column .......................... T-63
Approve ........................................ T-93
Audit Trail .................................... T-77
Autogenerate Peak Table ........... T-60
B
Backup .......................................... T-91
Browser .. T-9, T-11, T-87, T-90, T-91
Appearance ................................T-9
Functions ....................................T-9
Structure ..............................T-9, T-10
C
Calibration.................................T-77
Details ...................................... T-67
Internal/External Standard .. T-100
Changes.................................T-71, T-80
Chromatogram ......................T-71, T-76
Chromatography Server ................ T-110
Chromeleon....................................... T-1
Client................................................. T-5
Column ......................................... T-63
Amount.......... T-63, T-64, T-65, T-66
Standard ..................T-63, T-64, T-66
Contents ............................................ T-2
Context-Sensitive Help .............. T-13
Control File ............... T-26, T-27, T-38
Create ......................................... T-27
Edit .................................. T-26, T-38
Program Wizard ........... T-27, T-37
Properties ................................. T-26
Control Panel ..... T-17, T-19, T-22, T-98
Connect to Timebase...................T-21
Modify ........................................T-98
Open..................................T-18, T-19
Use ....................................T-24, T-25
Create.............................................T-27
Control File ..............................T-27
PGM File.....................................T-37
D
Data ................................................T-91
Backup ................ T-91, T-92, T-93
Exchange ..................................T-91
Restore ........................... T-91, T-93
Data Analysis............. T-71, T-74, T-76,
........................................ T-79, T-80
Database.........................................T-108
Datasource ........................ T-87, T-108
DEFAULT RDF (Template) ......T-81
Detect Peaks........................... T-53, T-58
Detection ............................ T-53, T-58
Peaks ............................. T-53 - T-58
QNT File ....................... T-53, T-58
Detection Parameter........ T-58, T-60
E
Edit ...................................... T-53, T-58
Electronic Signature ....................T-93
Index
T-ii
F
O
File .................................................. T-11
Copy ........................................... T-12
Move .......................................... T-12
Open ........................................... T-11
Functions........................................... T-9
Basic............................................. T-9
Special........................................ T-87
ODBC ............................................T-108
H
Help.......................................T-13, T-14
Context-Sensitive ......... T-13, T-14
Online......................................... T-13
Systematic Online Help......... T-14
Hotkeys ......................................... T-105
Online Help
Context-Sensitive ....................T-13
Systematic ................................T-14
Online Help........................... T-13, T-14
Overview .......................................... T-2
P
Peak..................................... T-53, T-62
Library ........................................ T-101
Detection...........................T-53 - T58
Graphical Input of Param. .....T-58
Identify..............................T-62, T-63
Peak Table ............................ T-51, T-62
Autogenerate .....................T-60, T-61
Peak Identification ..................T-62
PGM File............................. T-26, T-38
Create ........................................T-26
Edit .................................. T-26, T-38
Properties .................................T-26
Port ................................................T-110
Print Template ..............................T-81
Printer Layout...............................T-81
Printing ..........................................T-83
Automatic Batch Operation ...T-83
Results........... T-83, T-84, T-85, T-86
Samples.............................T-85, T-86
with own Report Templates ..T-83
Program Start .................................... T-5
Program Wizard ...........................T-27
M
Q
Method .......................................... T-67
Modify ........................ T-71, T-76, T-98
Chromatogram........ T-71, T-73, T-76
Control Panel .......................... T-98
Report......... T-71 - T-73, T-79, T-80
QNT File..........................................T-51
Autogenerate Peak Table .......T-60
Calibration ...............................T-63
Calibration Details ..................T-67
Create ..........................................T-51
Edit................................... T-53, T-58
Graphical Input of Param. .....T-58
Internal/External Standard .. T-100
Peak Detection.........................T-53
I
Identify ......................................... T-62
Integration Window .................... T-74
Interface ........................................ T-110
K
Keyboard....................................... T-105
Hotkeys .................................... T-105
Shortcuts................................... T-105
L
N
Network......................................... T-110
Index
Peak Identification ................. T-62
Spectra Lib Screen. .. T-101-T-103
Substance Quantification ...... T-63
Window ................................... T-52
QNT Method ................................... T-51
Autogenerate Peak Table ...... T-60
Calibration Details ................. T-67
Create ...............................T-51, T-52
Graphical Input of Param...... T-58
Internal/External Standard .. T-100
Peak Detection ........T-54, T-55, T-56
Peak Identification ................. T-62
Spectra Library Screening .. T-101
Substance Quantification ...... T-63
Quantification Method .................... T-51
Autogenerate Peak Table ...... T-60
Calibration ............................... T-63
Calibration Details ................. T-67
Create ......................................... T-51
Graphical Input of Param...... T-58
Internal/External Standard .. T-100
Peak Detection ........................ T-53
Peak Identification ................. T-62
Spectra Library Screening .. T-101
Substance Quantification ...... T-63
Quantify ........................................ T-63
Calibration...............T-64, T-65, T-66
Calibration Details ................. T-67
Substances ...................... T-63, T-64
Query ............................................ T-90
R
Raw Data....................................... T-109
RDF ..................................... T-80, T-81
Report....................................T-71, T-79
Modify ..................................... T-79
Tables ....................................... T-77
Report Definition ........................ T-80
Restore .......................................... T-91
Review .......................................... T-93
T-iii
S
Sample .................................. T-11, T-90
Find .............................................T-90
Open............................................T-11
Sample List ....................... T-39, T-109
Functions.....................................T-39
Open ..........................................T-39
Sample Processing .......................T-47
Automatic..........................T-47, T-48
Manual ......................................T-47
Save ...................................... T-71, T-80
Sequence ...... T-39, T-41, T-93, T-109
Edit..............................................T-47
Flow Chart ................................... T-4
Sign ...................................T-93, T-96
Sequence Wizard .........................T-41
Server.....................................T-5, T-110
Shortcuts ........................................T-105
Signature .......................................T-93
Special Functions.............................T-87
Spectra ........................................ T-101
Spectra Library Screening ....... T-101
Standard ..................................... T-100
External .....................T-100, T-101
Internal.......................T-100, T-101
Standard Column .........................T-63
Start................................ T-5, T-41, T-47
Analysis .......................... T-47, T-48
Sequence Wizard .... T-41, T-43, T-47
System........................... T-5, T-6, T-7
Structure............................................ T-9
Submit ...........................................T-93
Substance ......................................T-63
Summary .......................................T-77
System .............................................. T-5
T
Template........................................T-81
Timebase........................................T-111
Tutorial ......................................T-1, T-2
Index
T-iv
U
Use Control Panel ....................... T-22
Use ................................................ T-22
V
View ............................................ T-103
W
Workspace ................................. T-103
WSP File .................................... T-103
CHROMELEON
®
Chromatography Management System
User Manual
Doc.: CM_UserManual_660.doc
Contents
I
Contents
Introduction .....................................................................1
The User Help and Manual ................................................................. 1
The Administrator Help and Manual ................................................. 1
The Online Help (Overview) ............................................................... 2
The User Manual (Overview).............................................................. 3
Activating Online Help........................................................................ 5
Installation, Operation, and Data Management............7
Chromeleon (Overview) ......................................................................... 9
Chromeleon (Overview) ..................................................................... 9
Chromeleon Windows ................................................................ 10
Chromeleon Features ................................................................. 12
Chromeleon Licenses ................................................................. 13
Installing Chromeleon ................................................................. 15
Components of a Chromatography System....................................... 17
Components of a Chromatography System................................... 17
Chromatography Instruments ......................................................... 18
The Chromatography Data System................................................. 19
The PC................................................................................................ 21
The Operating System...................................................................... 21
The Network ...................................................................................... 22
Local Client/Server Installation ................................................... 23
Network Installation..................................................................... 24
Basic Operation .................................................................................... 27
Operation via the Keyboard ............................................................. 27
Keyboard Shortcuts.......................................................................... 28
Operation via the Mouse .................................................................. 30
Undo/Redo Commands .................................................................... 31
II
Contents
Starting the Program ........................................................................ 31
The Client User Interface ................................................................. 33
The Windows..................................................................................... 34
The Table Editor................................................................................ 35
Working with Several Windows ...................................................... 36
User Profiles (Workspaces) ............................................................. 36
Report Definitions............................................................................. 37
Create/Open Files, Windows, and Templates ................................ 38
Printing............................................................................................... 40
Control ................................................................................................... 43
Control Concept................................................................................ 43
Control Requirements ...................................................................... 44
The Control Program ........................................................................ 45
The Control Program (Details).................................................... 46
The Program Wizard................................................................... 47
The PGM Editor (Overview) ............................................................. 48
The Device Views ....................................................................... 49
The Surveyor MSQ or Finnigan AQA Views .............................. 49
The Commands View ................................................................. 50
The Post-Acquisition Steps View................................................ 52
The Program Syntax (Experts Only)........................................... 52
The Control Panel ............................................................................. 56
Control Panel: Appearance......................................................... 56
Control Panel: Function .............................................................. 57
Control Panel: The Signal Plot ................................................... 58
Control Panel: The Audit Trail..................................................... 58
Control Panel: The Trend Plot .................................................... 59
Data Management ................................................................................. 61
Data (Overview)................................................................................. 61
The Browser ...................................................................................... 62
Common Features with the Windows Explorer .......................... 63
Differences from the Windows Explorer ..................................... 63
Function ...................................................................................... 65
The Datasource ................................................................................. 65
Data Acquisition................................................................................ 69
Contents
III
Data Acquisition with Detectors without Separate Drivers.......... 70
Data Storage...................................................................................... 71
Data Export ........................................................................................ 72
Backup ............................................................................................... 73
Raw Data............................................................................................ 73
Raw Data Storage ............................................................................. 74
Raw Data Compression.................................................................... 76
Restoring a Chromatogram from Raw Data................................... 77
Raw Data Export ............................................................................... 77
Raw Data Import................................................................................ 78
Raw Data Storage in Case of Power Failure .................................. 79
Audit Trails ........................................................................................ 80
The Daily Audit Trail ................................................................... 81
The Sample Audit Trail ............................................................... 82
Samples and Sequences...................................................................... 83
Sample Preparation .......................................................................... 83
Sample Processing........................................................................... 83
Sample Definition.............................................................................. 84
The Sample List (Sequence) ............................................................ 85
The Sequence Wizard....................................................................... 86
Automatic Batch Processing........................................................... 87
Sample Evaluation ............................................................................ 88
Electronic Signature ......................................................................... 89
Theory of Calibration............................................................................ 91
Calibration (Overview)...................................................................... 91
Calibration Principle.................................................................... 92
Calibration Types (Linear) .......................................................... 94
Calibration Types (Non-linear).................................................... 95
Using the Calibration Curve........................................................ 97
Calculating the Calibration Curve ............................................... 98
Standard Methods ...................................................................... 99
Evaluation with Various Standard Methods.............................. 100
Implementation ......................................................................... 103
IV
Contents
Validation, AutoQ, and System Wellness......................................... 105
Validation and Qualification........................................................... 105
AutoQ Equipment Qualification ................................................ 107
Instruments Operational and Performance Qualification ......... 109
Operational and Performance Qualification for HPLC Systems109
Performance Qualification (PQ) for IC Systems....................... 111
The System Suitability Test (SST)............................................ 112
System Wellness for IC Devices (Overview)............................ 112
System Wellness for HPLC Devices (Overview)...................... 114
Data Representation and Reprocessing .......................................... 115
Data Representation and Reprocessing ...................................... 115
Post-Acquisition Steps (PGM Editor) ........................................... 116
The QNT Editor.................................................................................... 117
The Quantification Method ............................................................ 117
The QNT Editor................................................................................ 118
The General Tab Page............................................................. 120
Detection Parameters (Detection) ............................................ 122
Peak Table, Amount Table, Peak Tracking, MS Tracking ....... 124
Calibration Settings (Calibration).............................................. 127
Spectra Library Screening........................................................ 127
The System Suitability Test...................................................... 127
Mass Spectra (MS, MS Tracking) ............................................ 128
UV Spectra (UV))...................................................................... 128
Integration............................................................................................ 129
The Integration Window ................................................................. 130
Opening a Sample .................................................................... 131
Operation .................................................................................. 132
Manual Re-Integration .............................................................. 133
Chromatogram Comparison ..................................................... 134
Data Smoothing ........................................................................ 136
Combination of Channels ......................................................... 137
Peak Ratio ................................................................................ 138
The Spectra Plot ....................................................................... 140
The Mass Spectra Plot ............................................................. 143
Contents
V
Reports................................................................................................. 145
Reports (Overview) ......................................................................... 145
The Integration Report.............................................................. 147
The Calibration Report.............................................................. 148
The Peak Summary Report ...................................................... 148
The History Report.................................................................... 150
Special Report Tables .............................................................. 151
The Calibration Curve......................................................................... 153
The Printer Layout .............................................................................. 155
The Printer Layout (Overview)....................................................... 155
Appearance and Function ........................................................ 156
Creating Templates and Worksheets ....................................... 158
Printing ...................................................................................... 159
Peak Purity Analysis........................................................................... 161
Spectra Libraries................................................................................. 165
Spectra Libraries (Overview) ......................................................... 165
Creating a New Library ............................................................. 167
Comparing Spectra................................................................... 167
How to ...: .....................................................................169
Actions in the Browser....................................................................... 171
Moving and Copying Elements...................................................... 172
Creating a Sample List (Sequence)............................................... 174
Creating User-defined Columns ............................................... 176
Creating a Sequence Report Column ...................................... 179
Displaying Sequence Columns................................................. 184
Handling Files and Datasources ................................................... 186
Datasources on Removable Media .......................................... 186
Opening Audit Trails ................................................................. 187
Creating Backup Files............................................................... 188
Restoring Backup Files............................................................. 191
Exporting and Transferring Data .............................................. 193
VI
Contents
Exporting Data During or After a Batch .................................... 193
Exporting Data from Different Samples to a Single File ........... 195
Transferring Sequences Automatically..................................... 196
Signing Sequences Electronically ................................................ 197
Individual Steps of Electronic Signature ................................... 199
Checking the Signature Status and Undoing the Signature..... 202
Performing a Query ........................................................................ 204
Entering the Sample Query Using the Wizard.......................... 205
Selecting Search Criteria for Samples and/or Sequences ....... 206
Examples (Wizard) ................................................................... 210
Specifying the Sample Query Using the Dialog Box ................ 211
Examples (Dialog Box) ............................................................. 213
Editing a Query in SQL Syntax ................................................. 216
Saving and Performing a Query ............................................... 216
Answering Frequently Asked Questions .................................. 217
System Wellness............................................................................. 219
Opening a Wellness Control Panel........................................... 219
Viewing and Restoring Calibration Data................................... 220
Uploading ICS-1500/2000 Calibration Data ............................. 221
Performing Device Calibrations ................................................ 221
Calibrating the Leak Detector ................................................... 222
Calibrating the Pump Flow Rate............................................... 223
Calibrating the ICS-1000/1500/2000 Flow Rate....................... 223
Calibrating the Pressure Transducer Offset ............................. 224
Calibrating the ICS-90 Pressure Transducer ........................... 225
Degas Calibration ..................................................................... 226
Calibrating the ICS-1000/1500/2000 Degas Pump .................. 226
Wavelength Calibration............................................................. 227
Calibrating the Conductivity Cell .............................................. 227
Calibrating the ICS-90 Conductivity Cell .................................. 229
Calibrating the ICS-1000/1500/2000 Conductivity Cell ............ 230
Calibrating the pH Reference Electrode................................... 232
Entering the AS50 Inject Port Volume ...................................... 233
Performing Device Diagnostics ................................................ 233
Testing the Leak Detector ........................................................ 234
Wavelength Verification ............................................................ 234
ICS-90 Pressure and Conductivity Diagnostics........................ 235
Conductivity Verification (ICS-1000/1500/2000) ...................... 236
Entering Device Parameters..................................................... 236
Applying a Sodium Correction .................................................. 236
Importing PeakNet (Releases 4.5 Through 5.2) Method Files .... 237
Importing PeakNet (Releases 4.5 Through 5.2) Data Files ......... 239
Contents
VII
PeakNet (Release 4.5 Through 5.2) Translation Tables .............. 241
AS50 Setup and Timed Event Parameter Translation ............. 242
Pump and Eluent Generator Setup and
Timed Event Parameter Translation ......................................... 243
AD20 Setup and Timed Event Parameter Translation ............. 244
AD25 Setup and Timed Event Parameter Translation ............. 245
CD20 Setup and Timed Event Parameter Translation ............. 246
ED40 Setup and Timed Event Parameter Translation ............. 247
IC20 Setup and Timed Event Parameter Translation............... 248
DX-120 Setup and Timed Event Parameter Translation .......... 249
UI20 Setup and Timed Event Parameter Translation............... 250
Detector Component Table Parameter Translation.................. 251
Importing Agilent/HP ChemStation Data Files............................. 254
ChemStation Translation Tables................................................... 256
Translating ChemStation Sequences and Batches.................. 257
Translating ChemStation Samples ........................................... 258
Translating ChemStation Methods ........................................... 259
Translating ChemStation Spectra Libraries.............................. 261
Actions on the Control Panel ............................................................ 263
Opening a Control Panel .......................................................... 263
Connecting a Control Panel with a Timebase .......................... 265
Creating and Starting an Online Batch ..................................... 267
Modifying a Control Panel......................................................... 270
Modifying a Control................................................................... 271
Linking a Control to a Device.................................................... 273
Creating a Script Button............................................................ 274
Linking a Control to a Command .............................................. 275
Creating a Command Button .................................................... 276
Creating Hidden Windows ........................................................ 276
Using/Recording Demo Data.................................................... 277
Displaying Sample and Sequence Information ........................ 279
Answering Frequently Asked Questions .................................. 279
Actions in the PGM Editor.................................................................. 281
Creating a Program......................................................................... 282
Automatically Creating a Program............................................ 283
Editing PGM Files in the Device Views of the PGM Editor....... 284
Manually Editing a PGM File in the Commands View .............. 284
Calculating in a Program .......................................................... 288
Program Example ..................................................................... 290
Creating a Shutdown Program ................................................. 292
VIII
Contents
Creating an Emergency Program ............................................. 293
Creating a Power Failure Program ........................................... 294
Program with Variable Run Time.............................................. 295
Converting Units ....................................................................... 296
Using User-defined Columns in a Program.............................. 298
Adding Post-Acquisition Steps ..................................................... 300
Manual Injections with Application Templates............................ 302
Actions in the QNT Editor .................................................................. 305
Creating a Peak Table..................................................................... 306
Identifying Peaks ...................................................................... 307
Identifying Peaks via Their UV Spectra (Peak Tracking) ......... 309
Identifying Peaks via Their Mass Spectra (MS Tracking) ........ 311
Defining Retention Times and Reference Peaks ..................... 312
Defining the Retention Index and the Kovats Index ................. 314
Entering Amount Values (Amount Column) ............................. 314
Selecting the Standard Method (Standard Column)................. 316
Selecting the Calibration Function ........................................... 317
Weighting and Averaging Calibration Points ............................ 319
Defining the QNT Method for Several Detectors...................... 320
Entering Reference Spectra (Reference Spectrum Column) .. 321
Autogenerating the Peak Table ................................................ 322
Defining Detection Parameters ..................................................... 325
Reducing the Number of Evaluated Peaks .............................. 326
Excluding Certain Peaks .......................................................... 329
Inhibiting Peak Integration ........................................................ 329
Modifying the Baseline.............................................................. 330
Modifying the Peak Recognition Algorithm............................... 331
Defining Peak Start or Peak End.............................................. 332
Defining Rider Peaks ................................................................ 333
Defining the Area for PPA......................................................... 334
Grouping Peaks ..............................................................................334
Subtracting a Blank Run Sample .................................................. 335
Disabling Calibration Samples ...................................................... 336
Calibration ....................................................................................... 337
Introduction and Example: 1 Standard and 1 Substance ......... 338
Several Standards with Several Substances Each .................. 340
Multiple-Point Calibration Using 1 Single Standard.................. 342
Calibrating Using Standards of an Old Sequence.................... 343
Standard Addition ..................................................................... 345
Calibrating Unstable Substances ............................................. 348
Contents
IX
No Pure Substance - Known Relative Extinction Coefficient ... 349
Calibrating with an Internal Standard Substance ..................... 351
Compensating Measurement Errors (Internal Calibration)...... 352
Internal Calibration: Calculation................................................ 354
Correcting Sample Preparation Errors (Int./Ext. Calibration) .. 359
Internal/External Calibration: Calculation ................................. 361
Using Different ISTD Amounts (Variable ISTD) ....................... 366
Calibrating without Standard Sample ....................................... 368
Calibration Modes for External Calibration............................... 370
Calibration Mode: Total............................................................. 371
Calibration Mode: Additional.....................................................376
Calibration Mode: Group .......................................................... 378
Calibration Mode: Bracketed .................................................... 380
Calibration Mode: Fixed............................................................ 384
Entering the Concentration/Amount of the Validation Sample . 386
Validating the Calibration Curve ............................................... 387
Inverting Dependent and Independent Variables ..................... 389
Spectra Library Screening ............................................................. 392
Entering Criteria for the Spectra Library Screening.................. 392
Starting Library Screening and Viewing the Results ................ 395
Inserting Screening Results in Reports and Peak Labels ........ 396
Defining the System Suitability Test............................................. 398
SST Wizard: Overview and Start Conditions............................ 399
SST Wizard: Sample and Test Conditions ............................... 400
SST Wizard: Other Wizard Pages ............................................ 402
Modifying the System Suitability Test....................................... 403
SST Example: Is the Amount in the Calibrated Range? .......... 403
SST Example: Amount Deviation on Reinjection ..................... 405
Inserting SST Results in the Printer Layout ............................. 407
Processing Mass Traces ................................................................ 407
Processing UV Channels ............................................................... 409
Actions in the Chromatogram ........................................................... 413
Using Hotkeys in the Chromatogram ........................................... 414
Manual Re-Integration .................................................................... 416
Moving Peak Delimiters ............................................................ 417
Modifying the Baseline Manually .............................................. 417
Inserting or Deleting Peaks ...................................................... 418
Splitting Peaks .......................................................................... 419
Changing the Peak Type .......................................................... 420
Defining an Averaged Baseline ................................................ 420
X
Contents
Manual Peak Assignment............................................................... 421
Comparing Chromatograms .......................................................... 423
Selecting the Samples and Channels....................................... 423
Displaying Several Chromatograms ......................................... 424
Mirroring Chromatograms......................................................... 426
Normalizing Chromatograms.................................................... 426
Performing Data Smoothing .......................................................... 427
Copying a Channel ......................................................................... 429
Combining Channels via Arithmetic Operations......................... 430
Subtracting Background Spectra.................................................. 432
Displaying Peak Information in a Separate Area ......................... 435
Moving a Retention Window Graphically ..................................... 436
Defining Detection Parameters Graphically................................. 437
Actions in the UV Spectra and/or Mass Spectra ............................. 441
Displaying a Peak Spectrum (UV or MS) ................................. 441
Displaying and Overlaying Single (UV or MS) Spectra ............ 442
Match Factor, Difference Spectra, 1st/2nd Derivatives .............. 443
Starting a UV Spectra Search .................................................. 443
Actions in the Calibration Curve ....................................................... 447
Displaying Values ..................................................................... 448
Indicating the Confidence Interval ............................................ 449
Actions in the Report Table ............................................................... 451
Displaying a Report .................................................................. 451
Defining the Contents of a Report ............................................ 452
Defining the Appearance of a Report ....................................... 453
Adjusting the Line Height Automatically ................................... 454
Saving a New Report Definition File......................................... 454
Linking Report Variables .......................................................... 455
Calculating the Peak Variable "Amount" .................................. 456
Adding and/or Renaming a Worksheet .................................... 457
Displaying the Peak Summary ................................................. 459
Displaying an Audit Trail ........................................................... 460
Creating a History Report ......................................................... 462
Displaying MS Reports ............................................................. 463
Selecting Other Special Reports .............................................. 464
Setting Parameters for Variables.............................................. 465
Contents
XI
Actions in the Printer Layout............................................................. 469
Opening and Editing the Printer Layout.................................... 470
Inserting a Chromatogram........................................................ 471
Inserting a Trend Plot ............................................................... 472
Inserting and Editing a Table.................................................... 472
Inserting a Column into an Existing Table ................................ 474
Inserting and Deleting Individual Variables .............................. 476
Inserting Text ............................................................................ 477
Saving or Loading a Report Definition File ............................... 477
Specifying the Pages to be printed........................................... 478
Specifying the Printout.............................................................. 479
Printing the Results of a Single Sample ................................... 480
Printing the Results of a Sequence or a Sample Batch ........... 482
Setting Print Area and Print Title(s) .......................................... 485
Automatically Repeating the Output for the Selected Objects . 485
Automatically Repeating the Output: Example ......................... 487
Saving the Contents of a Report Definition File ....................... 490
Entering User-defined Formulas............................................... 491
Calculating the Amount Percentage (for Identified Peaks) ..... 494
Calculating the Concentration Percentage............................... 495
Creating Dynamic Columns That Contain Flexible Formulas .. 497
Creating Dynamic Links to Lines Other than the Current Line. 500
Creating Charts......................................................................... 500
Changing the Page Format ...................................................... 501
Notes on the Page Setup.......................................................... 502
Answering Frequently Asked Questions .................................. 503
Actions in the Peak Purity Analysis (PPA) Window ........................ 507
Analyzing Peak Purity ....................................................................507
Visual Check of Iso Line Plot.................................................... 508
PPI and PPI Match Factor ........................................................ 508
Normalized Spectra Overlay..................................................... 509
Peak Inhibition via Virtual Channels ......................................... 510
Multiple Ratio ............................................................................ 511
Selecting the Optimum Integration Path ...................................... 511
Extracting/Exporting Spectra, Chromatograms, and 3D Fields. 513
Answering Frequently Asked Questions ..................................... 515
XII
Contents
Actions Related to Mass Spectrometers.......................................... 517
Creating an MS Program and Sequence.................................. 517
Creating a PGM File for the aQa MS........................................ 520
Creating aQa MS Channels with the aQa PGM File ................ 523
Creating a PGM File for the MSQ............................................. 526
Creating MSQ Channels with the MSQ PGM File .................... 528
SIM Mass Lists for IC-MS ......................................................... 531
Acquiring MS Data in MCA Mode ............................................. 535
Extracting Mass Traces Online................................................. 537
Extracting Mass Traces Afterward............................................ 539
Extracting a Temporary Mass Trace......................................... 541
Showing Mass Spectra ............................................................. 542
Minimizing the Noise of Mass Spectra...................................... 544
Defining Further QNT Settings for MS...................................... 545
Actions Related to Fraction Collection............................................. 547
Setting up Fraction Collection................................................... 548
PGM Wizard: Fraction Collection - General Options ................ 551
Setting the Peak Detection Parameters.................................... 554
Checking the Fraction Collection Status on the Control Panel. 557
Recognizing the Peak Start, Peak Maximum, and Peak End .. 559
Defining the Reactions to Certain Events ................................. 561
Tracking Fraction Collection in the Chromatogram .................. 563
Tracking Fraction Collection in the Report ............................... 565
Program Example (One Detection Channel) ............................ 567
Program Example (Two Detection Channels) .......................... 569
Fraction Collection Control via an MS ...................................... 572
Fraction Collection Control via an MS for Different Samples .. 576
Actions in the Virtual Column Separation Simulator ...................... 579
Selecting the Analysis Parameters ........................................... 580
Viewing the Results Table ........................................................ 584
Viewing the Resolution Response Surface .............................. 585
Viewing the Virtual Chromatogram ........................................... 586
Selecting a Resolution Criterion ............................................... 588
Finding the Fastest Chromatogram .......................................... 592
Finding the Global Optimum ..................................................... 593
Selecting Product Test Conditions............................................ 594
Manually Selecting an Eluent Condition ................................... 594
Saving and Reloading Virtual Column Settings........................ 596
Contents
XIII
Device Control..................................................................................... 597
Device Control (Overview) ............................................................. 597
Control: Pumps ............................................................................... 598
Pump Commands ..................................................................... 598
Setting the Flow Rate................................................................ 601
Determining the Solvent Composition ...................................... 601
Determining a Gradient............................................................. 602
Determining Pressure and Pressure Limits .............................. 605
Starting and Stopping the Pump Flow ...................................... 606
Holding the Pump Flow............................................................. 606
Recording the Pump Pressure.................................................. 607
Setting Automatic Pre-Compression Control (P580) ................ 609
Viewing Leak Sensor and Workload Status ............................. 610
Peak Parking............................................................................. 610
Control: Autosamplers ................................................................... 611
Autosampler Commands (GINA 50) ......................................... 612
Autosampler Commands (ASI-100 Series) .............................. 615
Autosampler Commands (AS50) .............................................. 619
User-Defined Programs (FAMOS Autosampler) ....................... 623
Injecting a Sample .................................................................... 627
Setting Up Remote Injection ..................................................... 629
Priming the Syringe (ASI-100 Series)....................................... 632
Defining Sample Preparation Steps (AS50) ............................. 636
Entering Sample Preparation Vial Positions ............................. 637
Overlapping Samples................................................................ 638
Injecting Two Samples Simultaneously .................................... 639
Opening the AS50 Door during Operation................................ 640
Monitoring the Status of the AS50 ............................................ 641
Control: Detectors .......................................................................... 641
Detector Commands ................................................................. 642
Starting Data Acquisition........................................................... 643
Defining Signals, Signal Parameters, Axis Decoration, etc...... 643
Modifying Signal Parameters (Overview) ................................. 644
Modifying the Signal Parameters of a UV Channel .................. 645
Modifying the Signal Parameters of a 3D Field ........................ 646
Determining Wavelength Switching .......................................... 646
Determining the Optimum Emission Wavelength (RF2000)..... 647
Recording Fluorescence Spectra (RF2000) ............................. 648
Displaying the Signal Parameters of a Mass Channel ............. 650
Defining a Waveform ................................................................ 651
Selecting the Reference Electrode Mode with Alkaline Eluents653
Defining Step and Average ....................................................... 656
XIV
Contents
Viewing or Resetting the Lamp Age ......................................... 657
Controlling a Suppressor .......................................................... 657
Setting Atlas Suppressor Currents ........................................... 659
Setting SRS Suppressor Currents ............................................ 660
Setting SRS-MPIC Suppressor Currents.................................. 663
Controlling an MMS Suppressor............................................... 665
Control: IC........................................................................................ 666
Controlling a DX-120 Ion Chromatograph ................................ 666
Controlling an ICS-1000/1500/2000 Ion Chromat.System ....... 667
Controlling an ICS-90 Ion Chromatography System ................ 669
Controlling the Eluent Generator Concentration....................... 671
Monitoring the Eluent Generator Cartridge Lifetime ................. 672
Monitoring the DX-120 Operating Status.................................. 672
Monitoring the ICS-1000/1500/2000 Operating Status............. 673
Control: GC and Temperature ....................................................... 675
Flow/Pressure Control for Gas Chromatographs ..................... 675
Determining a Gradient (GC).................................................... 676
Injecting Two Samples Simultaneously (Dual Inject)................ 677
Temperature Control (On/Off)................................................... 678
Controlling the Temperature ..................................................... 679
Controlling the Column Temperature........................................ 680
Control: Component Controller..................................................... 681
Valve Control for CC81: Concentration .................................... 684
Valve Control for CC82: Dilution............................................... 685
Valve Control for CC83: Dilution with Reagent......................... 686
Valve Control for CC84: Concentration with Reagent .............. 687
Control: Special Commands, Relays, and Others....................... 688
Virtual Channel Commands......................................................688
Program Examples for Virtual Channels................................... 691
Trigger Commands ................................................................... 694
Mixed Commands ..................................................................... 697
Relay, TTL, and Remote Input Commands .............................. 699
Switching a Relay ..................................................................... 699
Device Successfully Connected ............................................... 700
The Ready Signal .....................................................................701
Determining the CRP Value...................................................... 701
Contents
XV
Commands and Tips for Device Control ..................705
Commands and Tips for Device Control .......................................... 707
General Commands for Device Control............................................ 709
System Commands .................................................................. 709
General Commands.................................................................. 710
General Device Commands .....................................................711
Commands for Controlling Dionex Devices..................................... 713
Dionex Pumps ................................................................................. 713
Dionex P680 HPLC Pumps ...................................................... 716
Dionex/LC Packings UltiMate Capillary/Nano HPLC Pump ..... 717
Dionex/LC Packings UltiMate Pump: Columns ........................ 719
Controlling Pumps without a Separate Device Driver .............. 720
Dionex Autosamplers ..................................................................... 721
Dionex ASI-100 Autosamplers ................................................. 722
Dionex AS50 Autosamplers...................................................... 725
Dionex FAMOS Capillary/Nano HPLC Autosamplers .............. 728
Dionex FAMOS Autosampler: Sample Preparation ................. 732
Dionex GINA 50 Autosamplers................................................. 734
Dionex Detectors ............................................................................ 735
Dionex UVD 170/340 UV/PDA Detectors ................................. 736
Dionex PDA-100 Photodiode Array Detectors ......................... 738
Dionex AD20/AD25 Absorbance Detectors.............................. 740
Dionex UltiMate Capillary/Nano HPLC UV Detectors .............. 742
Dionex RF2000 Fluorescence Detector ................................... 743
Dionex RF2000 Detector: Excitation Wavelength .................... 744
Dionex RF2000 Detector: Emission Wavelength ..................... 744
Dionex RF2000 Detector: Gain ................................................ 744
Dionex RF2000 Detector: Response........................................ 745
Dionex RF2000 Detector: Sensitivity........................................ 745
Dionex RF2000 Detector: Scanning ......................................... 745
Dionex RF2000 Detector: (Scan)Start and End Wavelengths . 747
Dionex RF2000 Fluorescence Detector: ScanSpeed .............. 747
Dionex RF2000 Detector: Scan With Analog Output ............... 747
Shodex RI-101 Refractive Index Detector ................................ 748
Dionex Electrochemical Detectors............................................ 749
XVI
Contents
Dionex Ion Chromatography Systems.......................................... 752
Dionex DX-120 Ion Chromatograph ......................................... 752
Dionex ICS-90 Ion Chromatography System ........................... 753
Dionex ICS-1000/1500/2000 Ion Chromatography System ..... 754
Dionex Eluent Generators.............................................................. 756
Dionex Thermostatted Column Compartments........................... 757
Dionex TCC-100 Thermostatted Column Compartment .......... 758
Dionex TCC-100: Column ID.................................................... 761
Commands and Tips for Third-Party Devices ..........763
Commands and Tips for Third-Party Devices.................................. 765
Agilent .............................................................................................. 766
1100 HPLC System .................................................................. 766
1100 HPLC System: General ................................................... 767
1100 HPLC System: Pump....................................................... 768
1100 HPLC System: Autosampler............................................ 770
1100 HPLC System: Wellplate Sampler................................... 772
1100 HPLC System: Column Compartment............................. 775
1100 HPLC System: UV Detectors........................................... 776
1100 HPLC System: Program Tips .......................................... 781
1100 HPLC System: Derivatization Program ........................... 784
1100 HPLC System: Operating Modes (Fluoresc. Detector) ... 785
1100 HPLC System Fluoresc. Detector: Lamp Commands..... 786
1100 HPLC System: Checking the Solvent Liquid Level ......... 788
1100 HPLC System: Troubleshooting ...................................... 789
1050 HPLC System: UV Detector ............................................ 790
6890/6850 GCs......................................................................... 791
6890/6850 GCs: General.......................................................... 792
6890/6850 GCs: Detectors ....................................................... 795
6890/6850 GCs: ECD ............................................................... 798
6890/6850 GCs: FID and FPD ................................................. 800
6890/6850 GCs: NPD ............................................................... 802
6890/6850 GCs: TCD .............................................................. 805
6890/6850 GCs: µ-ECD............................................................ 806
6890/6850 GCs: Column .......................................................... 807
6890/6850 GCs: Inlet (General) ............................................... 809
6890/6850 GCs: Inlet (Purged Packed EPC,
Cool On-Column EPC, ACI, PCM, Volatiles)............................ 811
6890/6850 GCs: Inlet (Split/Splitless EPC
(Split/Splitless EPC, Gerstel PTV, PTV, CIS3, CIS4) ............... 812
Contents
XVII
6890/6850 GCs: Inlet
(Purged Packed, Cool-On-Column, Unknown)......................... 815
6890/6850 GCs: Inject .............................................................. 816
6890/6850 GCs: Auxiliary Devices ........................................... 819
6890/6850 GCs: Application..................................................... 820
6890/6850: PGM File - Entering Pressure or Flow? ................ 823
HP 5890 GC: Application.......................................................... 825
CTC Analytics.................................................................................. 827
PAL Sampler Commands ......................................................... 827
PAL Samplers: Tips for Injection .............................................. 830
PAL Samplers: PGM File for LC_Inject Mode .......................... 832
PAL Samplers: PGM File for Custom Inject Mode ................... 833
PAL Samplers: Tips for Headspace Operation ........................ 834
PAL Samplers: PGM File for HS_Progr Inject Mode................ 837
PAL Sampler: Troubleshooting................................................. 838
Fisons AS800/ThermoQuest AS2000 Autosampler ................. 839
Fisons AS800/ThermoQuest AS2000: Program Example ....... 840
Fisons AS800/ThermoQuest AS2000: Troubleshooting .......... 841
Gilson ............................................................................................... 843
202 Fraction Collector (Ext.)/ 204 Fraction Collector ............... 843
Injection Modes......................................................................... 844
333 and 334 Pumps.................................................................. 845
Kontron ............................................................................................ 848
465 Autosampler....................................................................... 848
SFM25 Fluorescence Detector................................................. 849
Merck Hitachi................................................................................... 850
LaChrom HPLC System: Tips .................................................. 850
Pumps: Program Example........................................................ 850
Autosamplers............................................................................ 852
AS4000: Program Example ...................................................... 856
Perkin Elmer Autosystem GC ........................................................ 858
Split Valve Control for PSS and CAP Inlets ............................. 858
Pressure and Flow Ramps ....................................................... 859
Rheodyne Valves ............................................................................ 861
Shimadzu LC-10A HPLC System................................................... 862
Program Example for the Standard Injection Mode ................. 862
Program Example for the Reagent Injection Mode .................. 864
Program Example for the Dilution Injection Mode .................... 866
XVIII
Contents
Thermo Finnigan/TQ/TSP............................................................... 868
Trace GC .................................................................................. 868
Trace GC: Application............................................................... 871
Trace GC: Setting the Temperature ........................................ 871
Surveyor MSQ Mass Spectrometer.......................................... 873
Surveyor MSQ: Troubleshooting .............................................. 875
aQa Mass Spectrometer........................................................... 877
AS3500/AS3000 Autosamplers: Sample Preparation .............. 879
UV1000 Detector ...................................................................... 886
UV2000 Detector ...................................................................... 887
UV3000 Detector ...................................................................... 888
Troubleshooting ........................................................................ 889
UV6000 PDA Detector.............................................................. 890
UV6000 Detector: Troubleshooting .......................................... 893
FL2000 and FL3000 Fluorescence Detectors .......................... 893
FL2000 and FL3000 Detectors:
Different Names in Firmware and Chromeleon ........................ 896
FL2000 and FL3000 Detectors: Troubleshooting..................... 898
Valco Valves .................................................................................... 899
Varian ............................................................................................... 900
3800 GC: Troubleshooting ....................................................... 900
3400 and Varian 3600 GCs ...................................................... 901
3400 and 3600 GCs:
Different Names in Firmware and Chromeleon ........................ 905
3400 and Varian 3600 GCs: Detectors .................................... 907
3400 and 3600 GC Detectors:
Different Names in Firmware and Chromeleon ........................ 909
3400 and 3600 GCs: Tips for Operation .................................. 910
3400 and 3600 GCs: Troubleshooting ..................................... 911
Waters .............................................................................................. 914
Alliance 2690/2695 HPLC Modules.......................................... 914
Alliance 2790/2795 HPLC Modules.......................................... 920
Alliance 2790/2795: HPLC System .......................................... 920
Alliance 2790/2795: Pump and Degasser ................................ 922
Alliance 2790/2795: Injector ..................................................... 925
Alliance 2790/2795: Column Heater/Sample Temp. Control .. 929
Alliance 2790/2795: Overlapping Sample Preparation ........... 931
996 and 2996 PDA Detectors................................................... 933
2487 UV Detector ..................................................................... 935
Waters Instruments: Troubleshooting ...................................... 936
GPIB-Connected Devices: Troubleshooting................................ 938
Contents
XIX
Reference Manual .......................................................941
The Sequence.................................................................................. 943
Sample Variables ............................................................................ 945
The Program .................................................................................... 969
Control Commands......................................................................... 975
The QNT Editor..............................................................................1055
QNT Parameters............................................................................1057
QNT: Detection..............................................................................1105
Detection Parameters ...................................................................1107
The Report Definition File (RDF) .................................................1141
Report Categories.........................................................................1142
Glossary.......................................................................A-1
Index..................................................................................i
XX
Contents
Introduction
1
Introduction
The User Help and Manual
Although the User Help and the User Manual differ only slightly in content,
they are intended for different situations.
•
Refer to the user online Help to look up the meaning of unknown terms
or to learn how to use the application in a specific situation on-screen.
The easiest and quickest way to access the online Help is to press the
Activating Online Help.
¾F1 Key. For more information, refer to
•
Look up information in the User Manual to become familiar with the
fundamental features and operating procedures of the system. We
recommend that you read the first two sections and look up unfamiliar
terms in the glossary.
In addition, a separate Administrator Help is available. For information
The
about the topics described in the Administrator Help, refer to
Administrator Help and Manual.
The Administrator Help and Manual
In addition to the User Help and the User Manual, a separate Administrator
Help and an Administrator manual are available. The User Help/Manual
provides useful information for the Chromeleon end user. The Administrator
Help and Manual are intended for system administrators and Dionex
Service. For an overview of the topics described in the User Help and/or
Manual, refer to the Table of Contents.
The Administrator Help and Manual provide information about the different
Chromeleon management tools and the hardware installation procedures:
Software Installation and Communication
Validation and Qualification
Actions in the Browser and in the Operating System
Actions Related to Mass Spectrometers
Actions in the Server Configuration Program
2
Introduction
Actions in the Server Monitor Program
21 CFR Part 11 and Electronic Signature
Chromeleon User Management (covering the ¾User Manager
(CmUser) and Security Activation Tool (CmSecure))
Installation Instructions (Hardware). This topic provides among others
Installing Dionex Devices and
Installing and
the following sections:
Controlling Third-Party Devices.
The Online Help (Overview)
As with most Windows programs, the online Help features various windows
and levels. These largely correspond to the various parts of the User
Manual, such as the table of contents, main section, appendix, and index.
In addition to the manual, the online Help provides many context-sensitive
tips that open only from the respective program window.
The Contents tab provides an overview of the different Help sections. This
information corresponds to the table of contents in the User Manual:
Introduction
3
•
Double-click a book symbol to display the topic titles.
•
Double-click a question mark symbol to open the corresponding
topic.
•
The Index tab page enables you to find a specific term by searching
the index entries.
•
The Find tab page enables you to search for specific words and
phrases in the Help topics by searching the entire text of the Help
system.
The selected topic appears in a separate Help window. The window has a
white background if the topic deals with questions regarding theory,
installation, and operation. The background color is yellow if the topic
provides tips that are more practical (How to ...:). Both windows can be
displayed simultaneously.
Tip:
Online Help is automatically displayed in the language (German or English)
of the active language setting (see Windows Control Panel > Regional
Options). The English version is loaded by default.
The User Manual (Overview)
The User Manual is divided into different sections. In addition, the Table of
Contents provides an overview of the various topics described in the
manual. The Index allows you to search for specific terms.
There are many references to related topics. These references are
indicated as follows:
¾
Jump term
References technical terms in the Glossary section.
⇒
Jump term
References topics in the Reference Manual.
Shortcut
link
References topics in the Theory, Installation, Operation or How
to .... sections.
CmUser link
References topics in the Chromeleon User Management section
of the Administrator Help.
Installation
link
References topics in the Installation Instructions of the
Administrator Help.
4
Introduction
1. Section: Theory, Installation, and Operation
This section describes the structure and functions of Chromeleon, as well
as basic chromatographic facts and methods.
2. Section: How to ...:
The How to ... section provides helpful answers to frequently asked
questions, such as:
"How do I perform external calibrations?"
"How do I generate program files?"
"How do I re-integrate chromatograms manually?"
3. Section: Reference Manual
The reference manual describes commands, parameters, variables, and
report Categories in table form.
Appendix: Glossary
The glossary is an alphabetical list of chromatographic and program-related
terms. Consult the glossary for the exact definition of
•
A technical term; for example, Blank Run Subtraction.
•
A command; for example, Draw.
•
A parameter; for example, Skewness.
Index
Consult the index to locate information about a specific term. Ordinary page
numbers refer to the first four manual sections, while page numbers
preceded by the letter A refer to the glossary section; for example:
Command
Reconnect.......... A-93
Wait.................. A-147
Wash................ A-147
Comment................. A-30
Communication .....11; 21
Introduction
5
Activating Online Help
There are several ways to access online Help.
¾F1 Key
Press the F1 key
information.
to
display context-sensitive
Select How to ... on the context menu to display
detailed information about how to perform a particular
task. Select What’s this? to display a short
description of the corresponding control or the active
window.
Help menu
Select ¾Index on the Help menu to display the table
of contents of the online Help.
Select Using Help on the Help menu for more
information about how to use the online Help.
Click this icon on the toolbar to display a question
mark appended to the mouse cursor. Then, click the
item of interest for more information.
Click the Help button in a dialog box for more
information about the dialog box.
Online Help topics often include links that jump to other Help topics. To
¾jump to another Help topic, click the green underlined hyperlink or click a
shortcut symbol.
6
Introduction
Installation, Operation, and Data Management
7
Installation, Operation, and Data
Management
Installation, Operation,
and Data Management
8
Installation, Operation, and Data Management
Chromeleon (Overview)
9
Chromeleon (Overview)
Chromeleon (Overview)
Chromeleon is a modern Chromatography Management System that
allows you to control and monitor chromatography installations and to
backup, evaluate, and reprocess data. Chromeleon provides various subprograms, making it very flexible and ¾GLP conforming, and offering you
numerous options for operating effectively and productively:
The various sub-programs provide the following functions:
Icon
Description
Function
Chromeleon (Client)
Device control, data backup, reprocessing and
evaluation, validation (user)
Installation Qualification
Verification and documentation of the installation
(administrator)
¾Server Configuration
Device configuration (administrator)
¾Server Monitor
Interface between the installation and the
Chromeleon Client (administrator)
The Server Configuration and Server Monitor programs allow you to create
a client/server structure, thus adding to the networking capability of
Chromeleon.
10
Chromeleon (Overview)
In addition, there are two User Management programs available to the
administrator in the Chromel\CmUser directory:
Program
Description
Enabling and disabling User Management (administrator)
Installing User Management (administrator)
As a user, you will almost exclusively work with the Chromeleon Client
program. The different windows support all required functions.
Also, refer to:
Chromeleon Windows
Chromeleon Features
Chromeleon Licenses
Installing Chromeleon
Chromeleon Windows
You can open the various windows of the Chromeleon Client Program via
the associated icons or by double-clicking the respective directory:
To open the window of interest, click the associated icon:
Symbol
Description
Function
¾Browser
Data administration and storage
Integration
Chromatogram display and reprocessing
¾PPA
Peak Purity Analysis:
3D Field (only for PDA data)
QNT Editor
Quantification Method
¾Printer
Printer Layout creation and/or modification
Layout
Signed
Results
Electronically signed sequence (¾SOR File)
Chromeleon (Overview)
11
To open the following windows double-click the associated directory:
Symbol
Description
Function
¾Panel
Device control
PGM Editor
Processing of a control file
The Integration window and QNT Editor provide different partial windows;
click the associated icon on the Method toolbar:
Symbol
Description
Function
Report
Display of various report tables
¾Trend Plot
Display of trends in data series
Spectrum
UV spectrum (only for PDA data)
Calibration
Curve
Calibration curve
MS Spectrum
¾Mass Spectrum
For more information about the different windows, refer to:
The Browser
The Control Panel
The QNT Editor
The PGM Editor
The Integration Window
The Printer Layout
12
Chromeleon (Overview)
Chromeleon Features
Chromeleon provides a wide variety of features, including:
•
Modern control technology via client/server architecture and Windows
The Network.)
RPC technology. (For more information, refer to
•
Complete
•
Separate ¾User Manager (CmUser program) for user management
(refer to the Administrator Manual
Chromeleon User
Management).
•
Query-based data access via an integrated ¾Database.
•
Connection to standard ¾ODBC and/or SQL databases.
•
Tele-service (remote maintenance).
•
Freely definable workspace on the
•
¾Mass Spectrometer control and MS data evaluation.
•
¾Sample-Oriented Operation.
•
¾Electronic Signature of sequences.
•
Compliance with GLP through automated ¾Instrument Qualification
(IQ) and ¾Operational Qualification (OQ), as well as Instrument OQ
and ¾Performance Qualification (PQ).
Network Installation.
Control Panel.
The user interface and operation correspond to the standard Windows
requirements:
•
Comprehensive ¾Online Help.
•
Easy operation based on ¾Toolbars and assisting ¾Wizards.
•
Comprehensive context menus via the ¾Right Mouse Button.
•
Drag & Drop functionality (refer to How to …: Actions in the Browser
Moving and Copying Elements).
•
Real ¾Multi-Tasking and ¾Multi-Threading.
Chromeleon (Overview)
In addition, the
enhancements:
chromatographic
interface
features
13
these special
•
Optical representation of the gradient profile.
•
Online zooming beyond the current time.
•
Grid in online window.
•
Additional peak variables.
•
Determination of reference wavelength for individual channels.
•
Wavelength compensation via holmium oxide filter.
•
Enhanced data compression and restoration of old data.
•
Baseline subtraction can be undone at any time.
•
Base area correction/base area recognition.
Chromeleon Licenses
Chromeleon can be adjusted to the requirements of a specific application.
In addition to the basic software package, various options are available
from Dionex.
The purchased scope of performance is determined by means of a serial
number and the corresponding license key. The serial number is coded on
a software protection device that is installed locally, together with the
license key, or managed by a ¾License Server. (For more information,
refer to the Administrator Manual: Software Installation and
Communication
The Software License.)
Chromeleon can only be started with the full range of features if a
protection device (such as a ¾Dongle on the parallel or USB PC interface
or a ¾PAL on the A/D converter card) is detected, or if a license provided
by the license server is detected.
14
Chromeleon (Overview)
The following Chromeleon licenses are available from Dionex:
Server Features
Server
License:
The server license (without additional features) allows signal acquisition
without spectra from one timebase for any number of synchronous
channels.
Timebases
Class 1 to 3:
The ¾Timebase licenses allow you to control chromatography systems.
The Timebase Class 2 and Timebase Class 3 licenses allow you to
control additional chromatography instruments that are not supported
by the Timebase Class 1 license. Please note:
License
Includes
Timebase Class 2
Timebase Class 1
Timebase Class 3
Timebase Class 1 and Timebase Class 2
Up to six timebases can be configured on one server PC. However,
only two Timebase Class 2 or Timebase Class 3 are allowed.
Multiple Network
Control:
This license allows you to use Chromeleon on a network. In addition,
this license supports the ¾Online Transfer Agent (OTA) and ¾Network
Failure Protection (= NFP). One separate license is required for each
installed chromatography ¾Server.
PDA License:
Additional single-user license for ¾Photodiode Array Detectors on a
chromatography server. It supports digital data acquisition of a threedimensional data field. The PDA license is required for spectra
acquisition. One license is needed on each server.
MS Control:
Additional single-user license for a ¾Mass Spectrometer on a
chromatography server: digital data acquisition of a three-dimensional
data field. The MS Control license is required for spectra and channel
acquisition. One license is needed for each chromatography server.
IC Control SE:
Additional control license for the DX-120, ICS-90, and ICS-1000 standalone ion chromatography systems and for the AD25, AS50, and UCI
modules.
ICS-2000 Gradient Additional single-user license for the ICS-2000. This supports creation
Generation
of an eluent gradient.
Client Features
Client License:
License for data reprocessing on a network PC without control. The
license supports multitasking and reports from the following partial
methods: single- and multiple-point calibration with various fit models,
integration, ratio test, user programs, etc.
Server Control:
Single-user license for data reprocessing on a network PC or a local
PC, including control of any number of systems. In addition, for network
control, make sure that the Multiple Network Control license is enabled
on the chromatography server.
Note: This license is only available with a Client License.
Chromeleon (Overview)
Concurrent
Clients:
15
Number of client licenses on a PC. A maximum of three Concurrent
Clients can be established and licensed on one PC. For a ¾License
Server, the number of floating licenses allowed on one PC depends on
the INI file (see the Administrator Manual: Installation Instructions
Installing the License Server). You can open a maximum of three
clients on one PC.
Report
Publisher:
This license extends the report and calculation features. In addition, it
supports calculation using formulas and ¾Additional Functions.
GLP Features
License:
This license supports additional ¾GLP features:
•
¾File History
•
¾Electronic Signature
•
Library License:
User mode (see ¾User Management: User Manager and Security
Activation Tool)
Additional single-user license for access to special ¾Spectra Libraries.
These libraries must be purchased.
Virtual Column
Additional single-user license for the ¾Virtual Column Tool
ASAP:
Additional single-user license for ¾SDK applications.
Analyzer:
Additional single-user license for controlling the DX800 process
analyzer.
Installing Chromeleon
Usually, Dionex Service or a network administrator installs the Chromeleon
software. Therefore, refer to the Administrator Manual: Software
Software Installation and
Installation and Communication
Communication for a more detailed description of the installation
procedure.
16
Chromeleon (Overview)
Components of a Chromatography System
17
Components of a Chromatography
System
Components of a Chromatography System
Modern HPLC or IC systems consist of the following components (HPLC:
High
Pressure/Performance
Liquid
Chromatography;
IC:
Ion
Chromatography):
The pump (b) draws up to four solvents from one or several reservoirs (a),
mixes them as defined, and then directs this mixture through the system.
The solution of interest (= the sample) is injected into this flow via an
¾Autosampler (c) and separated into its individual fractions or substances
on the column (d). Using a thermostatted column compartment can
optimize the separation process. When a substance reaches the detector
flow cell (e), a signal is produced that is proportional to the concentration of
the substance. The signal whose profile corresponds to a Gaussian
distribution is referred to as peak. The exact quantity of each substance
can be calculated by determining the peak area and by means of a
previously acquired calibration curve (quantitative analysis).
In suppressed conductivity mode IC, a ¾Suppressor is installed before the
detector. As an option, a fraction collector (f) can be installed after the
detector to distribute individual substances or fractions to different
containers (g).
18
Components of a Chromatography System
The peak area is determined by the chromatography data system that is
installed on a PC (h). In addition, the data system
•
Controls and monitors all connected chromatography instruments.
•
Collects data and status messages.
•
Enables quantitative and qualitative evaluation of the data, using
¾Photodiode Array Detectors or ¾Mass Spectrometers.
The chromatography instruments communicate with the computer and the
data system via special interfaces, such as the ¾UCI Universal
Chromatography Interface, via serial interfaces, or via additional cards,
e.g., via the A/D converter card.
If several PCs are connected via a network, the systems can be controlled
from remote locations on the network. In addition, data can be managed
centrally and retrieved from any workstation.
For more information about the components of a chromatography system,
refer to:
Chromatography Instruments
The Chromatography Data System
The PC
The Operating System
The Network
Chromatography Instruments
Chromeleon is especially designed to control and monitor the following
Dionex devices:
Eluent Generator
EG40 or EG50
HPLC Pump
P680
IC Pump
GP40, GP50, GS50, IP20, IP25, IS25
Autosamplers
ASI-100, AS50, AS3500
Column Thermostats
TCC-100, LC25, LC30, AS50 Thermal Compartment
UV/VIS Detectors
Single wavelength
AD20, AD25
Multiple wavelength
UVD 170U
Full-spectrum (PDA)
UVD 340U, PDA-100
Components of a Chromatography System
19
Electrochemical Detectors
CD20, CD25, CD25A, ED40, ED50, ED50A
Fluorescence Detector
RF2000
Refractive Index Detector
Shodex RI-101
Instrument/PC Interfaces
UI20 Universal Interface, UCI Universal Chromatography
Interface
System Modules
DX-120, IC20, IC25, IC25A, ICS-90, ICS-1000, ICS-1500,
ICS-2000
Appropriate instrument control options may need to be purchased and
installed in order to control these instruments.
Third-party analytical instruments are also supported. See the following
examples:
Gas Chromatographs
Such as the Agilent (or HP) 6890 GC
HPLC Systems
Such as the Agilent (or HP) 1100 HPLC System
HPLC Modules
Such as the Waters Alliance 2690 HPLC Module
Radioactivity Detectors
Such as the Berthold LB507A detector
Appropriate instrument control options may need to be purchased and
installed in order to control these instruments.
For more installation information, refer to the Administrator Manual:
Installing Dionex Devices.
Installation Instructions
The Chromatography Data System
The data system is the control center of a modern chromatography system.
Tasks of a Data System
•
It converts user input into time-precise control commands.
•
It monitors the state of the connected chromatography instruments.
•
It logs all user entries and modifications to the system.
•
It saves and archives all data.
•
It graphically represents data and allows you to check the system
status and system results.
•
It allows you to thoroughly check and evaluate data.
20
Components of a Chromatography System
Components
•
The basis for precise control is a real-time capable
System.
•
Installation and configuration is the task of the ¾Server Configuration
Program.
•
Device Drivers and other drivers, e.g.,
Virtual
The various
Channel Drivers, enable communication with the different instruments
and device types (refer to the respective sections in the Administrator
Manual: Special Drivers).
•
Each operation, such as execution of a command, or the display of an
Audit Trail.
error message, is logged in the
•
Depending on the required scope of performance, Chromeleon
supports various options. (For more information, refer to Chromeleon
Chromeleon Licenses.)
(Overview)
Operating
Chromeleon User Interface
•
Regarding the menu structure, screen elements, and operation,
Chromeleon supports all typical Windows properties. A homogeneous,
situation-related menu structure is as evident as the Windows
technology, toolbars, and context-sensitive use of the right mouse
The Client User Interface.)
button. (For more information, refer to
•
Each user can save and activate a "personal" screen. (For more
User Profiles (Workspaces).)
information, refer to
•
The user can modify the graphical representation of the online control
The Control
windows. (For more information, refer to Control
Panel.)
Operation
•
Starting the Program), verify that
Before starting Chromeleon (see
the hardware configuration is correct and that the PC is connected with
the system and with a datasource.
•
The Network), the client PC can control
In network operation (see
systems and datasources that are not directly connected with the PC.
•
Various assistants, the ¾Wizards, facilitate operation of Chromeleon.
Components of a Chromatography System
21
The PC
Overview of the minimum PC requirements
Windows 2000
Windows XP
MS and PDA Server
Service Pack
SP3
SP1
--
CPU
Pentium III
Pentium III 800
Pentium III 800
RAM
128 MB
256 MB
256 MB
Hard Disk
10 GB
20 GB
20 GB
Display
1024 x 768 x, minimum 256 colors
For better software performance, we recommend a Pentium 4 with a CPU
of at least 1 GHZ and 512 MB RAM.
For applications with several windows and control panels, e.g., in mass
spectrometry, we recommend using a higher screen resolution.
The Operating System
Chromeleon supports the following operating systems:
•
Windows XP SP1
•
Windows 2000 SP3
These operating systems are 32-bit ¾Multi-Tasking operating systems,
which means that several programs can be executed simultaneously. This
is especially important for instrument control, data acquisition, and
comfortable printer support.
Tip:
For more information, refer to the Administrator Manual: Software
Installation and Communication
Chromeleon and Windows
Operating Systems.
22
Components of a Chromatography System
The Network
The network capability of Chromeleon allows you to operate the data
system within local and global networks, also referred to as LAN ("local
area network") or WAN ("wide area network").
Chromeleon can be operated either locally in a single-user installation or on
a network. This includes data transfer and remote operation via ISDN. The
Chromeleon stations can be linked around the world (Wide Area Network).
To use all advantages and possibilities provided by network operation, such
as centralized data storage, backup, and administration, shared access to
methods and worldwide availability, safe and fast data exchange is
essential.
Sometimes, even state-of-the-art networks have difficulty coping with the
enormous amount of data. That is why client/server systems provide
decisive advantages by specifically selecting transferred data.
Client/Server System
On a decentralized PC (client), ¾RPC commands (Remote Procedure
Calls) are used to start sub-programs on a central computer (¾Server or
Chromatography Server). The server performs the actual "work." For
example, the server searches for data in a database or runs an application.
The client only receives the search result or the status of the application.
This means that the client provides the user interface while the actual
operation is remotely performed on the server. Chromeleon, too, uses this
"division of labor."
After starting Chromeleon on a local PC, the so-called ¾Client, the user
can perform all server-independent tasks; for example, re-process raw
data, create sequences, or search for individual spectra in a library.
If a chromatography ¾Server has been started, it is also possible to control
and monitor the chromatography instruments connected to this server.
Theoretically, each client connected to a running server via a ¾Control
Panel can do this. In practice, only the first client is allowed to control the
instruments connected to the timebase. All other Chromeleon users can
only monitor the system status.
Via device drivers, the server converts the control commands entered on
the first client PC for the analytical instruments. Inversely, the server
receives information from the system and forwards it to the appropriate
locations. Thus, status information, such as the current flow rate, appears
on all client PCs connected with this chromatography system.
Components of a Chromatography System
23
Raw data is automatically stored in the directory of a ¾Datasource and the
underlying database.
Depending on whether the client and the server are located on the same
PC or on different PCs in a network, a distinction is made between the
following installation types: local client/server installation and network
installation. For more information, refer to
Local Client/Server Installation
Network Installation
Local Client/Server Installation
The ¾Client and the (Chromatography) ¾Server are located on the same
PC. They must be started separately.
The controlled instruments in the chromatography system can be
connected to the PC, e.g., via RS-232 ports, a ¾DX-LAN, or USB
(Universal Serial Bus). Additional interface cards can supply a sufficient
number of ports. Each chromatography server can control a maximum of 6
controlled¾Timebases (chromatography systems).
The data is saved is on the local PC. During the initial installation of
Chromeleon, a local datasource is installed on each local computer.
If the PC is part of a local network (LAN = Local Area Network or WAN =
Wide Area Network), data can also be saved externally. In the same way,
external data can be used for data editing.
Client
Operating
System
Server
Data
Sources
RS232
If the local PC is connected to a network, all options of a
Installation are available.
Network
24
Components of a Chromatography System
Network Installation
The ¾Client, the Chromatography ¾Server, and the ¾Datasource can be
installed on different computers. They are connected via the network and
the corresponding network server. Each chromatography server can
operate up to 6 chromatography systems (¾Timebases).
The Client, Server, and Datasource are independent units on the network.
WAN
ISDN
Client
Network
Server
Server
Chrom. System
On each PC, a server, a client, and a local datasource are set up during the
installation of Chromeleon. Independently of this, each PC can act as
"server only" or "client only." Theoretically, each client can access each
datasource and each server. In practice, this may not be desirable for
safety reasons. Therefore, various options are available to restrict user
access.
•
The ¾Server Configuration program allows you to define the extent of
network operation for each ¾Server and each timebase. A server can
be made available for the entire network or for certain ¾Access Groups
only. The server can be locked either partly or completely. If the server
is partly locked, for example, it is still possible to monitor the server, but
control of the connected instruments is disabled. In this case, control is
reserved for the local client. This distinction also applies to the
timebases. If three timebases (TIME1/2/3) are installed on a server,
TIME1 could be completely shared, TIME2 could be excluded from
network operation, and TIME3 could be shared for monitoring only.
•
Datasources and/or their subdirectories can also be protected from
undesired access. Depending on the location of the corresponding
database (on the local hard disk or a network PC), the respective user
or the network administrator decides (by "sharing" a directory) which
data can be accessed and by whom. In addition, datasources shared in
Windows can be locked in Chromeleon.
Components of a Chromatography System
•
25
In addition, access can be restricted via ¾Passwords or the
Chromeleon User Management. If the administrator has enabled
access control (in the Security Activation Tool (CmSecure program)),
the user must enter a password before being allowed to perform
specific operations. The system administrator determines these
operations in the User Manager (CmUser program). In this way, the
administrator can deny controlling rights or prohibit "locking" of
datasources.
Important data are often stored on central data server PCs. If, during data
acquisition, the network connection is interrupted or the data server PC
crashes, data acquisition should be continued, nevertheless. All data that
are relevant for the Chromeleon server are locally stored on the server's
hard disk, thus ensuring that the data acquisition will not be interrupted in
case of a network failure (see ¾Network Failure Protection).
In addition to allowing data exchange within a local area on a Windows,
Novell, DEC, or UNIX network (LAN), it is possible to transfer data across
huge distances (WAN) via ISDN. The basic requirement for any type of
network operation is the availability of the corresponding network drivers for
Windows 2000/XP.
Tip:
For more information, refer to the Administrator Manual: How to …:
Actions in the Browser and in the Operating System:
Sharing the Local Datasource on the Network
Creating a Network Datasource
Saving Chromatography Data on the Network
26
Components of a Chromatography System
Basic Operation
27
Basic Operation
Operation via the Keyboard
All commands and menu options are accessible from the keyboard. Press
the Alt key to display the underlined hotkey letters. To open a menu via the
hotkey, press Alt and the hotkey. To select a command or option on the
menu, enter another hotkey. Alternatively, use the arrow keys to move to
the desired option and then confirm your selection by pressing the Enter
key.
Example:
To left align several objects on a control panel, select
Align on the Edit menu, and then select Left.
Alternatively, press Alt, and then press the A and L keys.
Or else, press the Alt and E keys, and then select the
desired option, using the Ð and Î arrow keys. Confirm
your selection by pressing <Enter>.
In addition, important commands can be accessed via shortcuts. The
shortcuts are displayed to the right of commands and menu options. Enter
the key combination to directly execute the corresponding function.
Example:
Press the Control and C (Ctrl+C) keys to copy a selected
item.
For a list of available shortcuts, refer to Operation
Shortcuts.
Keyboard
28
Basic Operation
Keyboard Shortcuts
Shortcuts are provided for many operations, especially in online control:
General
Action
Esc or
right-click
Where
F1
Description
Aborts the drag/move action.
Opens the context-sensitive Help;
F2
Enables the Edit mode.
F3
¾Browser +
¾QNT Editor
F4
Chromatogram
Shift + F4
Renames files (following the Find
command: Find Next).
Takes you to the next sample.
Takes you
sample.
to
the
previous
F5
Updates the window.
F6
Takes you to the next partial
window.
Shift + F6
Takes you to the previous partial
window.
F10
Takes you to the next channel.
Shift + F10
Takes you
channel.
F7
Shift + F7
Browser
(F7 key only) +
to
the
previous
Optimizes the column width.
Optimizes the line height.
QNT Editor
F8
Opens the dialog box.
F10 or Alt
Shows the hotkey underlines.
Shift + F5
Browser
Alt + Enter
Browser + ¾Control Opens the respective Properties.
Panels
Ctrl + Tab
Ctrl
Displays the chromatogram.
Toggles between open windows.
Signal plot in the Allows zooming (the cursors is
¾Report and in the changed to the zoom cursor).
QNT Editor
Ctrl + N
Creates a new file.
Ctrl + O
Opens the file.
Ctrl + S
Saves the file.
Ctrl + R
Ctrl + P
Opens the batch report.
Report + ¾Printer Prints the selected object(s).
Layout
Basic Operation
Action
Edit
Sample List
Where
29
Description
Ctrl + Z
Undoes the previous action.
Ctrl + Y
Repeats the previous action.
Ctrl + X
Cuts the selected object(s).
Ctrl + C
Copies the selected object(s).
Ctrl + V
Pastes the selected object(s).
Ctrl + F
Finds a string of characters.
F3
Finds
the
characters.
Ctrl + H
Replaces the entry in the field.
F9
Fills the column and/or selected
cell(s) with the first value of the
selection.
next
string
Ins
Inserts the selected object(s).
Del
Deletes the selected object(s).
Ctrl + I
Inserts a sample.
Ctrl + D
Deletes a sample.
Ctrl + A
Adds a sample.
PPA
Alt + F4
Copies the
clipboard.
Control
Ctrl + F
Changes the pump's settings
Ctrl + I
Injects
Ctrl + Break
Stops the pump.
Break
Turns on the Hold mode.
Ctrl + A
Turns on data acquisition.
Ctrl + B
Edits the batch.
Signal plot
Gauge/Slider
Double-click...
spectrum
- Overview window
Unzooms.
- Time axis
Auto Plot Speed
- Signal axis
Autoscale
- Plot range
Signals...
to
of
the
(- or else:…)
Axis/decoration
Shift
- when zooming
Retains the scale ratio between
signal and value axis. (The shape
of chromatograms is maintained.)
Ctrl key
Press
dragging
when Toggles the Snap To Scale
option.
30
Basic Operation
Action
Where
Description
Script Button
Click
Button
Indicates whether the program is
still running. Stops the program
upon confirmation.
Edit Field
Tab/Enter
Sends the new value.
Esc
Aborts the input.
Layout Mode
Alt + Click
On the
panel
selected Draws a selection frame on the
control panel.
Alt + Drag
Temporarily toggles the Snap To
Grid option.
Esc
Deselects all.
Arrow keys
Drags the selection pixel by pixel.
Shift +
Arrow keys
Increases/reduces the selection
pixel by pixel.
Shift +
Extends the selection.
Select
Ctrl + Select
Highlights the selected control
(the highlighted control is used in
Align..).
Ctrl + Drag
Double-clicking
Copies controls.
On the online signal Opens the Properties... dialog
plot on the control box.
panel
Especially in the chromatogram, many additional hotkeys are available. For
information, refer to How to ...: Actions in the Chromatogram
Using
Hotkeys in the Chromatogram.
Operation via the Mouse
Chromeleon features all windows-typical properties and capabilities of a
two-button mouse.
The left mouse button serves to mark and select menus, menu
commands and options, and icons, to operate display and control elements,
and to modify windows (increase or reduce the window size, zoom, modify
display and control elements, etc). Double-click the left-hand mouse button
(= left-click) to execute the selected function.
Simultaneously left-click and press the SHIFT key to select several cells
in a table. Left-click the first cell, press the SHIFT key, and then select a
new cell. All cells between these two cells will be selected.
Basic Operation
31
Simultaneously left-click and press the CTRL key to select non-adjacent
cells in a table. Left-click the first cell, press the CTRL key, and then select
a new cell. Keep the CTRL key depressed and repeat until all required cells
are selected.
Left-click an object and drag it to the desired position (Drag and Drop).
This action allows you to move selected text or samples. To copy the
corresponding object, press the CTRL key while performing the Drag and
Drop operation.
Use the right mouse button (= right-click) to open context-sensitive
menus. These menus provide functions that are required or allowed in the
current situation.
For more information, refer to your Windows manual.
Undo/Redo Commands
Chromeleon "remembers" the user's last modification. Select the Undo
command on the Edit menu to cancel the operation. Select Redo to repeat
the last operation.
Starting the Program
Chromeleon runs on client PCs equipped with Windows 2000 or Windows
XP as operating system. To start Chromeleon:
•
•
Click Start to open the Start menu. Move the mouse cursor to
Programs and wait until the submenus display.
Move the mouse cursor to Chromeleon. A submenu displays with
several choices:
32
Basic Operation
Client
•
Select Chromeleon to start the Chromeleon software and to start data
evaluation.
•
If Chromeleon has been installed with integrated access control, the
user must enter his/her user name and password for identification. If
you have any questions regarding system access, please contact your
system administrator.
Tip:
To control a chromatography system under Chromeleon, you must also
start the Chromeleon server.
Server (Server Monitor)
Tip:
During installation, Chromeleon usually includes a link to the Server
Monitor program in the Autostart group. Thus, the program is started when
you start your computer. The corresponding icon appears on the taskbar.
•
If the icon does not appear on the taskbar, click Start and select
Server Monitor on the Chromeleon menu to start the ¾Server Monitor
Program. The Chromeleon icon will appear next to the Windows
system clock on the Windows taskbar.
•
Place the mouse cursor on the icon. The Chromeleon Server is not
running quick info message appears.
•
Select the Start Server command on the context menu (or double-click
the icon and then click Start). The color of the icon indicates the status
of the server monitor. Gray coloring indicates that the server is running
(the quick-info message reads: Chromeleon Server is running idle).
You can now open a control panel and directly access the devices
installed in the selected timebase. If the devices have been installed
correctly, you can operate them online.
Tip:
Any problems occurring when controlling single instruments may be caused
by an incorrect server configuration. Start the ¾Server Configuration
program to check the configuration.
Basic Operation
33
The Client User Interface
The Chromeleon user interface features all known Windows elements such
as menu bar, ¾Toolbar, and ¾Status Bar.
To display of the individual bars listed below, select them on the View
menu.
Standard
Toolbar for
various standard
functions
Online Tools for
using the control
panel
Layout Tools for
designing the
control panel
Method Tools for
selecting a
method window
Integration Tools
for the most
important
operations in the
Chromatogram
34
Basic Operation
Move the mouse cursor on one of the buttons to display its description. The
space between the task bar and the status bar represents the workspace,
Windows supported by
allowing you to display the different
Chromeleon.
The Windows
The appearance of the different windows can vary considerably. A window
can have various window sections of which the borders can be moved (a).
If the window section is too small to display the entire information, display
the hidden area, using the scroll arrows or scroll bars (b).
The window content depends on the type of the represented data. It is also
possible to integrate table editors as a window section in a method window.
For window sections with graphical representation, the coordinates of the
mouse cursor are indicated on the status bar.
Window Types
In addition to the Browser and the Control Panels, Chromeleon supports
the following method windows:
•
⇒QNT Editor
•
Integration
•
¾Peak-Purity-Analysis (PPA)
¾Printer Layout
¾Signed Results
¾Virtual Column
•
•
•
Basic Operation
35
Only one method window is enabled by default. To use several method
windows simultaneously, select Preferences on the File menu of the
Browser. On the Browser tab page, clear the Open only one method
window check box. This allows you to open each window any number of
times.
The Table Editor
Table Editors serve to enter and represent various parameters and
variables. For example, one table editor is available in the Browser. This
editor allows you to list or edit single samples or sample data or to display
numerical results in a report. It also serves to determine integration,
calibration, and peak table parameters.
•
Appearance and use of all tables correspond to the Windows standard.
Editing is by cell, column, or line.
•
It is possible to move or hide selected columns or to make them visible
again at the active cursor position. Adjust the column width by moving
the left or right delimiter. Or else, select Optimum column width, e.g.,
on the View menu in the Browser.
•
You can add additional lines or remove selected lines anywhere in the
table.
•
Most of the described functions are available on the context menu.
•
You can change the font size. For example, select a smaller size to
display more on-screen information.
•
Press the F1 key for online Help information about a specific column.
•
Press the F8 key to open an edit dialog box for a specific field. This
prevents input of incorrect or invalid values and names.
•
Press the F9 key to fill an entire column with the value of the current
field. In this way, you can also extend logical rows of numbers. For
example, if the input in the first three fields is 2, 4, and 6, the row is
continued with 8, 10, 12, etc.
36
Basic Operation
Working with Several Windows
Chromeleon not only supports working efficiently with several open
windows, as is typical in Windows programs, but it also introduces what has
become possible with object-oriented programming of applications and was
not common before:
Data representation is always updated in all windows!
Below please find two examples of samples with chromatograms. Specific
detection parameters were used for integration and analysis.
Example 1:
If the ⇒Minimum Area detection parameter is corrected
in the QNT Method window by entering a smaller value,
the corresponding chromatogram is immediately
adjusted. Peaks with an area smaller than the minimum
area are not considered.
Example 2:
Similarly, the result of a baseline that has been modified
manually is displayed immediately in the integration
report window.
User Profiles (Workspaces)
Chromeleon allows you to save the window arrangement of any work
situation, i.e., a combination of the different windows, in a workspace.
This facilitates opening single files or windows and allows you to work in
whatever work environment you prefer.
The information about the windows is stored in a WSP file. If you want to
use a specific workspace when you start working, open the corresponding
WSP file, or generate a new WSP file by storing the screen contents. The
following commands are available on the Workspace menu:
•
Select Open Workspace on the Workspace menu to open an existing
workspace.
•
Select Save Workspace to save the current workspace.
•
Select Save Workspace as to save the current workspace with a new
name.
•
Select Autosave Workspace to always save the most recent
workspace as the default workspace.
Basic Operation
37
There is no restriction as to the number of windows that can be saved with
The Windows.)
each workspace. (Also, see Basic Operation
A useful workspace arrangement might combine, for example, the report, a
control panel, and the Browser:
The appearance of each individual window is stored in the corresponding
file. For example, the appearance of the on-screen report and the Printer
Layout is stored in the
Report Definition file.
Tip:
When Chromeleon is started, the most recently used workspace is loaded.
Report Definitions
The ¾Report Definition File (RDF) comprises the current settings such as
the names and scaling of axes, the representation of chromatograms and
spectra, the display of additional information or auxiliary lines, the setting of
various fonts, font styles, and sizes, as well as the column arrangement in a
table.
38
Basic Operation
The report definition also determines how the screen contents (hardcopy)
or online batch results are printed. (For more information, refer to Basic
Printing.)
Operation
Unlike a workspace, a report definition can be used for individual windows.
If no workspace is loaded, each new window is opened based on the most
recently used report definition. If you have not yet stored a report definition
file, the default Chromeleon RDF is used. The default report definition file
(DEFAULT.RDF) is located in the Report directory.
•
Select Save Report Definition on either the context or View menu to
save the current settings.
•
Select Load Report Definition on either the context or View menu to
open a previously saved Report Definition File.
Create/Open Files, Windows, and Templates
There are several ways to open or create files, windows, and templates.
Frequently, other information is required for this. For example, when a
control panel is opened, it searches "its" timebase, that is, the correct link
between the client PC and a certain chromatography system (¾Timebase)
and each method window searches data from a specific ¾Datasource.
Tips:
If problems arise, they are mainly caused because the user renamed
timebases or datasources that were correctly installed before by the Dionex
Service Representative.
Similar problems may occur if databases are located on a network PC to
which the user currently cannot connect or for which (s)he has no share
authorization.
In this case, refer to the Administrator Manual: How to ...: Actions in the
Browser and in the Operating System
Connecting a Datasource.
Automatically Loading the Most Recent User Profile
Chromeleon automatically loads the most recently used workspace. If this
is not possible, the Browser is started.
Basic Operation
39
Opening Existing File Types and Windows
Select the Open command on the File menu; select the datasource and the
directory containing the file to be opened and the file type. Refer to the
online Help for more information about this dialog box.
Or else, double-click to open a file in the ¾Browser or select Open on the
File menu.
Open Most Recent Files and Templates
The lower section of the File menu and the Workspace menu lists the
most recently used templates and files. Click to open a template or file. This
is the simplest and quickest way to continue an interrupted task.
New
Select New on the File menu to receive a list of all possible file types and
chromatographic operations.
•
Select Control Panel to open a window from which you can control the
chromatography devices of a specific timebase.
•
Select Sequence File (using Wizard) to start the
Sequence
Wizard. The Wizard guides you through the process of creating a
¾Sequence and lets you include the samples into the sequence.
•
Select Sequence (from LIMS Worklist) to include data and a
¾Sequence of a ¾LIMS in the ¾Worklist format.
•
Program Wizard. The Wizard
Select Program File to start the
Control Program
guides you through the process of creating a
(PGM File). A ¾PGM File includes all ⇒Control Commands that must
be transferred to the different chromatography devices to process a
sample or a series of samples.
•
Select Method File to create a new quantification method.
•
Select Spectra Library to create a new spectra library.
40
Basic Operation
Printing
The Chromeleon ¾Printer Layout provides numerous options for the
presentation of the results. Similar to a word processor, you can choose
between printing the on-screen contents and printing based on defined
templates.
Printing from the Browser
If you have selected one or several samples or sequences in the Browser,
you can select Batch Report… on the File menu to start the printout. Use
the Batch Report dialog box to determine which ¾Report Definition File
(RDF) shall be used and which pages of the printer layout shall be printed
for which sample type and for which channel.
The single pages are created in the Printer Layout: (See Data
Reprocessing
The Printer Layout for more information).
Note:
Click Setup in the Printer Layout to define the page format. (For more
information, refer to How to ...: Actions in the Printer Layout
Changing the Page Format.)
Printing in an Online Batch
If several samples are processed in an automatic sample batch (¾Batch
Processing), you can determine which pages of the Printer Layout are
printed. Printing can be started either immediately after a sample has been
processed or after the entire sequence has been processed.
Note:
The report template used for printing is stored with other settings (such as
screen settings) in the Report Definition File (RDF). Future Chromeleon
versions will save the screen and the print settings separately.
Basic Operation
41
Printing the Screen Contents
To print the contents of the active window, select the corresponding
Print ... command. Select Print Sequence, to print the current sample list
from the Browser, with either the corresponding PGM File or QNT File.
If a ¾PGM File is open, you can print the required views by selecting the
Print... command. In the same way, you can print the selected pages of a
¾Quantification Method (QNT Method) by selecting Print QNT Method in
the QNT Editor.
42
Basic Operation
Control
43
Control
Control Concept
The chromatographic equipment in an analytical laboratory usually
comprises instruments from different manufacturers and of various
generations. Depending on whether individual devices can be controlled via
a PC, they are referred to as either controlled or non-controlled devices.
Controlled Devices
Controlling devices makes high demands on the adaptability of a modern
chromatography data system, as operation of individual components must
be easy and thus device-independent. This means that input and
representation of a specific control command (for example the pump flow)
must always be performed in the same way, regardless of whether the
instrument is by manufacturer X or Y.
Chromeleon enables this with the Chromatography BIOS and the available
device drivers. (For more information about these items, refer to the
Administrator Manual: Device Communication
Communication
between the PC and the Chromatography System (Chromatography
Device Drivers.)
BIOS) and
The advantages of a uniform user interface are as follows:
•
Short training time for inexperienced users.
•
Uniform, device-independent operation: The same commands are used
for all systems.
•
Transfer of methods: Chromatographic methods can usually be
transferred from one system to another. This is why the corresponding
device functions must be available on both systems.
•
Clear and uniform documentation.
44
Control
Non-controlled Devices
In rare cases, non-controlled systems are used, and the individual
instruments are operated manually. Chromeleon only records the data. Two
conditions must be fulfilled:
•
The injection time is communicated to Chromeleon by connecting the
Inject contact of the injection valve with a ¾Remote Input of the
system. This guarantees time synchronization between sample start
and recording time.
•
Detector data is transferred to Chromeleon as digital or analog data
(serial interface or ¾UCI Universal Chromatography Interface).
In principle, there are two ways to control a chromatography system:
•
Many samples are usually processed in a ¾Sequence, via a program.
The Control Program. To edit the
For more information, refer to
The PGM Editor.
program, refer to
•
To create a new chromatographic method, you can control the
instrument directly from the ¾Control Panel, i.e., the single devices and
device functions are controlled directly and interactively from the
The Control Panel.)
control panel. (Also, see Basic Operation
Control Requirements
The requirements for controlling devices are as follows:
•
The appropriate
•
The controlled instruments must be correctly installed and configured in
the ¾Server Configuration program.
Chromeleon License must be available.
Tips:
Dionex Service or a network administrator usually performs installation.
For more information, refer to Administrator Manual: Installation
Instructions
Installing Dionex Devices
Installing and Controlling Third Party Devices
Control
45
•
A connection between the PC and the ¾Chromatography Server must
be established.
•
A connection between the chromatography server and the
chromatography system must be established via a serial interface, a
DX-LAN, or any other interface (TCP/IP, GPIB, USB). (For more
information, refer to the Administrator Manual: Software Installation,
The Serial (RS-232) Interface
Communication, and Validation
The DX-LAN.)
and/or
The Control Program
Modern analytical laboratories usually analyze many samples. These
samples are grouped in ¾Sequences and processed with a
chromatographic control ¾Program, the PGM File. When creating a
program, you include a list of commands and the times when the
commands will be executed (relative to the time of injection). Chromeleon
automatically adds the ⇒Inject and ⇒End commands. The finished
¾Program is displayed in the Commands view of the PGM Editor.
Tip:
Dionex recommends always editing programs in the associated Device
view. The Device view is easy to use and ensures correct command
syntax. Use the Commands view only if the desired parameters are
unavailable in the Device view.
When starting the program, all commands are executed precisely at the
defined time.
For more information, refer to:
The Control Program (Details)
The Program Wizard
The PGM Editor
The Program Syntax
46
Control
The Control Program (Details)
The control program (often referred to as the ¾Program or PGM File)
includes a list of time-precise ⇒Control Commands. The user creates the
actual control program. The aim is the automatic and repeated execution of
specific routine tasks such as processing samples automatically or
conditioning a column by rinsing with various solvents. Monitoring certain
parameters or limits, or triggering reactions when these limits are
exceeded, can also be performed via a program.
A ¾Wizard (see
Program Wizard) assists you in creating a program by
automatically converting your entries into the respective Chromeleon
program commands. In this way, you can create a program even if you do
not know the command syntax.
To edit an existing program, open the program by double-clicking the
program name in the Browser. This displays the view of the PGM Editor
that you opened last.
•
To edit the control settings for a device, click the corresponding device
icon on the left pane. This view provides the corresponding pages of
the Program Wizard. Enter the desired parameters or change existing
parameters according to your requirements.
Tip:
Dionex recommends always editing programs in the associated Device
view. The Device view is easy to use and ensures correct command
syntax. Use the Commands view only if the desired parameters are
unavailable in the device view.
Tip:
Similar to a control panel, you can edit a PGM File only if it is connected to
a ¾Timebase while the server is running. Therefore, first verify that the
server is running, and then if necessary, start the server in the Chromeleon
¾Monitor Program. To connect the PGM File to a timebase, select
Connect to Timebase on the Control menu.
If the PGM File is connected correctly to a timebase, all icons
corresponding to the devices for that timebase are available on the
respective PGM Wizard pages.
Control
47
For more information about how to create a program, refer to
The
Program Wizard. For information about how to edit a program with the
The PGM Editor.
PGM Editor, refer to
For practical tips, refer to How to ...:
Actions in the PGM Editor
Device Control
Creating a Program
Device Control (Overview)
The Program Wizard
The Program Wizard guides you through ¾Program creation. To start the
Program Wizard, select New on the File menu, and then select Program
File. The Program Wizard systematically collects all of the information
required to generate a basic program. Each step consists of a template in
which the user enters or selects data. Depending on the installation,
different steps are required.
For a typical HPLC or IC timebase, the Program Wizard includes the
following steps:
Step 1: Select a ¾Timebase
Step 2: Select the temperature settings (if supported)
Step 3: Define a flow system
Step 4: Determine a gradient profile (optional)
Step 5: Enter the autosampler settings (if supported)
Step 6: Determine channels and the duration of data acquisition
Step 7: Determine signal parameters for the individual channels
Step 8: Define the peak detection parameters for fraction collection
(optional)
Step 9: Complete the Program Wizard
Chromeleon completes the information entered in steps 1 to 9 by adding
the ⇒Inject and ⇒End commands, thus creating an operable program,
which is displayed in the Commands view of the PGM Editor. (For more
information about the PGM Editor, refer to
The PGM Editor.)
To edit your basic program, use the
Views in the PGM Editor.
48
Control
The PGM Editor (Overview)
The PGM Editor allows you to edit the control programs (¾PGM Files). The
PGM Editor includes different views, which are accessed via the
corresponding icons on the left pane (the shortcut bar):
View
Commands
Icon
Description
Display of the program
(see
Post-acquisition
steps
The Commands View)
Determination of steps for data reprocessing
(see
The Post-Acquisition Steps View)
Device
If the respective instrument is installed, the associated icon is available:
Finnigan AQA or
Surveyor MSQ
Input of AQA or MSQ parameters (see
Column Oven
Input of thermostat parameters
Surveyor MSQ or Finnigan AQA Views)
(see
Device Views)
Pump
Input of pump parameters
(see Device Views above)
GC
Input of GC parameters
(see Device Views above)
Sampler
Input of autosampler parameters
(see Device Views above)
UV
Input of UV detector parameters
(see Device Views above)
RI
Input of refractive index detector parameters
(see Device Views above)
Emission
Input of fluorescence detector parameters
(see Device Views above)
Relay and State
Devices
Input of relay and condition parameters
(see Device Views above)
Control
49
Note:
The name of the single device views corresponds to the Device Name
entered in the Server Configuration program. For example, if you have
named your pump HPLC pump, the device view is also named HPLC
pump; it is not named pump, as stated in the above table. The above table
lists the default device names.
The Device Views
The different device views in the
PGM Editor offer a user-friendly way
to edit an existing program at any time according to your requirements.
The Program
The device views contain the corresponding pages of
Wizard. To modify existing commands or to enter new commands for a
device, click the device icon on the left pane. In the device view, the
respective page of the Program Wizard is re-opened.
Neither the device view nor the Program Wizard supports some special
commands. Define only these commands in the Commands view. For
more information, refer to
The Commands View.
Tip:
Dionex recommends always editing programs in the associated Device
view. The Device view is easy to use and ensures correct command
syntax. Use the Commands view only if the desired parameters are
unavailable in the device view.
The Surveyor MSQ or Finnigan AQA Views
These views of the
PGM Editor are part of ¾Xcalibur and are used to
specify the method that is used by the Surveyor MSQ or the aQa mass
spectrometer.
AQA: On the Ionization Mode tab page, specify the mode of ionization
(Electrospray/APCI). On the Analysis tab page, specify the sensitivity and
fine-tune the mass spectrometer.
50
Control
Caution:
When using the Xcalibur method editor, disregard the Other detectors
section and perform data acquisition as usual. Do not use this section for
data acquisition with other detectors (such as the UV detector)!
On the Acquisition tab page, set the aQa-specific signal parameters for
¾Mass Spectra acquisition.
Surveyor MSQ: The view displays the selected chromatogram, if available.
Specify the Ionization Mode and, in the Full/SIM Scan Events section,
specify the device parameters for mass spectra acquisition.
Tip:
These views of the PGM Editor are part of the Xcalibur program. The
Xcalibur Help system provides detailed information about mass spectra
acquisition. If you use an aQa mass spectrometer, open the Xcalibur Help
system via the Help menu or by clicking the Help button. If you use the
MSQ, first click the question mark (at the top right) and then click the option
of interest to open the corresponding Help topic.
For more information about how to create an MS method, refer to How
to …: Actions Related to Mass Spectrometers:
Creating a PGM File for the aQa MS and/or
Creating a PGM File for the MSQ
The Commands View
The Commands view in the
PGM Editor shows the actual program with
the various commands in chronological order. To facilitate orientation within
a program, control commands are displayed in black, comments in green
and ¾Triggers in blue.
Tip:
Dionex recommends always editing programs in the associated Device
The Device Views). The Device
view (see Control/The PGM Editor
view is easy to use and ensures correct command syntax. Use the
Commands view only if the desired parameters are unavailable in the
Device view.
Control
51
Press <F8> to open a dialog box that allows you to edit the program. The
dialog box provides you with exactly those commands required for
controlling the devices in the current timebase:
Note:
If the PGM File and the timebase are not connected correctly, the F8 box is
not available and the program lines are displayed in gray print.
The input procedure via the respective device icons or the F8 key prevents
entering invalid command syntax. If Chromeleon finds a command with
unknown or incorrect syntax, the corresponding line is displayed in red
print.
•
Press <F4> or <Shift> + <F4A> to browse through the errors and
correct any wrong input.
If you are familiar with the
Program Syntax, you can change or extend
your file directly via the keyboard. After editing, save the ¾PGM File by
selecting Save on the File Menu.
For more information about how to create and/or edit a PGM File, refer to
Control:
The Control Program
The Program Syntax
The Program Wizard
For information about the different ⇒Control Commands, refer to How
to ...:
Actions in the PGM Editor
Device Control
Creating a Program
Device Control (Overview)
52
Control
The Post-Acquisition Steps View
Use the Post-acquisition steps view of the
PGM Editor to define
extraction and smoothing steps that are performed by the PGM File after
data acquisition. In addition, you can copy existing channels or combine
them using arithmetic operations. A new data channel is created for each
step. The individual steps can be performed online, after data acquisition,
or offline of the chromatogram, UV spectrum, or mass spectrum.
Therefore, the Post-acquisition steps view is one of many data
reprocessing tools in Chromeleon, but it is not part of the actual control. For
more information, refer to
Post-Acquisition Steps.
For more information about post-acquisition, refer to How to …: Actions in
Adding Post-Acquisition Steps.
the PGM Editor
The Program Syntax (Experts Only)
Tip:
Dionex recommends always editing programs in the associated Device
view (see Control/The PGM Editor
The Device Views). The Device
view is easy to use and ensures correct command syntax. Use the
Commands view only if the desired parameters are unavailable in the
device view. To enter the commands, use the F8 dialog box as described in
Commands View).
the Commands view (see Control/The PGM Editor
Enter commands directly only if you know the correct syntax.
For uniform operation by different users, ⇒Control Commands are always
entered in English. The syntax for the ¾Program commands is as follows:
Retention Time
DeviceName.Command
or
Retention Time
DeviceName.Property
=
Value
Program Wizard as well
If you are not familiar with the syntax, use the
as the different Device Views that guide you through program creation. For
a list of general commands and commands for Dionex and for third-party
Commands and Tips for Device Control.
devices, refer to
Control
53
Time Value (Retention Time)
The time value is entered at the beginning of the control command. It
determines when the command is to be executed. The information is
entered in ¾Decimal Minutes, for example,
2.500
This input is optional. If no time is entered, the time specified in the
previous program line will be used.
Device
Devices are all instruments, channels, relays, or remote inputs that are
available in the active timebase. They can be recognized in the F8 dialog
box by the device symbol (
). Each Device has a number of commands
and/or properties.
As various instruments can have the same commands or properties, adding
the device name in front of the command makes a distinction. The syntax is
as follows:
Retention Time
DeviceName.Command
or
Retention Time
DeviceName.Property
=
Value
If no confusion occurs with other commands or properties, the device name
can be omitted For example, you can omit the Flow command when only
one pump is installed. The syntax is as follows:
Retention Time
Flow
=
Value
Command
Commands are represented in the F8 edit box by an exclamation mark
( ). If a command can be clearly assigned to an instrument, the name is
sufficient for identification:
2.500
NeedleUp
The ⇒NeedleUp command exists for the Dionex Autosampler GINA 50,
only. In this case, the device name can be omitted. This is in contrast to:
2.500
UV_VIS_1.AcqOn
54
Control
The ⇒AcqOn command by itself is not unique (if there is more than one
channel in the system). To address one specific channel, the channel name
must be added to the command.
In addition, commands can be extended by additional parameters, for
example:
2.500
2.500
⇒Inject
Relay1.On
Position = 20, Volume = 30
or
Duration = 20
The possible command extensions and their order are predefined. They are
listed in the F8 edit box. As confusion is impossible, you can also use the
following syntax:
2.500
2.500
Inject
Relay1.On
20, 30
20
or
Device-Independent Control Commands
If a command cannot be assigned to a Device, it is listed in the F8 edit box
⇒Branch, ⇒Log, ⇒Message,
by itself. This applies to the commands
⇒Protocol, ⇒Wait, ⇒Delay, ⇒Trigger, ⇒EndTrigger and ⇒End.
For more information, refer to How to ...: Device Control
Trigger Commands
Mixed Commands
Property
Properties are distinguished by their value. An I/0-symbol ( ) in the F8 edit
symbol indicates
box represents values predefined by the system. The
that the values are freely selectable. A command string is also considered a
property ( ). For example:
2.500
2.500
2.500
UV.Lamp
UV_VIS_1.Wavelength
%A.Equate
= On
= 300
= "%A"
or
Control
55
If a property gives an actual value (for example, Pressure (bar), %A (%),
Signal (mAU), etc.), this is indicated by a separate symbol ( ). Properties
in connection with the actual value are subordinate to it. For example,
Chromeleon enables the output of the current system pressure
( pressure) and the definition of an upper and lower pressure limit
LowerLimit). Assigning a solvent name is via the
( UpperLimit and
Value, Equate). The corresponding syntax is:
same method ( %B,
2.500
2.500
2.500
2.500
pressure.UpperLimit
pressure.LowerLimit
%B.Value
%B.Equate
=
=
=
=
350
20
30
"Methanol"
Here the same applies: if the syntax is not clear, the device name must
precede the command or property. For example:
2.500
UV_VIS_1.Signal.UpperLimit
= 500
Text, Names
Comments on the program or individual commands can be included before,
after, or between individual commands. The comment lines are always
started by a semicolon ";." and are displayed in green print:
;
The following program ...
Text must be entered in quotation marks if it is displayed on screen
because of a command and is included in the audit trail (as with the
⇒Protocol and ⇒Message commands).
2.500
Protocol
"Test program"
If these commands are entered using the F8 edit box, the quotation marks
are added automatically.
56
Control
The Control Panel
You can directly control the different devices of a timebase using a
¾Control Panel (in short: panel; also referred to as online plot or online
window). A control panel controls and monitors the chromatography
instruments configured in a ¾Timebase. With regard to appearance and
function, it is a special type of window. In accordance with the Chromeleon
philosophy, you are free to design its appearance in any way to meet the
user requirements. For more information, refer to:
Control Panel: Appearance
Control Panel: Function
Control Panel: The Signal Plot
Control Panel: The Audit Trail
Control Panel: The Trend Plot
Control Panel: Appearance
¾Control Panels do not have a uniform user interface. The appearance is
determined by a combination of various default controls.
Control panels can include, for example:
•
A slider to change the pump flow or another variable parameter.
•
A separate field to display status information such as the running
retention time.
•
A screen LED to indicate whether the detector lamp is active.
•
A script button to execute the inject command.
•
A signal plot to monitor the detector signal.
•
The current ¾3D Field and the current ¾Mass Spectrum.
•
The Audit Trail to follow the execution of an operation.
You can determine the number of available controls and their functionality,
depending on individual requirements. Each user thus "designs" a personal
interface. The available functions depend on the functionality of the
analytical instruments that are combined in a timebase. For example, if a
controllable column oven is part of your system, you can control the oven
temperature.
Control
57
The system administrator assigns user-specific ¾Privileges in the ¾User
Manager (CmUser program) to determine whether a user is authorized to
create his "personal" user interface. The organization of the panel allows
you to lock certain functions on the user interface or to disable the display
of irrelevant information.
You can save a new user interface as a separate file (*.pan). Each user
who can access the directory containing the file can select and use the file
by clicking Open on the File menu.
If this type of screen arrangement seems confusing at first, the system
provides several default control panels. It is not possible to modify these
panels. They do not only cover all standard control functions but they can
also be used easily and intuitively.
For more information about how to create a control panel, refer to How
to ...:
Actions on the Control Panel.
Control Panel: Function
¾Control Panels serve to control and monitor individual ¾Timebases. The
timebase that will be controlled from the control panel is specified when the
panel is created. When you open the control panel, the system tries to
connect to the specified timebase. If this is not possible, for example,
because the corresponding timebase was renamed or because the
corresponding chromatography ¾Server is not running, a message
appears. If this occurs, change the assignment manually:
•
Select Open on the File menu to open the desired control panel.
•
Select Connect to Timebase on the Control menu. Enter the name of
the timebase to be connected with the control panel or select the name
from the list.
The new assignment is valid until you close the window. Save the window
to have the current assignment available when you open the window the
next time.
The currently selected timebase is displayed on the status bar.
Tip:
Select Integrate on the View menu to display the report for the running
sample from the control panel.
58
Control
Control Panel: The Signal Plot
The signal plot is an essential part of the
Control Panel. The ¾Signals
of the channels, which have been selected by the user, are displayed
online, i.e., during data acquisition. Different commands are available via
the context menu (right-click). They allow you to define how the signals
shall be displayed:
Autoscale
Each time when performed, the ¾Autoscale command adjusts the scaling
of the signal axis exactly to the open chromatogram or to a section
thereof.
Instead of executing the command from the context menu, you can also
double-click the signal axis.
Auto Autoscale
The ¾Auto Autoscale option automatically adjusts the scaling of the
signal axis exactly to the open chromatogram or to a section thereof
whenever the signal leaves the signal plot.
Auto Plot Speed
Select the Auto Plot Speed option to prolong the time axis automatically
by the period defined on the Axis/Decoration tab page as soon as the
end of the signal plot is reached.
Replot from
Beginning
Selecting the ¾Replot from Beginning command has the following effect:
When the signal leaves the right border of the signal plot, the window is
enlarged by the period defined on the Axis/Decoration tab page. Thus,
the entire chromatogram is always displayed.
Control Panel: The Audit Trail
The Audit Trail on a ¾Control Panel logs all commands performed during
sample processing and saves information regarding the entire system. This
includes graphical and text information. Chromeleon classifies ⇒Control
Commands, status information, and error messages.
After starting data acquisition, the Audit Trail window displays the start time
and any subsequently performed commands (⇒AcqOn/Off, ⇒Inject, etc.).
Of course, each event included in the Audit Trail window is stored. Storage
is very precise and comprehensive. This makes it possible to determine
later how a sample was processed and which events occurred during
sample processing. For more information, refer to Data Management
Audit Trails.
Sample Audit Trail is included in a
For documentation purposes, the
report, by default. For more information, refer to How to ...: Actions in the
Displaying a Audit Trail.
Report Table
Control
59
Control Panel: The Trend Plot
When included in a
Control Panel, the ¾Trend Plot provides the ability
to monitor impending problems by viewing a plot of module-specific data.
Tip:
The Dionex Templates > Panels > Wellness directory of the local
¾Datasource includes several examples of control panels with trend plots
of module-specific data.
The following commands are available via the context menu (right-click to
select a command). The commands allow you to define the appearance of
the trend plot.
Full Size
Displays the full-size plot of all trend data.
¾Autoscale
Scales the trend plot for optimal fit of the y values.
¾Auto Autoscale
Automatically adjusts the scaling of the trend plot for optimal fit
of the y values.
Unzoom
Restores the previous zoom position.
Flow Change
Marks
Displays vertical solid lines on the trend plot to indicate a change
in flow rate.
Eluent Change
Marks
Displays vertical solid lines on the trend plot to indicate a change
in eluent.
Module Change
Marks
Displays vertical solid lines on the trend plot to indicate that different serial
numbers were found in sample sets with the same device name.
Consumable
Change Marks
Displays vertical solid lines on the trend plot to indicate a change in a
consumable part (for example, a column or suppressor).
Calibration Marks Displays vertical solid lines on the trend plot to indicate that calibration
was performed.
Statistics From
Defines how statistics are calculated on the trend plot. Select one of these
options:
•
All Data: Statistics are calculated from all data points.
•
Data in Viewed Range: Statistics are recalculated as soon as the
currently viewed time axis changes.
•
Properties
Control Chart: Statistics are calculated from the target and 1s
values entered on the Statistics tab page in the Trend Properties
dialog box.
Opens a dialog box in which you can select the properties of the plot,
including the events, statistics, and data to display.
For information about how to add a trend plot to a control panel, refer to
How to ...:
Modifying a Control Panel.
60
Control
Data Management
61
Data Management
Data (Overview)
Input Data
For the analysis of a sample and for documentation and archiving
purposes, various types of input data are required, which the user must
determine or enter before starting the analysis.
These include, for example, the sample name, weight, injection volume,
chromatographic conditions (solvent, flow, detection wavelength, connected
devices, etc.), as well as the run time. A distinction is made between:
•
Data describing a sample (¾Sample Data),
•
Data describing a sequence (¾Sequence Data, usually entered
automatically), and
•
Data describing the chromatographic
(¾Chromatographic Methods).
treatment
of
a
sample
The user input data serves as the basis for the analytical process.
Sample Data,
Chromatographic Method
Analysis
Input Data
Raw Data,
Protocol Data
Output Data
Output Data
The data recorded during analysis is referred to as output data. Output data
includes:
•
Data provided by the analysis process itself (analysis and ¾Raw Data)
•
Protocol data on the analysis (¾Audit Trail).
Due to this variety of data types, systematic data organization and storage
Data Storage).
is especially important (see Data Management
62
Data Management
The Browser
The Chromeleon ¾Browser is the tool for data management. It displays the
directories in which chromatographic data is located and that can be
accessed.
The Browser allows you to open, move, and delete chromatographic data,
as well as search for specific data in various databases. To open and view
a file in a separate window, select the file by its name.
Caution:
Browser functions and structure are similar to the Windows Explorer.
However, do not confuse the Browser with the Windows Explorer. Do not
use the Windows Explorer for operations within Chromeleon
¾Datasources. Administrators can prevent these operations by selecting
the Protect Datasource Directory option on the General tab page (via the
Properties...option on the datasource context menu).)
For more information, refer to:
Common Features with the Windows Explorer
Differences from the Windows Explorer
Function
Data Management
63
Common Features with the Windows Explorer
The window has two separate sections. As in the Explorer, the directory
structure of all selectable directories is displayed on the left. Detailed
information about files and ¾Sequences is shown on the right. Operation is
also identical to the Explorer:
•
Click the + and - characters next to each directory name to expand or
collapse the subdirectory structure.
•
Select a directory to display its contents in the right-hand window
section. Files and single samples are displayed.
•
Double-click a file or sample in the right-hand window section to open
the appropriate editor for a file or the corresponding chromatogram for
a sample.
For convenient identification, each file type (sequence, ¾PGM File,
¾Quantification Method, ..) has a type-specific icon. Chromeleon
automatically recognizes the ⇒Type (Sample Type) (standard sample,
unknown sample) and/or the stored data format (3D field, etc.) and displays
the data appropriately, i.e., in the corresponding chromatographic
environment.
•
Select a subdirectory, a sequence, or a file to drag it with the mouse to
a different directory. If you drag the file while holding down the right
mouse key, a context menu is opened. Copy or move the file to the
new directory by selecting the corresponding option. If you drag the file
while holding down the left mouse key, the action, which has been
defined as default action (Ask, Copy, or Move), is performed.
Differences from the Windows Explorer
Structure
Only the chromatographic data that is part of a ¾Datasource is presented
in the Browser. (For more information, refer to the Data section.)
A datasource always represents the top level of the Browser hierarchy. The
datasource can be created exclusively with the Browser and cannot be
compared to the "normal" subdirectory in the Windows Explorer. A small
icon indicates the type of data on which it is based (Chromeleon,
GynkoSoft data, etc.). Users can only see those datasources for which they
have the appropriate access rights.
64
Data Management
Below the datasource level, there is the familiar Explorer directory structure
(yellow file folder). The directory structure helps to manage the different
¾Sequences in a datasource. A blue file folder represents a sequence. It is
not possible to create a subdirectory below the sequence level.
Operation
•
Select a datasource, a subdirectory, or a sequence to display its
contents on the right pane of the browser.
In the case of the datasource or subdirectory, you see the usual list of all
directories and files. If you select a sequence, the right-hand window half is
divided in two horizontal sections. These include the header with general
properties of the selected sequence and a list of all analysis and standard
samples and their sample data (Sequence Editor).
Caution:
The sample data can be edited directly here! GynkoSoft users recognize
the Sample (SMP) File. Additional samples can be included in the
sequence, existing sample data can be modified, or old samples can be
removed. For a detailed description of the sequence editor, refer to the
Sequences section.
•
Select Query on the context menu to search for specific sequences,
data or samples. It is also possible to search several datasources
simultaneously. This is a true ¾Query that can also search for specific
properties, for example, "all samples starting with PAK."
Moving, deleting, or copying directories, data, and files is very similar to
these actions in the Microsoft Explorer. (For more information, refer to The
Browser
Common Features with the Windows Explorer).
Caution:
Move, delete, or open chromatographic files only in the Browser! The
reason is that, except for the visible results, processes are performed below
the surface!
Data Management
65
Function
•
Set up ¾Datasources and create subdirectories, to copy or move files
via Drag & Drop, or to delete files via the Cut command.
•
Select a ¾Sequence in the left window (blue folder) to display a list of
its contents on the right.
•
Double-click a
chromatogram.
•
Double-click a ¾PGM File name to open the PGM Editor.
•
Select a file and right-click for more functions.
•
Select several files (as in the Windows Explorer) and right-click to
perform a function for several files simultaneously.
•
Select a file, right-click, and start a ¾Query for several sequences
and/or datasources. The result of the query, that is the different files or
samples with at least one common feature, is displayed on the right
pane of the Browser.
sample
name
to
display
the
corresponding
The Datasource
The term ¾Datasource is used for the top level of the directory structure
displayed in the ¾Browser. The Browser is the tool for handling
datasources. Each datasource is based on a separate database. When
setting up a datasource, a path to an existing database is entered or a new
database is created.
•
Select Datasources on the File menu to set up a datasource. For
more information about the required steps, refer to the Administrator
Manual: How to ...: Actions in the Browser and in the Operating
Setting up a Datasource.
System
The Browser indicates only the name of the datasource, but not the name
of the underlying database. The type and number of the datasources visible
to the user determine the data that can be accessed. This simplified
representation has the following advantages:
•
Data is always accessed in the same way. The user does not have to
worry about the data’s actual storage location on the network. It is not
necessary to enter the entire path. The location is specified when the
datasource is created.
•
Each user can take advantage of a database without having to deal
with special database programs.
66
Data Management
Database Formats of a Datasource
Chromeleon supports several database formats. In addition to the most
frequently used Access database format (mdb container), Chromeleon’s
¾ODBC Capability allows the handling of SQL ("Structured Query
Language") and database formats, such as Oracle and SQL servers. "Old"
GynkoSoft directories ("drives") and third-party data can be displayed as if
they were datasources with an underlying database. GynkoSoft and
Chromeleon are easily recognized by their different symbols:
GynkoSoft - Datasource
local CHROMELEON datasource
When Chromeleon is installed, a default datasource is automatically
created on each client PC. The datasource name is derived from the
computer name (assigned during the installation under Windows) and the
addition Local. In this way, each user has a separate datasource in which
(s)he can store his(her) "personal" results and data. For single systems and
for users who do not have additional access rights on a network, this is the
only way to store their data. In this datasource, the raw data of each
manually performed analysis is stored. Therefore, do not delete the <PC
NAME_Local> datasource!
Directory Structure of a Datasource
Each datasource can have any number of hierarchically organized
subdirectories. To create a subdirectory, select the datasource in the
Browser, and then select New Directory on the File menu. The data tree
structure is similar to the MSDOS data tree.
Caution:
Do not use special characters (such as the umlaut) for new directory names
or sequences. This may cause problems in Novell networks!
Actions performed in the Browser, for example, creating datasources or
directories, require complex operations below the user interface and cannot
be compared to or performed by the Windows Explorer! The representation
of directories and data also differs considerably from the Explorer.
Data Management
67
See the image below examples of datasources and their different directory
structures:
Subdirectories
Sequences
Default Sequence
The SOURCE1 datasource has the three subdirectories: LAB201, LAB202,
and DYES. The DYES directory contains three ¾Sequences: SEQ1, SEQ2,
and SEQ3.
The default PC Name_local datasource has one subdirectory. The name of
the subdirectory corresponds to the name of the timebase installed on the
computer. The default sequence (named manual) is located in this
subdirectory.
When a sequence is selected, its "inner life" is visible in the right Browser
pane: the file structure (control ¾Program ( ), ¾Quantification Method
(QNT Method) ( ), ¾Report Definition Files (RDFs), etc.), the sequence
information, and the samples (standard samples ( ), and unknown
samples ( ), etc.)
For information about the representation, refer to Data Management
The Browser.
For information about the functions and significance of sequences, refer to
The Sample
the Samples and Sequences section; especially, refer to
List (Sequence).
68
Data Management
Drag & Drop allows you to move subdirectories, sequences, and
chromatographic methods in a datasource but also between datasources of
different types. However, this copy process may also change the underlying
database! If you want to actually move a sample, delete the original sample
in the Browser after you have copied the sample to the new location.
Caution:
Execute drag & drop operations only in the ¾Browser. Drag & drop
operations outside Chromeleon, for example, in the Windows Explorer, will
result in the loss of data!
Locking Datasources, Directories, and Sequences
To protect data and results, you can lock datasources, directories, or
sequences. It is not possible to modify Locked objects or any object under
the locked one. For example, if a datasource is locked, all subdirectories
and all sequences therein are locked as well. Locked objects are identified
in the Browser by the red lock on the corresponding icon (
).
How To
•
Select the object in the Browser.
•
Select Properties... on the context menu.
•
Select the Locked check box.
Data Management
69
To remove the lock, return to the Properties dialog box and deselect the
Locked check box.
Note:
Locking and sharing objects is subject to access control. (Refer to the
Administrator Manual: Software Installation and Communication
Access Control for more information). Only users who have the
corresponding privilege can lock and/or share objects.
In addition to locking datasources and directories via the Locked check
box, access to these items can be controlled by adding them to ¾Access
Groups or removing them.
•
Select the datasource or directory in the Browser.
•
Select Properties... on the context menu. On the Access Control tab
page, define the Access Group assignment, using the Add and
Remove buttons.
Only users who are members of an Access Group listed in the Access
Groups are authorized to access datasources and directories.
Data Acquisition
Even the best method of
Data Storage is only as good as the quality of
the stored data. Therefore, data acquisition plays a very important role.
Data acquisition starts with the quality of the used detector, it comprises all
components participating in the data flow and is concluded with processing
the data in Chromeleon.
A distinction has to be made between detectors supplying digital data and
detectors supplying analog data. The best results are obtained with
detectors that are capable of communicating digital signals via a serial
interface. (Refer to the Administrator Manual: Software Installation and
Communication
The Serial (RS-232) Interface for more information.)
Detectors that supply analog signals have to rely on precise conversion of
the signals. The product range available from Dionex includes an extremely
sensitive and low-noise ¾UCI Universal Chromatography Interface for
converting analog signals to digital signals. The UCI guarantees highest
precision with minimum noise.
70
Data Management
The following terms are important for data acquisition: ¾Data Collection
Rate, ¾Sampling Rate, and ¾Step. However, sometimes it may be difficult
to understand the relationship between these terms.
If the detector directly supplies digital data to the PC, this is referred to as
Data Collection Rate. In contrast, the term Sampling Rate is used if an A/D
converter, such as a UCI Universal Chromatography Interface, supplies the
data. Both terms describe the number of data recorded per second.
The Step describes the time interval between two data points. By default,
the step is the reciprocal value of the data collection rate or the sampling
rate. Nevertheless, it is possible to select a different step.
There is nothing particular you need to observe if an A/D converter supplies
the data. However, for detectors supplying digital data make sure to define
the Step command after the Data Collection Rate command.
Data Acquisition with Detectors without Separate
Drivers
In addition to the ¾Device Drivers for Dionex detectors, Chromeleon
provides many drivers to control third-party detectors.
For an overview of the different manufacturers whose devices can be
controlled under Chromeleon, refer to the Administrator Manual:
Installation Instructions
Installing and Controlling Third-Party
Devices.
In addition, it is also possible to acquire data using detectors for which
separate device drivers are not available. In this case, install the
¾Integrator Driver.
For information about how to install the device drivers, refer to the
Administrator Manual: How to ...: Actions in the Server Configuration
Adding, Configuring, or Deleting Components
Program
Data Management
71
Data Storage
For fast and efficient access to specific data, intelligent storage and
organization of the complete data is very important. Chromeleon solves this
problem by storing data and files in different locations. Databases and
sequence directories are available for this purpose. They are part of the
Datasource.
Storage in a Database
Data that can be compared across sequences is stored and managed in a
relational, ¾ODBC-capable database. This applies to the entire ¾Sample
and ¾Sequence Data. The advantage of this type of data management is
not only the comfortable integration in other applications such as Excel,
Access, dBase, etc., but also the efficient searching and sorting
capabilities.
Perform a ¾Query to find all samples processed on a certain day, created
by a certain user, and/or carrying a certain name.
Storage with the Sequence
The entire data describing the chromatographic treatment of a sample or
data recorded during the analysis is stored in a ¾Sequence. This includes
control files (¾PGM File) and evaluation parameters (QNT Method), but
also the entire raw and protocol data (see Data Management
Raw
Data Storage).
History (Modification History)
For the datasources, you can enable the modification history (in short:
¾History). This allows you to document all modifications together with the
user name and the object name.
Objects can be samples, sequences or datasources, ¾Control Panels,
¾Report Definition Files (RDFs), ¾PGM Files and/or ¾QNT files and
modified chromatograms.
For more information, refer to the Administrator Manual: How to ...:
Actions in the Browser and in the Operating System
Tracking File
Modifications (History).
72
Data Management
Data Export
Chromeleon provides various data export options for communication with
other programs:
1.
You can export report pages from the ¾Browser, by selecting Batch
Report on the File menu. In the Export Options section, select the
Export check box. Click the Export Settings button to open the Export
Wizard. You can now export the corresponding report pages in five
different formats:
a)
ANDI/Chromatography - ¾AIA (*.cdf)
b)
ASCII text format (*.txt)
c)
Excel file format (*.xls)
d)
Adobe Acrobat file format (*.pdf)
e)
Chromeleon Archive format (*.cmb = ¾Backup files)
For more information refer to How to…: Actions in the Browser
Exporting Data During or After a Batch.
2.
In the ¾Printer Layout, you can open a dialog box that corresponds to
the Export Wizard. First, enable the Layout Mode on the Edit menu.
Then, select Batch Report Setup on the File menu to open the Batch
Report Setup dialog box. . Via the Printer Layout, you can export data
from different samples in a single data file (refer to How to…: Actions
Exporting Data from Different Samples to a
in the Browser
Single File).
3.
You can also open the Export Wizard from a ¾Control Panel. Select
Reporting on the Batch menu and select the Print/Export Report
check box.
4.
In addition, you can also export raw data in the AIA format. To do so,
select Export/Backup on the File menu in the and then select the
ANDI/Chromatography (AIA) option. (Also, refer to Data Management
Raw Data Export).
5.
If you wish to send Chromeleon data to another laboratory, for example,
Backup first and then
via e-mail, we recommend that you execute a
transmit the compressed data as *.cmb file, which is the Chromeleon
archive format.
Data Management
73
You can also start the Chromeleon export function from a separate
program, using the command line under Start > Run or a DOS command
prompt. For more information, refer to the Administrator Manual: How
Using
to ...: Actions in the Browser and in the Operating System
Chromeleon Data in an External Program.
Backup
To avoid unforeseen data losses (for example, due to a defective hard
disk), we recommend that you back up your saved data to a different data
medium at regular intervals, using the ¾Backup command on the File
menu of the ¾Browser.
Backup data is compressed; that is, the data is "packed" and stored in a
different location. For security reasons (GLP does not allow modification of
backup data), direct access to the data is not possible. To unpack the data,
select the ¾Restore command on the File menu of the Browser.
The backup logs each single file that is copied and issues warnings if errors
occur. The directory structure is maintained.
For more information, refer to How to ...: Actions in the Browser:
Creating Backup Files
Restoring Backup Files
Raw Data
Data generated by the system are referred to as raw data, whereas data
entered by the user are referred to as user data. The user is not allowed to
change the raw data, e.g.:
•
•
Sample data acquired on different channels (see ¾Signal)
Audit Trails
•
Injection times (see ⇒Inj. Date/Time)
•
¾History
74
Data Management
In a narrower sense, all analog or digital values measured by a detector
and stored digitally on the PC are referred to as raw data. Raw data only
exists for those signals or channels that were selected by the user before
data acquisition.
The extent and precision of the stored raw data depend on the selected
¾Sampling Rate or ⇒Step.
For more information, refer to
Raw Data Storage
Raw Data Compression
Raw Data Storage
Raw data storage refers to saving the signals received from a detector in
digital form. Other important data (such as the analysis time, signal unit,
number of data points, etc.) is also stored. If a detector is only supplied with
an analog output, the data must be converted into digital signals. The A/D
converter performs this task.
Storage Procedure
With conventional data systems, an analog value is digitized at a fixed time
interval. For example, a digital value of defined accuracy is stored every
second. The number of stored values per second is normally referred to as
the ¾Sampling Rate. The inverse of the sampling rate (the time interval
between two data points) is referred to as ⇒Step.
The higher the sampling rate, that is, the smaller the step, the more data
points are stored, and the more exactly the original signal can be restored
from the stored data. However, a higher sampling rate has a higher
memory requirement. Chromeleon solves this problem by the step setting
step=auto. This type of storage requires high algorithmic resources in real
time (!), which is justified by the following advantages:
•
Raw data files are as small as possible, as fewer data points would
result in a loss of precision! If an analysis requires a conventional step
width of 0.5 seconds, Chromeleon can typically acquire such
chromatograms with an average step width (= chromatogram length
divided by the number of data points) of 2 seconds. The compression
factor of 4:1 is thus achieved, making optimum use of the available
storage capacity.
Data Management
75
•
Despite this minimal file size, maximum integration accuracy is ensured
for the given chromatographic conditions, as the continuous signal is
approximated to the optimum. Generally, more data points are stored
below peaks than with conventional acquisition methods.
•
The processing speed, for example, for peak detection, re-integration,
graphical output, etc., is significantly higher due to the reduced number
of data points.
Storage Location
Raw data is stored in the directory of the current sequence. For each
channel that is specified during the Chromeleon installation, a separate
subdirectory is created. In addition, an audit trail directory is created. The
directories are not visible in the Browser.
The reason for this is: Chromeleon manages the entire raw data
automatically. At no time, the user must access the raw data directly.
Viewing this type of data is only possible via the Windows Explorer
provided the datasources are not locked. As, however, the datasource
names are not displayed in the browser, you must follow the path to the
corresponding sequence directory (see figure).
In this example, the 3dfield, Ext228nm. Uv_vis-1, and Uv_vis-2 channels
were defined. The audit trail directory (Audit_Tr) was added. If a directory
is expanded, the raw data of the corresponding channel is visible.
76
Data Management
A separate raw data file is created for each sample in a sequence, for
which raw data of a specific channel was recorded.
Caution:
Do not modify these directories! Operations outside Chromeleon are not
permitted! Therefore, we recommend that you protect your datasources to
prevent that they are accessed via the Windows Explorer. Select
Properties... on the context or File menu of the datasource and then select
the Protect Datasource Directory check box.
Raw Data Compression
Storing raw data automatically compresses the data. For the signal value,
this is achieved by storing the difference to the next data point instead of
storing each data point. Only from time to time, the actual value is stored.
This way, the compression is increased by 50%. This effect is especially
noticeable in the case of ¾3D Fields.
The size of the raw data file of a 3D field increases with the number of
recorded data points. These depend on the ¾Optical Resolution of the
detector, the field size (area between the upper and lower limit of the 3D
field) as well as the selected ¾Sampling Rate.
At a sampling rate of, for example, two spectra per second (step = 0.5) and
an optical resolution of 2nm, this means that 2 x 60 x 70 = 8400 data points
per minute must be recorded for the UV range from 200 to 340nm. As each
absorption value is recorded with an accuracy of 25bits, a hard disk storage
capacity of m x (N+1) x 4 = 70 x ((2x60)+1) x 4 = 33.88kByte per minute is
required. The storage requirement for the 3D field of a "normal" (20-minute)
chromatogram is thus 0.678 MByte!
However, by skillful data compression procedures, it is possible to reduce
the required storage capacity by approximately 50-60%. This is possible by
completely storing approximately each eighth spectrum. Of all other
spectra, only the difference to the previous one is stored and is recalculated, when needed. This procedure is a good compromise between
optimum data compression and the required time for restoring a 3D field.
Note:
The compression procedure is not destructive, that is, the complete data is
stored. The 3D field thus contains the complete information provided by the
detector. The data can be restored at any time.
Data Management
77
Further, there are three ways to minimize storage capacity requirements:
•
Limit the wavelength range to the necessary range.
•
Reduce the sampling rate (step) so that no more than 10 to 20 spectra
are below the narrowest peak, or select an automatic sampling rate
(step).
•
Use the possibilities of the ⇒Diode Bunching.
Restoring a Chromatogram from Raw Data
When restoring a chromatogram from the raw data, equidistant data points
are joined with straight lines. A diagram "resembling" the recorded analog
signal is thus created.
Clearly, the resemblance (and thus the precision of integration) is increased
with an increasing sampling rate. However, a higher sampling rate requires
more storage capacity. When using a fixed sampling rate, the sampling rate
must be set so that a minimum of 10 datapoints is stored during the
smallest peak in order to integrate the smallest peaks of a chromatogram
(generally the earlier peaks) with the same precision as the larger peaks.
This results, however, in huge data volumes especially in the case of wide
peaks and long baseline sections.
Using a dynamic sampling rate can solve this problem. Chromeleon is
capable of continuously optimizing the sampling rate during an analysis.
That is few data points are stored during baseline sections, whereas many
are stored below peaks. The local sampling rate is set according to the
actual information volume such that the deviation between the resulting
diagram and the actual analog signal is never greater (or smaller) than the
actual noise component of the signal. This method ensures that neither too
many nor too few data points are stored, but always the optimum. The
⇒Step values vary between 0.01 and 5 seconds (sampling rate: 0.2 to
100 Hz).
Raw Data Export
Chromeleon supports exporting raw data by conversion into ¾AIA and
ASCII formats. In addition, you can export raw data as ¾Backup file
(*.cmb). Select Export/Backup on the File menu in the Browser to export
the data as an AIA or backup file.
78
Data Management
Format
AIA Cat.
Description
2
AIA Cat. 1+2
Samples and peak variables are stored in the AIA format.
In addition to samples and peak variables, the raw data of a
chromatogram (each stored data point) is stored in the AIA format.
Chromeleon
All data of a ¾Sequence or a ¾Datasource are stored in the cmb format.
backup (*.cmb):
ASCII
Raw data may be exported in the ASCII format as well. Select Batch
Report on the File or context menu. Click Export in the dialog box and
then select the ASCII export format. All raw data from the channels
selected for the actual sequence are stored in the ASCII format.
Tip:
If you intend to export raw data to other applications or other computers, it
may be necessary to use a fixed ⇒Step (= equidistant raw data storage)
instead of a dynamic step.
It is also possible to export report data sheets in different formats (see Data
Data Export).
Management
Raw Data Import
Importing the following raw data is possible:
•
¾AIA data (*.cdf)
•
Agilent/HP ChemStation
•
GynkoSoft data
•
¾LIMS/¾Worklist data (*.wle)
•
PeakNet (up to version 5.2) data
•
Files in the Chromeleon archive format (*.cmb)
On the File menu of the Browser, select Import/Restore and the
corresponding option. Select Import to import any of the above raw data.
Select ¾Restore to import Chromeleon ¾Backup data.
In addition, it is possible
chromatography data systems.
to
install
databases
from
third-party
Data Management
79
How To
•
Open the Browser and select Datasource on the File menu.
•
Follow the instructions described in the Administrator Manual: How
to ...: Actions in the Browser and in the Operating System
Connecting a Datasource.
For details on importing PeakNet 5.2 or earlier data files, refer to How
Importing PeakNet (Release 4.5
to ...: Actions in the Browser
through 5.2) Data Files.
Raw Data Storage in Case of Power Failure
The raw data of a sample interrupted by a power failure is not lost, as a raw
data autosave is performed continuously during sample processing.
Autosave ensures that the raw data is stored on the hard disk in short
intervals. The user can reduce the time intervals to approximately 30
seconds. In the case of a power failure, the maximum data loss is thus 30
seconds.
If a power failure interrupts the automatic sample batch, the ¾Power
Failure Protection and the power failure handling ensure that processing is
continued at the same position after starting the system anew. In addition,
you can run a power failure program before to reset the system to a defined
state.
If processing is interrupted by warnings or error messages, it is possible to
react with an appropriate ¾Emergency Program. The system is then in a
defined state that is recorded in the ¾Audit Trail.
For information about how to develop appropriate programs for both cases,
refer to:
How to ...: Actions in the PGM Editor:
Creating an Emergency Program
Creating a Power Failure Program
80
Data Management
Audit Trails
In addition to raw data, Chromeleon also records the Audit Trail. An audit
trail includes the following information:
Audit Trail entries for ¾Preconditions:
Device settings before a sample run, such as the temperature of a column oven.
Note:
These entries can be displayed only in the daily audit trail and in the
sample audit trail.
Audit Trail entries for a sample run:
System messages, such as
•
Restart of Chromeleon after booting the computer
• Start of sample processing
Warning
Error
Abort Error (batch or program is aborted)
Next command in the batch
Executed instruction. The color indicates the filter level. The message is displayed
from this level on:
Green: Normal
Yellow: Advanced
Red: Expert
This color code is not yet valid for manual and triggered commands.
Command executed manually from the control panel or the F8 box.
Triggered command
Fulfilled ⇒Trigger condition
⇒Protocol; comments program steps or describes chromatographic conditions
⇒Message on the screen that must be confirmed by the user
⇒Log (performed by either the user or the ¾Device Driver)
Data Management
81
•
Each item is stored with the current time. Information gathered during
recording the data and/or processing a sample batch, also contain the
associated retention time.
•
The Audit Trail information is stored continuously both over a whole
workday and for the duration of processing a single file. Two different
Audit Trails are available:
The Daily Audit Trail
The Sample Audit Trail
Audit trails can be displayed either in the Browser (as daily or sample audit
trails), on a control panel, or in a report or Printer Layout (only as sample
protocol).
To define the extent and the type of the audit trail display, select the display
filter (Normal, Advanced, Expert, and Error, or Warning) on the context
menu. The Advanced and Expert display options are available only for
audit trails created with Chromeleon 4.0 or higher.
In addition, you can select one of the following options on the context
menu:
•
Run only to display only the entries for the sample run.
•
Preconditions only to display only the conditions before a sample run
•
Preconditions and Run to display all entries
The Daily Audit Trail
The daily
Audit Trails store the entire GLP-relevant data that is related
to the status of a specific timebase. The information is displayed in the
The Audit Trail). The
Audit Trail on the control panel (refer to Control
Daily Audit Trail also includes the preconditions before the sample run.
When the Chromeleon ¾Server is started, Chromeleon creates an AUDIT
directory for the corresponding timebase in the server datasource. The
server saves the daily audit trail for the timebase to this directory. The
name under which the file is saved is the current date. For example,
200403051027.slg is the file for the daily audit trail of March 5, 2004. A
separate file is created for each day.
82
Data Management
On the right, the Browser displays the daily audit trails indicating their
names and the time of their last change. Double-click a file name to open
the corresponding audit trail in a separate window. The information is the
same as in the audit trail section of the control panels including the
preconditions. It is also possible to print the contents of the daily audit trail
file.
Copy, move, rename, and delete daily audit trails in the Browser. The user
rights required to perform these actions are defined in the ¾User Manager
(CmUser program).
Note:
Chromeleon generates a new file for each daily audit trail. Therefore,
periodically transfer files that are no longer required to an external storage
device.
In addition to the daily audit trail, a
sample.
Sample Audit Trail exists for each
The Sample Audit Trail
The sample audit trail contains the entire data of the corresponding sample
Audit Trails). For more
and is part of each default report (see
information, refer to How to …: Actions in the Report Table
Displaying an Audit Trail.
You can display the sample audit trail as follows:
•
To display the audit trail data of the current sample, display a report in
any method window and select the Audit Trail worksheet.
•
You can also enable the sample audit trail in the Browser. Select the
sample in the Browser, select Open on the context menu, and then
select Audit Trail.
If you do not find a specific entry in the sample audit trail, check the
Daily Audit Trail.
Samples and Sequences
83
Samples and Sequences
Sample Preparation
Sample preparation is a major part of the chromatographic analysis. It can
include simple procedures as weighing, solving, and diluting a sample, as
well as more complicated physical (filtration, centrifugation etc.) and
chemical separation procedures (liquid-liquid-extraction, fixed phase
extraction). Generally, the careful performance contributes substantially to
the quality and the reproducibility of chromatographic separations.
In addition, Chromeleon provides two correction factors: ¾Sample Weight
Factor (Weight) and ¾Dilution Factor. They allow you both to use the
"approximates weight" and to define dilution steps. Thus, they can be used
to consider sample preparation during data evaluation.
Sample Processing
Sample processing includes three major steps:
•
Sample definition (single samples and ¾Sequence and/or ¾Batch)
•
Analytical procedure (manual or automatic control - ¾PGM File)
•
Evaluation (¾QNT Methods and reports)
The performance of each step depends on the methods that are used and
the available instruments. The working environment could range from a
fully automatic sample laboratory with large quantities of samples to singleuser applications in a research lab. Easy and quick analysis procedures
may be the focus in the first case, while special methods and parameters
for peak recognition may have priority in the latter case. Thus, it is not
surprising that functions crucial to one group of users may be irrelevant to
others.
Keep this in mind when you read the information in the following sections.
84
Samples and Sequences
Sample Definition
In Chromeleon, the term sample has a more specific meaning than in
normal colloquial use.
Each injection is defined as an individual "sample"!
Multiple injections from the same sample vial under similar conditions are
considered several samples.
Defining a sample means the process of determining how much of a
substance is injected from which vial and under which conditions, and
which evaluation parameters are used. A distinction is made between a
single sample and a sample series.
Single Sample
A sample can be analyzed individually by entering all required information
and user commands via the keyboard or the mouse.
The user selects the Inject command, enters the volume to inject, and
performs the injection via a hand-operated valve. If an ¾Autosampler is
available, the user determines the sample location with the ⇒Inject
command. Then, data acquisition is started (via the ⇒AcqOn/Off
command). When the end of the sample is reached, the user completes
data acquisition (Acq Off command) and specifies where to save the
acquired data.
The recorded data is temporarily saved to the manual sequence of the
default ¾Datasource of the system. In network operation, this datasource is
Network.
designated with the computer name of the user on the
As soon as the user completes data acquisition, the user is prompted to
select the final storage location for the temporarily saved data.
Sequence/Batch
If several samples are to be processed successively, they are included in a
sample list (sequence), together with the instrument control and evaluation
information.
The samples are then processed automatically. (For more information, refer
to ¾Batch.)
For more information about how to create a sample list, refer to
Sample List (Sequence).
The
Samples and Sequences
85
The Sample List (Sequence)
The sample list is part of the Browser. (For information about the Browser,
The Browser). When you select a
refer to Data Management
¾Sequence the sample list is displayed on the lower right of the Browser
window. One line corresponds to one sample. When a chromatographic
analysis is started, the samples that shall be analyzed are processed from
the top to the bottom of the sample list. Thus, the sample list also
determines the order (= sequence) in which the analysis is performed:
A sample is characterized by various column entries. The entries are
managed in a database and are referred to as ¾Sample Data. For a short
explanation of the column, press the F1 key.
Before the analysis, the user has to enter all samples to be processed and
the characteristic sample data into the sample list. There are two options:
1. Change an existing sequence manually and then save it under a new
name.
2. Create a new sequence automatically using the
For more information, refer to
How to ...: Actions in the Browser
Sequence Wizard.
Creating a Sample List.
86
Samples and Sequences
In addition to "real" sample data (such as the sample name, the injection
volume, the vial, and the sample type (unknown or standard)), the sample
list also contains the Program File and Method columns. The entries
made in these columns refer to specific ¾Chromatographic Methods
determining the performance of the analysis. They include the control
program (see Control
The Control Program) required for fully
automatic control of analytical instruments as well as evaluation instructions
The Quantification Method (QNT Editor))
(see The QNT Editor
determining the integration and calibration. In addition, the sample list can
contain ¾User-defined Columns and ¾Sequence Report Columns.
The number of samples per sequence is virtually unlimited, but more than
100 samples should be an exception. The fewer samples are added to a
sequence, that is, the more sequences are created, the faster single
samples can be accessed and the easier it is for the user to keep track of
the processed samples. Criteria for combining several samples in one
sequence could be, for example, the same analysis conditions, the same
origin, the samples of the same day, etc.
The Sequence Wizard
The Sequence ¾Wizard helps you to quickly create a basic sample list
consisting of analysis and standard samples. To open the Sequence
Wizard, select New on the File menu in the Browser. Follow the steps
below to create a ¾Sequence:
•
Step 1: Select the timebase
•
Step 2: Generate the analysis samples
•
Step 3: Generate the standard samples
•
Step 4: Determine the ¾PGM File and the analysis method
•
Step 5: Save and name the sequence
Refer to the online Help for more information about the above steps.
Each step is performed on a separate wizard page. Clicking <Back or
Next> to takes you to the previous or next page.
In the fifth step, click <Finish> to save the sequence and close the wizard.
Samples and Sequences
87
Caution:
Do not use special characters (such as the umlaut) for new directory names
or sequences, as this may cause problems on Novell networks!
Note:
In future Chromeleon versions, it will be possible to include validation
and/or blank run samples with the Sequence Wizard.
For information about how to create a sample list, refer to
How to ...: Actions in the Browser
Creating a Sample List.
Automatic Batch Processing
The enormous technical complexity of modern chromatography systems,
the resulting high purchasing costs and the constantly increasing number of
samples in analysis laboratories make continuous operation even outside
of regular working hours a necessity. Thanks to ¾Autosamplers, very
efficient PCs, and modern data systems, this has become routine. The user
merely provides "replenishment."
When the actual sample preparation is completed, the chromatographic
conditions of processing, the samples to be processed and in which order
must be communicated to Chromeleon. This is performed in the sample list.
The result is stored as a ¾Sequence.
Independently processing one or several sequences is known as ¾Batch.
To start processing, the following steps are required:
Starting the Automatic Batch
After data input is completed, the analytical process can be started in an
online batch.
•
Open a control panel and select Edit on the Batch menu. (This
command is also available on the Batch menu in the Browser.)
•
Enter the names of the sequences containing the samples to be
analyzed.
•
Perform a ¾Ready Check.
•
Start the analysis process by clicking Start.
88
Samples and Sequences
Online Batch: During the chromatographic analysis of the batch
As soon as the online batch is started, all samples of the sequence with the
status single or multiple are analyzed successively. If a sequence
contains a sufficient number of samples, sample processing "around the
clock" is possible. Instead of including all samples in one sequence, they
can be distributed among several sequences. Accordingly, more
sequences are entered in the batch dialog (maximum 16). This list is
considered a batch; it is also referred to as online sample batch or online
batch. The order of the sequences determines the order of processing:
When starting the batch process, samples 1 to n of the first sequence, then
samples 1 to n of the second sequence, etc. are analyzed.
Offline Batch: After the chromatographic analysis
The data acquisition results of the batch are saved with the individual
samples. When the results are processed offline, e.g., printed, exported,
signed, etc., after data acquisition is finished, a batch is called an offline
batch.
Sample Evaluation
In spite of largely automated work processes and intelligent pre-settings, it
is within the responsibility of each user to set the framework conditions for
sample evaluation.
Calculations
Sample processing is performed based on a ⇒Program (¾PGM File) that
was previously created and included in the sample list. Similar to this, the
analysis results are calculated based on the evaluation method indicated in
the sample list. The method itself is created in the ⇒QNT-Editor. For more
information, refer to How to ...:
Actions in the QNT Editor.
Result Output
The result of sample processing can be represented in graphics and tables,
either on the screen or in a printed output. Chromeleon provides method
windows for generating this output. Use the Printer Layout to define
templates standardized presentations of the sample results. For more
information, refer to How to ...:
Actions in the Report Table and
Actions in the Printer Layout.
Samples and Sequences
89
Overview of the Most Important Results in the Browser
To take an overview of the sequence results already in the sample list, add
the desired ¾Sequence Report Columns to the list. For more information,
refer to How to ...: Actions in the Browser
Creating a Sequence
Report Column.
Data
For more information about the available method windows, refer to
Representation and Reprocessing.
Electronic Signature
During the last decades, quality assurance and ¾GLP have become
increasingly important. Data verification is one of the key aspects. That is
why it is especially important to ensure that ¾Raw Data is not modified
later. In addition, the results generated from this raw data must not be
modified without authorization once they have been accepted. Contrary to
a data system, printed records can ensure this in part only.
If ¾User Mode is enabled, Chromeleon allows you to electronically sign the
results generated from your raw data. This is an important aspect for quality
assurance and GLP. Electronic Signature allows you to sign and to protect
¾Sequence reports that have been accepted as correct and thus, to
'freeze' the current state of your results.
Note:
Electronic signature is only available for user databases that were created
with a User Manager (CmUser) program version 6.10 or higher. Update
your database if an error message notifies you that electronic signature is
not supported.
Electronic signature includes three steps:
•
Submit
•
Review
•
Approve
Typically, the user who created the report signs and submits it. Afterward,
for example, the laboratory manager reviews the report and signs it as well.
Finally, the quality assurance manager approves the results. For
information about how to sign reports electronically, refer to How
Signing Sequences Electronically.
to ...: Actions in the Browser
90
Samples and Sequences
Theory of Calibration
91
Theory of Calibration
Calibration (Overview)
If the signal of a chromatography detector is proportional to the
concentration of a substance in the flow cell, it is suitable for quantitative
determination. This is a characteristic, for example, of the absorption
supplied by a UV detector in the scope of the Lambert-Beer law.
The proportionality constant depends on the chemical quality of the
substance of interest and on the physical properties of the used detector.
For UV detectors these are mainly the optical wavelength and the spectral
bandwidth. As integration programs can only determine the area (and
height, respectively) below a peak, conversion into absolute amount or
concentration units is possible only if a calibration was executed before the
analysis.
For more information, refer to:
Calibration Principle
Calibration Types (Linear)
Calibration Types (Non-linear)
Using the Calibration Curve
Calculating the Calibration Curve
Standard Methods
Evaluation with Various Standard Methods
Implementation
At the end of a calibration, Chromeleon creates calibration curves from the
available calibration points for each calibrated substance. Representing
and evaluating the curves is performed in the calibration curve method
window (see Data Reprocessing
The Calibration Curve).
92
Theory of Calibration
Calibration Principle
The principle of the calibration is based on that one or several samples of
known composition are analyzed by chromatography and a conversion
factor amount (or concentration)/ area is calculated from the detected areas
below the individual peaks and the known amounts or concentrations. This
factor can then be used to multiply the area of the respective peak of an
unknown sample. The result is the corresponding amount of the substance
(or concentration of the substance).
However, this simple method will work only,
•
If the relation between amount and area is strictly linear, i.e., if the
Lambert Beer Law is applicable for UV detectors)
•
If the area zero equals the amount zero, i.e., if the calibration line leads
through the origin (no offset) and
•
If matrix effects can be neglected
If the detector signal S is proportional to the concentration (K) of a
dissolved substance, the proportionality factor c1 applies:
S = c1 * K
Under certain conditions, the area F(x) corresponding to a certain amount
(x) is proportional to the contained amount.
F(x) = c1 * x
If a sample of the substance A of known concentration (the standard or
calibration sample) is analyzed chromatographically, the result is a specific
ratio between the injected amount and the determined area value. The
result can be graphically presented by entering the value pair in an
amount/area diagram. In this diagram, each injection corresponds to one
¾Calibration Point.
Ideally, all calibration points are located on a straight line, and there is a
direct ratio between the amount and the determined area. The "conversion
factor" corresponds to the slope of the calibration line (left fig. "ideal").
Theory of Calibration
93
Determined Area Value
e.g. mAU x min
1500
Calibration Line
Calibration Points
Calibration Line
1000
500
real
ideal
0
0
25
100
150
200
0
25
100
150
200
Injected Amounts
e.g. µg
During each calibration, deviations from the ideal behavior might occur
which are above all caused by weight and/or dilution errors. This causes
scattering of calibration points. Therefore, the Gaussian method of the least
Calculating the Calibration Curve) is used to calculate a
squares (see
regression line. This line is defined as the best approximation to the
existing calibration points and, usually, it does not go through the origin
(right fig. "real").
If the various calibration points are not located on a straight line, but show a
parabola or exponential shape, the slope of the curve and the distance to
the zero point (offset) describe the corresponding (approximate) curve
(calibration curve). The basic mathematical function is referred to as
¾Calibration Function; the coefficients are the calibration coefficients.
By selecting the ⇒Calibration Type peak table variable, the user decides
whether a linear or a non-linear calibration curve is calculated from the
existing calibration points. Distinguish between the following calibration
types:
Calibration Types (Linear)
Calibration Types (Non-linear)
94
Theory of Calibration
Calibration Types (Linear)
If one calibration sample of a standard substance is analyzed for calibration
only, the user enters exactly one concentration value in the first ⇒Amount
column of the peak table. The result is exactly one ¾Calibration Point.
Connecting the calibration point with the origin then forms the calibration
curve. It is described by the function derived from the Lambert Beer law:
F ( x) = c1 * x
The slope of the line corresponds to the proportionality factor c1 (left partial
fig.). C1 is also called RF value.
Area
1-Point Calibration
2000
3-Point Calibration
1-Point Calibration
(5 Replicates)
2
1500
3
1000
4
500
}Offset
0
0
50
100
150
0
50
100
150
0
50
100
150
Amount
If one calibration sample is analyzed several times, several points can be
entered in the amount/area diagram. The points of one concentration are
called replicates. With an increasing number of available replicates, the
impact of imprecision decreases after averaging. In spite of several
replicates, only one amount/area ratio is determined. This is referred to as
multi-point calibration on one calibration level (in the middle of the
partial fig.).
The result is better secured if several concentrations are measured instead
of one. Of course, several replicates can be used per concentration. As a
result, calibration points at different concentrations are received in addition
to the replicates of one concentration. This is called a multiple point
calibration on several levels (for example, 3-level calibration (see right
partial fig.). The calibration curve does not necessarily have to go through
the origin. The linear ¾Calibration Function is therefore corrected by an
offset.
F ( x) = c0 + c1 * x
Theory of Calibration
95
Caution:
The decision whether a calibration type differing from the linear default is
physically sensible, is within the responsibility of the user, not of
Chromeleon!
Note:
If you calibrate using the ¾Standard Addition method and if no calibration
points are available with Amount = 0 (only Spiked samples), Chromeleon
calibrates with an offset.
If calibration points with Amount = 0 are available (also Unspiked samples)
and if you calibrate using the Linear calibration method, the calibration
curve does not go through the origin. Instead, it is forced through the mean
of all samples for which Amount = 0; i.e., all unspiked samples for this
substance. Please note that the results may be different from those
obtained by calibrating using the Linear with Offset calibration method.
Calibration Types (Non-linear)
In (the more general) case of a non-linear calibration, more terms are
added to the linear ¾Calibration Function.
Parabola-shaped curves are described as follows:
F ( x) = c1 * x + c 2 * x 2
(Quadratic)
F ( x) = c0 + c1 * x + c 2 * x 2
(Quadratic with offset)
To calculate curves of this type, a minimum of two (quadratic) or three
(quadratic with offset) calibration samples must be available (left partial
fig.).
96
Theory of Calibration
Area
Parabola
Point-to-Point
Exponential
2000
Quadratic
(Minimum
2 Points)
1500
1000
Quadratic
with Offset
(Minimum
3 Points)
500
0
0
50
100
150
0
50
100
150
0
50
100
150
Amount
Note:
If you calibrate using the ¾Standard Addition method and if no calibration
points with Amount = 0 are available (only Spiked samples), Chromeleon
also calibrates with an offset when the Quadratic calibration method is
used.
If calibration points with Amount = 0 are available (also Unspiked samples)
and if you calibrate using the Quadratic calibration method, the calibration
curve does not go through the origin. Instead, it is forced through the mean
of all samples for which Amount = 0; i.e., all unspiked samples for this
substance. Please note that the results may be different from those
obtained by calibrating using the Quadratic with Offset calibration method.
The power function is described as follows:
c
F ( x) = c0 * x 1
To calculate curves of this type, a minimum of two calibration samples must
be available (right partial fig.).
Note:
If you calibrate using the ¾Standard Addition method and if you use the
Exponential calibration function, the expected offset value is subtracted
from all calibration points with positive amount values (i.e., from all
¾Spiked Samples). Thus, the following formula applies:
c
F ( x) = c~ + c0 * x 1
Theory of Calibration
97
Distinguish between the following cases:
a) If no calibration points with Amount = 0 are available (only Spiked
samples), the expected offset is calculated using the Linear with Offset
method, i.e.:
c~ = c 0
b) If calibration points with Amount = 0 are available (also Unspiked
samples), the expected offset is the average of all samples with
Amount = 0.
If none of the above functions can be applied to the available ¾Calibration
Points, the calibration curve can be described as a polygon, that is, a linear
interpolation between two adjacent calibration points (Point to Point). If
several replicates of one calibration level are available, these are averaged
before interpolation.
Caution:
The decision whether a calibration type differing from the linear default is
physically sensible, is within the responsibility of the user!
A large number of replicates increases the precision and the reliability of
the curve at this point (on the calibration level), but is not decisive for the
entire curve. The more calibration levels are examined, that is, the more
standards of different concentrations are measured, and the more precise
is the area/amount allocation for a larger range. To be exact, the calibration
is valid for the range of the calibration samples only and not beyond it.
Using the Calibration Curve
If the calibration coefficients are known, the amount value can be
calculated for any area value by inserting the coefficients in the formula of
the respective calibration type. Within the range of the curve that is covered
by the calibration points, it is possible to convert any peak area into the
corresponding amount. This is shown in the following example:
Standard Sample
The user enters the amounts (x1 to x4) of the different standard samples
and determines the ¾Calibration Function by selecting the calibration type
(here: linear with offset). Depending on the selected integration type, area
values (F1 - F4) are established from the detected peaks. One area and
98
Theory of Calibration
one substance amount value form one calibration point. The positions of
the calibration points determine the curve that Chromeleon calculates with
an approximate method. The final course of the calibration curve is
determined by the calibration coefficients (here: c0, c1).
Analysis Sample
Standard Sample
Determined
Area
Calculated
Amount
“Calibration Function”
F = c0 + c1 * x
“Reverse Function”
x = c0 + 1 * F
c1
c1
F4
F3
F2
F1
c0
0
x1
x2
Known
Amount
x3 x4
c0
c1
0
Determined
Amount
Unknown Sample (Analysis Sample)
In the case of an unknown sample, the previously calculated calibration
coefficient and one or several area values are known. The area value is
now a known parameter and is thus drawn in x-direction. In the diagram,
the two axes must be exchanged for one another. As a result, the
calibration function must be converted in its inverse function. This is also
performed by Chromeleon. Now, the amount can be calculated by inserting
the calibration coefficients and area values.
Furthermore, the exclusion of outliers, the different weighting of calibration
points, and the formation of "averaged" calibration points from one
calibration level, are alternative ways of how to calculate the calibration
curve.
Calculating the Calibration Curve
Calculation of the calibration curve (¾Calibration Function) is based on the
method of least squares. With a given calibration type (linear, linear with
offset,...), the parameters of the calibration curve F(a), that is, c0, c1, and
c2, are determined so that the sum of the squared distances of all
measured points becomes negligible. For this purpose, the following
optimization problem is solved:
n
∑ w *( x
i =1
i
i
− F (ai )) 2 → min
Theory of Calibration
99
(xi- F(ai)) refers to the distance of the xi-value from the calibration curve
F(a), wi is the selected weighting (see ¾Weights, for example, wi=1,
wi=1/Amount or wi=1/Amount2) and xi is the actual value.
The formula used for calculating the calibration curve depends on the
Standard Method (External/Internal…).
For information about these formulas, refer to
Standard Methods.
Evaluation with Various
Standard Methods
Calibrations can be based on external or ¾Internal Standards ("ISTD").
External standard means that there is a separate standard sample. Using
an internal standard means adding the standard to the unknown sample.
This can be either before (External/Internal) or after (Internal) sample
preparation.
Standard
Description
external
= Default setting. Calibration is via one or several standard samples. Via the
amount values entered in the Amount column, a ratio is established between
the area and the amount. On this basis, the amount in samples of unknown
concentration is determined via the peak area.
With an increasing number of different amounts, the area/amount ratio
(=calibration curve) can be determined more exactly.
If different amounts are obtained by diluting the original substance, a ¾Dilution
Series is resulting. The amount of each concentration is entered in a separate
amount column in the corresponding line of the peak table.
If the calibration is performed with a single standard sample by injecting
different volumes (Var.InjectVol.), only the amount of the original sample is
stored in an amount column. The remaining amount values (for the different
injection volumes) are calculated by Chromeleon.
internal/
external
Choose a substance as ¾Internal Standard ("ISTD") whose retention time
behavior is similar to the behavior of the substances to be analyzed. Before the
sample preparation, an internal standard is added to all samples (unknown and
standard samples) in exactly the same amount so that the concentration is
identical in all samples. For example, diluting the sample or performing a precolumn derivatization later will change the concentration of the internal
standard. During calibration, the internal standard and the substances to be
determined are calibrated.
100
Theory of Calibration
Standard
Description
internal
In the pure internal standard method, calculation is via area and amount ratios
instead of absolute areas and amounts. For this procedure, it is necessary to
inject a constant amount of the ¾Internal Standard ("ISTD"). The internal
standard is added before the sample preparation. In a dilution series, the
standard does not have to be diluted (Const. Internal Standard). Due to the
equivalent amount of added internal standard, the same ISTD result should be
achieved for all samples. Forming the ratio of ISTD values allows you to draw
conclusions about the precision of the analysis and calculating the actual result.
Due to the intense experimental procedure, this type of calibration is rarely used
in HPLC.
For examples of the different standard methods, refer to
How to ...: Actions in the QNT Editor
Calibration.
Evaluation with Various Standard Methods
Below please find a description of how Chromeleon calculates calibration
points, which form the basis for any calibration function F. Please note that
Standard
the formula used for the calculation depends on the selected
Methods (External, Internal/External (with/without Var.ISTD), Internal
(with/without Var.ISTD)).
For evaluating unknown samples, that is, calculating the ⇒Amount values,
the inverted form of the corresponding ¾Calibration Function F is used
(=inverted function A). This means that the c0, c1, and c2 calibration
coefficients form function A that is inverted for amount calculation. The
result is F.
'External' Evaluation:
Calibration:
•
Y(i,k) = RESPONSE(i,k)
•
X(i,k) = AMOUNT_NOMINAL(i,k) ∗ (WEIGHT(k)/DILFAC(k)) ∗
(INJECTVOL(k)/REFINJECT)
Evaluation: Calculation of the Amount peak variable for peak i in the
sample x
•
AMOUNT(i,x) = F( RESPONSE(i,x) ) ∗ (DILFAC(x)/WEIGHT(x)) ∗ RSP-FACTOR(i)
Description: see below.
Theory of Calibration
101
'Internal' Evaluation:
Calibration:
•
Y(i,k) = 100 ∗ (RESPONSE(i,k)/RESPONSE(ISTD,k)
•
X(i,k) = AMOUNT_NOMINAL(i,k) ∗ (WEIGHT(k)/DILFAC(k))
Evaluation: Calculation of the Amount peak variable for peak i in the
sample x
•
AMOUNT(i,x) = F(100 ∗ (RESPONSE(i,x)/RESPONSE(ISTD,x)) ∗
(DILFAC(x)/WEIGHT(x)) ∗ RSP-FACTOR(i)
The ISTD peak itself will not be evaluated!
Description: see below.
'Internal' Evaluation (with variable ISTD):
Calibration:
•
Y(i,k) = AMOUNT_NOMINAL(ISTD) ∗ RESPONSE(i,k)/RESPONSE(ISTD,k)
•
X(i,k) = AMOUNT_NOMINAL(i,k) ∗ (WEIGHT(k)/ DILFAC(k)) ∗
(INJECTVOL(k)/REFINJECT)
Evaluation: Calculation of the Amount peak variable for peak i in sample x
•
AMOUNT(i,x) = F(AMOUNT_NOMINAL(ISTD) ∗ (RESPONSE(i,x)/RESPONSE(ISTD,x)) ∗
(DILFAC(x)/WEIGHT(x)) ∗ RSP-FACTOR(i)
The ¾Internal Standard ("ISTD") peak itself is not evaluated!
Description: see below.
'Internal/External' Evaluation:
Calibration:
'External' calibration, including the ISTD peak! (Also, refer to 'External')
Evaluation: Calculation of the Amount peak variable for peak i in the
sample x
•
The ISTD peak itself is evaluated 'Externally'!
•
FACTOR_IS(x) = AMOUNT_NOMINAL(ISTD)/AMOUNT(ISTD,x)
•
AMOUNT(i,x) = F(RESPONSE(i,x)) ∗ (DILFAC(x)/WEIGHT(x)) ∗ RSP-FACTOR(i) ∗
FACTOR_IS(x)
Description: see below.
102
Theory of Calibration
'Internal/External' Evaluation (with variable ISTD):
Calibration:
Calibration is 'External', including the ISTD peak! However, the nominal
amount for the ISTD peak from the sample list (Sample Amount) is used.
The sample weight of the ISTD peak is not considered.
•
Y(i,k) = RESPONSE(i,k)
•
X(i,k) = AMOUNT_NOMINAL(i,k) ∗ (WEIGHT(k)/DILFAC(k)) ∗
(INJECTVOL(k)/REFINJECT)
•
X(ISTD,k) = AMOUNT_NOMINAL(ISTD,k) ∗ (1/DILFAC(k)) ∗
(INJECTVOL(k)/REFINJECT)
Evaluation: Calculation of the Amount peak variable for peak i in the
sample x
The ISTD peak itself is evaluated 'Externally', but without Weight correction.
•
AMOUNT(ISTD,x) = F(RESPONSE(ISTD,x)) ∗ (DILFAC(x)) ∗ RSP-FACTOR(ISTD)
•
FACTOR_IS(x) = AMOUNT_NOMINAL(ISTD,x)/AMOUNT(ISTD,x)
•
AMOUNT(i,x) = F(RESPONSE(i,x)) ∗ (DILFAC(x)/WEIGHT(x)) ∗ RSP-FACTOR(i)
∗ FACTOR_IS(x)
Description:
F:
Calibration function
X(i,k):
X-coordinate of a calibration point for peak i for the
standard sample k
Y(i,k):
Y-coordinate of a calibration point for peak i for the
standard sample k
k:
Calibration sample (standard)
x:
Unknown sample (analysis sample)
RESPONSE(i):
Reference variable (Int.Type; that is, Area, Height, CEArea) of peak i in a sample
RESPONSE(ISTD):
Reference variable (Int.Type; that is, Area, Height, CEArea) of the corresponding ISTD peak of a sample
AMOUNT_NOMINAL(i):
Amount of peak i from the peak table for the standard
sample k
AMOUNT_NOMINAL(ISTD):
⇒ISTD Amount for the sample k from the sample list
AMOUNT(ik):
Calculated amount of the peak i for the sample k
WEIGHT:
⇒Weight (Sample Weight Factor) for a sample
DILFAC:
⇒Dil. Factor (Dilution Factor) for a sample
INJECTVOL:
⇒Inj. Vol. (Injection Volume)
REFINJECT:
Injection volume of the first sample in a calibration series
RSP-FACTOR(i):
⇒Response Factor of the peak i from the QNT peak table
Theory of Calibration
103
Implementation
Follow the description below to implement and perform calibration:
Sample List
•
Enter the available standard samples in the
(Sequence), similar to unknown samples.
•
To facilitate and automate the input, use the
•
Each sample in the sample list can be converted into a standard
sample by assigning the ¾Sample Type STD. Note that the position
(line number) in the sample list determines the order of processing. If a
standard sample is to be injected several times, a separate line is
created in the sample list for each injection.
•
In the Method column, specify the quantification method to be used for
evaluating the sample.
•
Input in the remaining fields of the sample list is analog to each
unknown sample (Position, Injection Volume, ..).
•
For a detailed description of the procedure, refer to How to ...: Actions
Creating a Sample List.
in the Browser
Sample List
Sequence Wizard.
Quantification Method (QNT Editor)
•
Open the General worksheet and check the current settings. Define
the calibration⇒ Mode to be used.
•
Enter the names and the retention times of the peaks to be determined
on the Peak Table sheet. If a processed sample refers to a QNT File
with an "empty" peak table, the peak table can be automatically filled
with the retention times of the integrated peaks after the analysis by
selecting the Autogenerate Peak Table command. Each peak
contains a successively numbered default name.
•
Enter the amount values of the standard substances in the amount
columns. Determine the standard method, the calibration type, and the
integration type for the calibration.
•
Determine the standard method, the calibration type, and the
integration type to be used for the calibration.
•
For more information, refer to How to ...: Actions in the Browser
Creating a Peak Table.
104
Theory of Calibration
Validation, AutoQ, and System Wellness
105
Validation, AutoQ, and System
Wellness
Validation and Qualification
Analysis data from various workstations or laboratories can only be
compared if it is possible to determine the quality of the results produced
with a chromatography system.
Validation (Definition)
The process of ensuring that a system and its analysis procedures supply
reproducible and reliable results is referred to as validation. This includes
above all procedures regarding the planning, implementation, and
documentation of an analytical method. Thus, validation is an integral part
of ¾GLP ("Good Laboratory Practice").
Qualification (Definition)
First, the manufacturer validates the single devices and the data system.
During qualification, the user checks whether a device or data system
works according to its specification. This includes procedures guaranteeing
the optimum technical condition of instruments (hardware and firmware)
and of computers (hardware and software). Thus, qualification, too, is an
integral part of Good Laboratory Practice.
When is qualification necessary?
Instruments should be qualified before setting them into operation and at
regular intervals thereafter; especially after exchanging worn-out parts,
performing repair work, or replacing an instrument by a new one. Also,
perform system qualification procedures after you have updated the
software of your data system.
In addition, the data system itself should be qualified at least after an
update. ¾Installation Qualification and ¾Operational Qualification are
available for this.
106
Validation, AutoQ, and System Wellness
When they are started, many instruments perform an automatic self-test to
ensure optimum function. For example, for the Dionex UVD 340U
¾Photodiode Array Detector, spectra calibration is performed automatically
via the ¾Holmium Oxide Filter whenever the detector is started.
When is validation necessary?
The analytical method and the ¾PGM File should be validated before they
are used in daily laboratory procedures. As modifying single parameters
can already be of great importance, validation is also necessary in the daily
routine. On the SST tab page of the ⇒QNT Editor, define ¾System
Suitability Tests to check whether your analytical method and your program
file are suitable for analyzing special samples.
How is validation performed?
The focus of an analysis procedure is on Calibration (see Theory of
Calibration
Calibration (Overview). Within the scope of validation, it is
then important to check whether calibration has been performed correctly.
The precision, ¾Limit of Detection, dynamic work range, and robustness of
a procedure and the involved components have to be determined. The
following features are available for this: ¾Validation Samples, ¾Blank Run
Samples, ¾Matrix Blank Samples, ¾Confidence Interval/Range, averaging,
normal distribution, outlier tests, detection of statistical and systematic
errors, ¾Correlation Coefficient, ¾Standard Deviation, ¾Relative Standard
Deviation, etc.
Chromeleon provides numerous options to meet all GLP, qualification, and
validation requirements. For more information, refer to:
AutoQ Equipment Qualification
The System Suitability Test (SST)
System Wellness for IC Devices (Overview)
System Wellness for HPLC Devices
Validation, AutoQ, and System Wellness
107
AutoQ Equipment Qualification
Dionex AutoQ is a comprehensive range of user-friendly Chromeleon
qualification tools. These tools help you quickly and easily perform
qualification tasks that would otherwise be troublesome and timeconsuming. In addition, they simplify compliance with qualification
standards and qualification rules. AutoQ qualification tests are available for
Chromeleon software and for several HPLC and IC systems. The following
tasks can be automated with AutoQ test procedures.
For Chromeleon software:
•
¾Installation Qualification (IQ)
•
¾Operational Qualification (OQ)
For instruments:
•
Installation Qualification (IQ)
•
Operational Qualification (OQ)
•
¾Performance Qualification (PQ)
What is unique about Dionex AutoQ?
Dionex AutoQ is a comprehensive suite of qualification procedures for
instruments from several manufacturers. AutoQ is available for the
following systems:
•
Dionex IC modules and Summit HPLC modules
•
Agilent 1100 HPLC System modules
•
Waters HPLC modules (including the Alliance 2690/2695 Systems
and the 996/2996 PDA's)
Which standards and regulations does Dionex AutoQ help you to
meet?
The following standards and regulations are important in a validated
environment:
•
Good Laboratory Practice (¾GLP)
•
Current Good Manufacturing Practice (cGMP)
•
21 CFR Part 11
•
ISO 9000
108
Validation, AutoQ, and System Wellness
What are the benefits of AutoQ?
•
Most AutoQ tests run automatically most of the time. Therefore, they
require very little operator time. For example, it only takes about 30
minutes to prepare the Instrument OQ, after which Chromeleon runs
the test automatically. In comparison, the conventional test routines
used in many validated laboratories typically require a full day of the
analyst's time.
•
The high level of automation reduces the risk of errors and ensures
comparable results.
•
Chromeleon automatically documents the results. The reports created
by the system include charts, calculations, and the single results
(Passed/Failed).
•
AutoQ is virtually identical for all instruments, regardless of the
manufacturer. This means:
•
-
Considerable time savings for the creation and maintenance of
SOPs (Standard Operating Procedures) for laboratories using
instruments from different manufacturers.
-
Only one test procedure to learn and work with.
-
Test reports have the same format for all instruments.
-
All reports can be easily managed, saved, and stored as electronic
documents, using Chromeleon electronic reports and ¾Electronic
Signatures.
AutoQ instrument qualification tests can be adapted for use with
instruments from other manufacturers.
Tip:
This requires advanced knowledge of report creation in Chromeleon.
•
AutoQ qualification procedures are included in every Chromeleon
software package.
Note:
Certified standard solutions, which are available from Dionex, are
needed to run AutoQ qualification tests.
Validation, AutoQ, and System Wellness
109
Instruments Operational and Performance Qualification
After you have validated the chromatography system, perform
¾Operational and/or ¾Performance Qualification for the instruments.
The Qualification menu in the Chromeleon Browser provides the following
options: Instruments PQ and PQ Setup and Instruments OQ and OQ
Setup. Select Instruments PQ (or Instruments OQ) to perform the
performance (operational) qualification. Select PQ Setup (or OQ Setup) to
create the templates required for performing the performance (operational)
qualification. Usually, this is necessary only during the initial installation of
the system or if the configuration has been changed.
Tip:
Only qualified Dionex service personnel should perform the Performance
and Operational Qualification checks. For more information, please contact
Dionex Service.
For information about performance qualification, refer to
Operational
and Performance Qualification for HPLC Systems and/or
Performance Qualification (PQ) for IC Systems.
In addition, you can perform operational qualification for the ¾UCI
Universal Chromatography Interface. For more information, refer to the UCI
Operational Qualification Operating Instructions.
Operational and Performance Qualification for HPLC
Systems
Chromeleon provides a datasource with a master template in the Template
directory on the Chromeleon software CD. This template is designed for
Performance (Operational) Qualification in a standard HPLC configuration.
In addition to various sequences, the template contains a ¾Report
Definition File (RDF) for OQ and PQ, providing the following pages:
SPECIFICATION: On the SPECIFICATION page, enter the system
specifications (instruments, fluidics, and limits).
COLUMN OVEN: The COLUMN OVEN page indicates whether the column
oven temperature corresponds to the selected temperature (within the
specified limits).
110
Validation, AutoQ, and System Wellness
INJ_REPRO_AND_RET_REPRO and ASI_REPRO_AND_RET_REPRO,
respectively: On the INJ_REPRO_AND_RET_REPRO page, serves for
checking the reproducibility of the injector and the retention time. If the
result is within the specified limits, the Result column indicates OK.
INJ_CARRY_OVER: The INJ_CARRY_OVER page supplies a measure
for the carry over in your system. If the result is within the specified limits,
the Result column indicates OK.
INJ_LINEARITY: The INJ_LINEARITY page supplies a measure for the
linearity of injection volume and peak area.
PUMP_GRADIENT: On the PUMP_GRADIENT page serves for checking
the gradient precision. The limits of the Specification page are included. If
the values are within the specified limits, the last column (Result) indicates
OK. The Result of all tests column indicates Test passed.
PUMP_GRADIENT_REPRO: The PUMP_GRADIENT_REPRO page
serves for checking the gradient reproducibility (with 3 repetitions in this
example). The limits of the Specification page are included. If the values
are within the specified limits, the last column (Result) indicates OK. The
Result of all tests column indicates Test passed.
DET_NOISE_AND_DRIFT: The DET_NOISE_AND_DRIFT page serves
for checking whether noise and drift in your system correspond to the limits
on the SPECIFICATION page. If the values are within the specified limits,
the Result column indicates OK.
DET_WAVELENGTH: The DET_WAVELENGTH page supplies a measure
for the wavelength precision of the corresponding detector.
DET_LINEARITY: The DET_LINEARITY page serves for checking the
detector linearity. From five different injections at different concentrations,
the correlation coefficient (supplied in %) between the peak height and the
concentration is determined. If the value is above the specified limit, Test
passed is returned as the result.
RF_DET_NOISE and RF_DET_WAVE: These pages evaluate the
noise and the wavelength precision of the fluorescence detector.
Audit Trail: The Audit Trail page displays the audit trail for the analyzed
sample.
Validation, AutoQ, and System Wellness
111
Caution:
Do not edit the report pages (except the Specification page), even if
editing is possible! Chromeleon automatically reads the corresponding
values. Within the report, individual data sheets are very often accessed via
references. If you insert or delete lines and columns, these references will
be lost. Thus, the calculations will be wrong!
The report must be printed as Batch Report from the browser to make
sure that, in the report, the data are read in and processed correctly. Select
the sequence for which to print the report. Make sure that no sample is
selected. Select Batch Report on the File menu and click OK to start
printing.
Tip:
Enter the actual concentrations of the used standards in the Amount
columns of the QNT File for evaluating the detector linearity.
For more information about Operational and Performance Qualification,
refer to the Operational Qualification/Performance Qualification
Operating Manual that is available from Dionex Service.
Performance Qualification (PQ) for IC Systems
Validation is becoming increasingly important to analytical laboratories.
Documented evidence must be provided to demonstrate the integrity of
data collected and validate the results obtained on laboratory
instrumentation.
The Qualification menu in the Chromeleon Browser includes options for
Instruments PQ and PQ Setup. Instruments PQ is used to perform the
performance qualification. PQ Setup is used to generate the templates
required for performing the performance qualification. (This is generally
necessary only after a new installation or after changes to the
configuration.) The Chromeleon CD provides a datasource with a master
template in the PQ\Templates\PQ directory.
PQ should be performed at regular intervals after the initial installation and
Operational Qualification (OQ). Dionex recommends performing PQ every
six months. A qualified Dionex Service Representative should perform all
tests, in accordance with the instructions in the IC System Operational and
Performance Qualification User’s Guide. The user’s guide is included in the
IC OQ/PQ Kit with Test Cells (P/N 057599) and the IC OQ/PQ Basic/Refill
Kit (P/N 057608).
112
Validation, AutoQ, and System Wellness
The PQ procedure used to qualify Dionex Ion Chromatography Systems
meets the requirements established by the National Institute of Standards
and Technology (NIST) and the American Society for Testing and Materials
(ASTM). This PQ procedure provides qualification testing for ICS-2500,
ICS-2000, ICS-1500, ICS-1000, DX-600, DX-500, DX-320, DX-120, and
BioLC systems.
The System Suitability Test (SST)
On the SST tab page in the ⇒QNT Editor, define a ¾System Suitability
Test (SST) to check whether the quantification method and the ¾PGM File
are suitable for analyzing special samples. (For more information, refer to
Defining the System
How to ...: Actions in the QNT Editor
Suitability Test.) The System Suitability Test can already be performed
while the chromatogram is recorded. The corresponding QNT Method
needs to be available in the sample list (in the Browser). As Fail Action,
select Abort Batch to automatically abort the sample ¾Batch if a test
condition is not met.
System Wellness for IC Devices (Overview)
What is System Wellness?
System Wellness monitors the overall "health" of a chromatographic
system. It provides built-in diagnostic and calibration features that help
prevent unscheduled system shutdowns and assure reliable operation of
system devices. Calibration and diagnostic commands are available from
Wellness control panels and Help topics are provided for performing the
various tasks.
Supported Devices
For System Wellness support, a device must have a version of
¾Moduleware installed that supports System Wellness. The following
devices are supported:
Device
Moduleware Version Required
IC Pumps
GP40/IP20
3.46 (or higher)
GP50/IP25
3.46 (or higher)
GS50/IS25
1.00 (or higher)
Validation, AutoQ, and System Wellness
Device
113
Moduleware Version Required
Detectors
AD25
1.02 (or higher)
CD20/ED40
3.05 (or higher)
CD25/ED50
1.05 (or higher)
CD25A/ED50A
1.00 (or higher)
PDA-100 (DX-LAN)
1.04 (or higher)
PDA-100 (USB)
1.0.0 (or higher)
¾Autosampler
AS50
1.05 (or higher)
¾Eluent Generator
EG40
2.23 (or higher)
EG50
2.26 (or higher)
System Modules
IC20
3.08 (or higher)
IC25
1.04 (or higher)
IC25A
1.00 (or higher)
ICS-90
1.00 (or higher)
ICS-1000
1.1.0 (or higher)
ICS-1500
1.1.0 (or higher)
ICS-2000
1.1.0 (or higher)
System Wellness Features
•
System Wellness control panels, which allow easy access to diagnostic
and calibration commands and data
•
Download of current, previous, or factory calibration data
•
Leak detector testing and calibration
•
Wavelength verification and calibration for UV and PDA detectors
•
Cell calibration for conductivity detectors
•
pH calibration for amperometry detectors
•
Pressure offset and degas calibration for pumps
•
Flow rate calibration for pumps
For instructions on setting up and using System Wellness features, refer to
System Wellness
How to ...: Actions in the Browser
114
Validation, AutoQ, and System Wellness
System Wellness for HPLC Devices (Overview)
Chromeleon provides several system wellness features for the Summit
HPLC modules. The user does not have to calibrate these modules
because either they have been calibrated in the factory (e.g., the pump
flow) or calibration is performed automatically (for example, for the UV
detector via a ¾Holmium Oxide Filter).
Device
Feature
P680
Leak detector
Reliable operation
Monitoring of piston seal tightness
Reliable operation
Pressure limits
Prolongs the life of HPLC columns,
reliable operation
Total workload monitoring
Allows you to schedule the next
service date.
Degasser vacuum level monitoring
Reliable operation
Active rear-seal wash system
Prolongs the life of the consumable
parts
Monitoring of the liquid level for rear-seal
washing
Reliable operation
ASI-100
Description
Leak detector
Reliable operation
Injection counter
Allows you to schedule the next
service date.
Needle seal wear monitoring
Allows you to schedule the next
service date.
Rotor wear and stator wear monitoring for
the internal motorized switching valve
Allows you to schedule the next
service date.
Needle port wear monitoring
Allows you to schedule the next
service date.
Synchronization of injection and pump cycle Injection reproducibility
TCC-100
UVD340U
System
Gas and leak detectors
Reliable operation
Column identification features
Tracking and troubleshooting
Temperature limit monitoring
Tracking and troubleshooting
Lamp ignition counter
Allows you to schedule the next
replacement date
Lamp age monitoring
Allows you to schedule the next
replacement date
Minimum lamp intensity monitoring
Reliable operation
Documentation when new wear parts have
been installed.
Tracking and troubleshooting
Documentation when the solvent has been
changed
Tracking and troubleshooting
Data Representation and Reprocessing
115
Data Representation and
Reprocessing
Data Representation and Reprocessing
Chromeleon allows you to represent your raw data under various aspects.
It depends on the aspect of interest and on the type of data reprocessing
you wish to perform, which window or partial window is most appropriate.
For example, you can reintegrate a chromatogram manually, compare
several chromatograms, analyze the peak purity, calculate calibration
curves, or search single spectra in a spectra library.
Each operation is displayed in a separate window. Each window is intended
for one specific task and has its own window arrangement and menu
structure.
Immediately after data acquisition, you can perform certain steps for data
reprocessing. You can define these steps in the ¾Program in the PGM
Editor; see
Post-Acquisition Steps.
You can perform all other steps of data reprocessing at any time. Use the
following windows (or partial windows):
The QNT Editor
Integration
Reports
Calibration (Overview)
The Printer Layout
PPA: Peak Purity Analysis
Spectra Libraries
After the raw data has been acquired, the Quantification Method
determines how the data is evaluated. Thus, the QNT Editor with the
Integration window is the most important window for data reprocessing.
116
Data Representation and Reprocessing
If, after the analysis, the detection and peak table parameters chosen in the
QNT Method prove to be inappropriate for the sample and/or sequence,
you can change them in the QNT Editor at any time. You do not need to
analyze the corresponding sample again.
All modifications are immediately and globally effective. All modified
variables are immediately re-calculated and the new values are displayed
on the screen. The modifications are finally accepted by selecting the Save
or Save Manipulations command.
If the modified quantification method is saved, the results of all samples
evaluated by this method will be adjusted.
Post-Acquisition Steps (PGM Editor)
Immediately after data acquisition, you can perform certain data
reprocessing steps. You can use the Post-acquisition steps view to
define these steps in the ¾Program via the PGM Editor. (Also, refer to
The PGM Editor).
Control
The following data reprocessing steps are available in the Post-acquisition
view:
•
Arithmetic combination of channels (2D channels)
•
Copy Channel
•
Extract MS channel (for extracting a ¾Mass Trace - only possible if
MS data is available)
•
Extract optimum integration path (for extracting the ¾Optimum
Integration Path - only possible if a 3D field is available)
•
Extract UV channel (for extracting a UV channel - only possible if a 3D
field is available)
•
Smooth data (for chromatogram ¾Smoothing)
For more information, refer to How to …:
Adding Post-Acquisition Steps.
Actions in the PGM Editor
The QNT Editor
117
The QNT Editor
The Quantification Method
In Chromeleon, all instructions and parameters representing the basis of
calculation for the sample evaluation are included in the Quantification
Method (QNT File). This refers for example to the following questions:
•
Below which height, width, or area a peak will be ignored?
•
Which course has the baseline?
•
Which peaks are classified as ¾Riders?
•
What is the ⇒Amount of the standard samples?
•
Which ¾Calibration Function is used for creating a calibration curve?
•
Which peaks are identified by name?
Before the actual analysis, these evaluation parameters are defined in the
Quantification method (QNT File).
•
Select File > New > Method File to generate a new QNT File.
•
Alternatively, select a sequence in the Browser and double-click the
corresponding QNT File to open it.
•
To open the QNT Editor from a method file, click the QNT Editor icon
on the toolbar. This allows you to display the QNT File data of the
current sample.
For more information about the QNT Editor, refer to
The QNT Editor.
118
The QNT Editor
The QNT Editor
The QNT Editor allows you to create a method for evaluating
chromatographic results of different samples. The ⇒QNT-Editor usually
provides:
•
The chromatogram of the current sample
•
The calibration curve of the current peak
•
Various tab pages:
Instead of the calibration curve, you can display the following items:
•
The Trend Plot (refer to the Control section; similar functionality as
the control panel)
•
UV spectrum (if available)
•
¾Mass Spectrum (if available)
In addition, you can include report tables in the tab pages.
The QNT Editor is divided in two window sections. The upper section
serves for information purposes. To create a QNT File, use only the lower
window section.
The QNT Editor
119
Upper Pane
The upper pane serves to display additional plots:
•
Select Show Chromatogram or Show Calibration Curve on the View
menu to enable or disable the display of the chromatogram and/or the
calibration curve.
•
Press the F4 key or the Shift+F4 keys or select the Next > Previous
Chromatogram option (on the File menu) to toggle between the
chromatograms of the individual samples.
•
Select the Show Spectra option to enable or disable the display of the
spectrum of a single peak.
Lower Pane
The lower pane serves to determine the evaluation parameters.
•
Select a specific tab to open a worksheet, similar to Microsoft Excel.
•
You can freely select the names of the worksheets. Double-click the
corresponding name, and enter a new name in the edit dialog box.
The following worksheets are available by default (unless you changed
their names):
The General Tab Page
Detection Parameters
Peak Table, Amount Table, Peak Tracking, and MS Tracking
Calibration Settings
Spectra Library Screening
System Suitability Test
Mass Spectra (MS)
UV Spectra (UV)
Tip:
These topics describe the structure and functions of the individual
worksheets. For detailed information about how to enter data, refer to How
to ...:
Actions in the QNT Editor.
120
The QNT Editor
The General Tab Page
The General tab page contains global settings for the following worksheets:
Peak Table ("Retention Time Settings"), Amount Table ("Amount
Calibration ("Global Calibration Settings"). Enter a
Interpretation"), and
name for the current QNT File in the Title field. The name appears in the
Browser and can be included in a Report when printing data of the QNT
Editor. Click Unidentified Peaks to determine how to quantify unidentified
peaks.
Retention Time Settings
•
Select the Use Recently Detected Ret. Times check box to use the
⇒Retention Time of the preceding sample to identify a peak via a
retention time window (⇒Window). You can also use the retention time
of the last standard. Select Standard from the of last drop-down list.
Click Options… to display more options. (For more information, refer to
⇒Use Recently Detected Retention Time.) This function allows the
system to automatically react to changing retention times that are, for
example, due to column trends. If the option is disabled, the actually
determined retention time listed in the peak table is used for identifying
the peak.
The QNT Editor
•
121
Use the Peak retention time determination section to specify how the
retention time of peaks shall be determined: Select Use absolute
greatest signal value to use the retention time of the greatest absolute
signal value. Select Use relative greatest signal value over the
baseline to use the retention time of the greatest relative signal value.
Dead/Delay time(s)
•
Enter the ⇒Dead Time in the Dead Time field. The dead time is used
for calculating the ¾Capacity Factor k' and the ⇒Kovats Indexes.
•
Select the name of any further detector from the 2nd Detector and/or
3rd Detector drop-down list and enter the ⇒Delay Time in the min
Defining
field. (Also, refer to How to ...: Actions in the QNT Editor
the QNT Method for Several Detectors).
Amount Interpretation
•
For documentation purposes (exclusively), the physical dimension
(amount or concentration) that is used for the amount values can be
included in the field ⇒Dimension of Amounts. Amount values are not
automatically converted into concentration values or vice versa.
•
In addition, the reference injection volume is defined on this page.
Either select the injection volume of the first standard (Use inject
volume of first standard) or enter any volume via Fixed.
Global Calibration Settings
•
Use the Mode field to determine how the samples of a sequence shall
be calibrated and based on which ⇒Calibration Mode calibration shall
be performed. For example, this allows you to calibrate certain samples
as a group or to include calibration samples for samples that are
analyzed later. For more information, refer to: How to ...: Actions in
the QNT Editor
Calibration.
•
When Auto Recalibrate is enabled, each modification within a
chromatogram such as moving peak delimiters results in automatic
recalculation of the ¾Calibration Coefficient and all derived calibration
data. Disable Auto Recalibrate in your peak table to include the c0,
c1, and/or c2 columns. Perform recalibration by clicking the Calibrate
button. Nevertheless, you can enter the corresponding values manually
as well.
122
•
The QNT Editor
Curve Fitting allows you to determine dependent and independent
variables for calibration: Select Normal to accept normal evaluation
and the axis settings of the calibration curve (x-axis = amount, y-axis =
measured value). Select Inverted to use inverted evaluation and to
invert the axes (x-axis = measured value, y-axis = amount).
For more information, refer to How to ...: Actions in the QNT Editor
Inverting Dependent and Independent Variables.
Blank Run & Matrix Blank Subtraction
In the Blank Run & Matrix Blank Subtraction section, determine whether
the absorption values of a ¾Blank Run Sample are considered
(= subtracted) in the sample evaluation (= ¾Blank Run Subtraction). You
can also subtract the results of a single ¾Matrix Blank Sample.
•
Select No Blank Run Subtraction if no correction is to be performed.
•
Select Subtract Recent Blank Run Sample in Corresponding
Sequence to use a finished blank run sample of the current sequence
for the subtraction. The chromatogram of the blank run sample is
subtracted point by point from the active chromatogram.
•
Select Subtract a Fixed Sample to perform the correction with any
sample. Click Browse to search for the sample.
•
Select Enable Matrix Blank Subtraction to enable the subtraction of
matrix blank samples. Contrary to the other options the resulting peak
areas or peak heights are subtracted.
Also, refer to How to ...: Actions in the QNT Editor
Blank Run Sample.
Subtracting a
Detection Parameters (Detection)
Detection parameters serve, for example, to recognize, classify, and
suppress peaks as well as to determine the baseline.
The default values are normally suitable for optimum integration of 90% of
all recorded chromatograms.
In critical cases (for example, with wavelength switching), the user can
improve the integration results by modifying the parameters.
The QNT Editor
123
Detection parameters are time-dependent. Each parameter can be
enabled, disabled, or changed in its value at a specific time. The time when
the parameter shall change, the parameter name and its value are entered
in the corresponding column of the first line. It is possible to change a
parameter several times in succession.
Detection parameters are defined in the QNT Editor. You can either enter
them in the table of the Detection tab page or define them graphically in
the chromatogram.
In the example above, integration is inhibited (⇒Inhibit Integration
command) at the time t = 0.000 (On). After three minutes (t = 3.000),
integration is enabled again (Off). It is also stipulated that peaks with a
minimum area of 1 x [Signal] *min only are recognized as peaks.
The last defined value of each parameter is valid until the sample run is
completed. After that, the parameters assume their preset (default) values.
The ¾Channel column indicates whether the settings are valid for a specific
channel (as in this example) or for all channels.
Usually, the chromatogram and the report table are immediately updated
when the detection parameters have been changed. However, you may
disable this function by deselecting Autom. Re-Integerate on the View
menu of the QNT Editor. If this option is disabled, save either the QNT
Method or select Start Integration on the View menu to start re-integration
with the new integration parameters.
Tip:
The setting for this option is saved in the corresponding ¾Report Definition
File.
124
The QNT Editor
For more information and examples, refer to
How to ...: Actions in the QNT Editor
Parameters and
Defining Detection
⇒Detection Parameters (Overview)
In many cases, it is easier to define the detection parameters graphically in
the chromatogram.
For more information, refer to How to ...: Actions in the Chromatogram
Defining Detection Parameters Graphically.
Peak Table, Amount Table, Peak Tracking, and MS
Tracking
The peak table contains all parameters required to identify a peak and to
determine the amounts of the substances serving as standards. The peak
table contains a minimum of 26 columns (the Amount, C0, C1, and C2
columns are duplicated as required, depending on the number of different
standards). By default, the peak table consists of four tabs: Peak Table,
Amount Table, Peak Tracking, and MS Tracking. Individual columns and
entire pages can be displayed and hidden.
The peak table is usually created manually; i.e., the user enters the names
and retention times of the expected peaks. In addition, peaks serving as
standard peaks must be labeled as such in the ⇒Standard column. The
(known) concentration of a standard is indicated as the ⇒Amount in the
Amount column. These values form the basis for any type of calibration.
Select Autogenerate Peak table on the Edit menu to automate peak table
creation. There are two ways to create a peak table:
•
Select Enumerate peaks of current chromatogram to include all
peaks integrated in the current sample in the peak table. The peak
name is the sequence name plus a consecutive number.
•
Select Use Spectra Library Screening results to use reference
spectra found via
Spectra Library Screening for automatic peak
table creation.
In both cases, the values in the Retention Time and Window columns are
recalculated and default settings replace all other entries (see How
Autogenerating the Peak Table by
to ...: Actions in the QNT Editor
Including Library Screening Results).
The QNT Editor
125
Caution:
All previous entries are overwritten. Thus, they are lost when you save the
peak table or the QNT File!
For more information, refer to
How to ...: Actions in the QNT Editor
Creating a Peak Table
Peak Identification/Peak Tracking and MS Tracking
Peak identification refers to the following procedure: A previously unknown
peak is recognized due to peak-specific properties, such as the retention
time or its UV spectrum, and is then labeled with a characteristic substance
name.
Peaks are usually identified by their retention times. If you know the exact
retention time of a peak, enter the retention time and the Substance name
in the corresponding columns of the peak table. If a peak is detected at the
specified time, the name is automatically assigned (Fig. a).
To ensure peak identification even if there are retention time fluctuations or
neighboring peaks, use the ⇒Window peak table parameter to define a
tolerance range (fig. b). If a peak is detected within the tolerance range, it is
identified, even if the set (nominal) and the actual retention time do not
match exactly (fig. c). If several peaks are detected within this range,
Chromeleon identifies the "greatest," the "first," or the "nearest" peak,
depending on the selected extension for the window parameter (fig. d).
set
peak name
actual
window
Tret (f)
Tret (n)
Tret (g)
Tret
a)
b)
c)
d)
The retention time value previously entered in the peak table is corrected
accordingly!
126
The QNT Editor
Peaks can also be identified by the UV spectrum. You can use the
Spectrum alone, or the spectrum and the retention time (Spectrum and
time). This method requires a ¾Photodiode Array Detector, such as the
Dionex UVD 340U. For each peak, the substance spectrum is recorded and
compared to the library spectra, using certain criteria (see How to ...:
Actions in the QNT Editor
Peak Tracking). If the spectra match, the
peak is identified. This method is very reliable.
If you use a ¾Mass Spectrometer, peak identification can be based on
¾Mass Spectra. Use the MS Tracking tab page to select different options
Identifying Peaks via
(see How to ...: Actions in the QNT Editor
Their Mass Spectra (MS Tracking)). This method is also very reliable.
Calibration/Amount Table
During calibration, the peak area of a known standard amount is
determined. The result is used for calculating the amount of unknown
samples via the area to amount ratio.
Enter the amount contained in the different substances. In addition,
determine the calibration function and specify whether external and/or
internal standards will be used.
This is performed via the Amount, Standard, Cal.Type, and Integration
Type columns in the Amount Table. For a description of the columns and
for details on the required column input, refer to How to ...: Actions in the
QNT Editor
Creating a Peak Table.
Tip:
For background information about calibration, refer to Theory of
Calibration
Calibration (Overview). For information about how to
perform calibration, refer to: How to ...: Actions in the QNT Editor
Calibration.
The QNT Editor
127
Calibration Settings (Calibration)
In the simplest case of a chromatographic analysis, first one or several
standard samples and then the unknown samples are analyzed. All
samples of a sequence are evaluated based on the same standard
samples. If the quality of the column changes between processing the first
and the last sample, this will not be considered.
That is why Chromeleon allows sample evaluation via specific patterns or
based on any selected standard samples. The settings are determined via
the ⇒Calibration Mode on the General tab page of the QNT Editor.
The Calibration tab page indicates which standard samples are used for
calibrating the current sample. When you change from the current sample
to another sample by pressing the F4 or SHIFT+F4 keys, the list of the
displayed standard samples is updated.
If you notice that a specific standard sample falsifies your calibration
results, you can disable this sample in the Enabled column. The standard
sample is then excluded from the calculation. The corresponding calibration
points are then highlighted in the curve by a different color.
For more information, refer to How to ...: Actions in the QNT Editor
Disabling Calibration Samples.
Spectra Library Screening
To identify substances, spectra can be compared to library spectra. The
spectra search can be performed via the Spectra Library Screening sheet
of the QNT Editor. For more information about Spectra Library Screening,
Spectra Library
refer to How to ...: Actions in the QNT Editor
Screening.
The System Suitability Test
The aim and objective of the ¾System Suitability Test (SST) is to ensure
that the operational conditions required for a specific measurement are
achieved. Specify the conditions for the SST on the SST tab page in the
QNT Editor. For more information about System Suitability Testing, refer to
Defining the System
How to …: Actions in the QNT Editor
Suitability Test.
128
The QNT Editor
Mass Spectra (MS, MS Tracking)
If you have a Thermo Finnigan ¾Mass Spectrometer installed, you can
acquire mass spectra using Chromeleon. For more information about how
Actions
to install the spectrometer and acquire data, refer to How to ...:
Related to Mass Spectrometers.
In HPLC MS, mass spectra especially serve for peak identification. Using
mass spectra for identifying substances is via the MS Tracking tab page.
For more information, refer to How to ...: Actions in the QNT Editor
Identifying Peaks via Their Mass Spectra.
As MS chromatograms normally show increased noise, you have to
process ¾Mass Spectra before you can use them. On the MS tab page,
define the number of single peak spectra to be averaged. Also, define how
many background spectra shall be aggregated into a total background
spectrum that is then subtracted from the averaged peak spectrum.
For more information, refer to How to ...: Actions in the QNT Editor
Processing Mass Traces.
UV Spectra (UV))
On the UV tab page, you can process UV spectra before you use them.
Define the number of single peak spectra to be averaged. Also, define how
many background spectra shall be aggregated into a total background
spectrum that is then subtracted from the averaged peak spectrum.
For more information, refer to How to ...: Actions in the QNT Editor
Processing UV Channels.
Integration
129
Integration
Integration (Overview)
The chromatographic process of converting peak areas below peaks in
amount or concentration values is referred to as integration.
Amount and concentration values are calculated based on the calibration,
which supplies the calibration curve and the ¾Calibration Coefficient. (For
more information about calibration, refer to Theory of Calibration
Calibration (Overview).)
To calculate the amount concentration for a single peak area, the
calibration coefficients are inserted in the ¾Formula for Amount
Calculation. Chromeleon automatically performs this process for all peaks
found.
A
Calibration
A
Standard sample/~s
(known amount)
Determine
peak area
Integration
A
Analysis sample/~s
(Amount = ?)
Calibr. Curve /
Calibr. coeff.
Determine
peak area
Amount
Substance A
It depends on the
Quantification Method (QNT File) specified for each
sample before the analysis how many peaks are detected and whether at
least part of these peaks can be identified. The results of the area
calculation and peak identification processes are represented graphically in
the Integration window (chromatogram) and as a table (Report) on the
Integration plot. For more information, refer to:
The Integration Window
Reports Overview)
The Calibration Curve
130
Integration
The Integration Window
Chromatographic results are displayed in the Integration window. In
addition, you can use the window to reprocess single chromatograms.
Usually, the following items are displayed:
•
The chromatogram of the current sample
•
Various report tables
In addition, you may as well display:
•
The calibration curve of the current peak (see the image above)
•
The Trend Plot (refer to the Control section; similar functionality as
in the control panel)
•
UV spectrum (if available)
•
¾Mass Spectrum (if available)
Integration
Settings in the Integration window are saved together with the
Layout in the ⇒Report Definition File (RDF File).
131
Printer
For more information, refer to:
Opening a Sample
Operation
Manual Re-Integration
Chromatogram Comparison
Data Smoothing
Peak Ratio
The Spectra Plot
The Mass Spectra Plot
Opening a Sample
When you open a sample, this action automatically opens the integration
method window and displays the sample chromatogram. There are three
options:
•
Select a sample and select Open on the File menu, or
•
Double-click a sample of a specific sequence in the ¾Browser, or
•
Select a sample in the Browser, right-click to open the context menu,
select Open, and then select a channel of the sample.
Samples are usually opened after they have been completely processed.
However, you may open samples while they are being analyzed, that is,
while they are in the Running ⇒Status, provided that a certain amount of
data has already been acquired. Depending on the ⇒Step and other
detector settings, the sample must have run for several minutes. There are
two ways to open a running sample:
•
In the Browser, using the different options described above, or
•
On a ¾Control Panel, by selecting Integrate on the View menu.
132
Integration
Operation
Modifying the Appearance
You can change the appearance of each chromatogram by enabling and
disabling the display of various elements and by modifying them.
•
Double-click a peak to display the Peak Properties.
•
Draw a frame around the chromatogram section to be enlarged.
•
Select Decoration ... on the View menu to modify the window
background, the different axes, the layout of individual peaks as well as
the color, shape, and size of their captions.
•
Select Spectra Tool on the context menu (or click the corresponding
icon on the Integration toolbar) to display a spectrum from the
chromatogram at the time t. As soon as the spectrum symbol is added
to the mouse cursor, you can click any position to extract and display a
spectrum on the spectra plot if the corresponding data exists. A
minimized representation of the spectrum itself is displayed in the
chromatogram. Repeat the operation while pressing the SHIFT key to
overlay single spectra. Alternatively, select Overlay Spectra on the
context menu. Hide Time Spectra will remove the overlaid spectra.
Opening Additional Window Sections
Select one of the following commands to display more window sections:
•
Select Split Zoom on the View menu to split the window. By drawing a
frame, you can zoom a window section. The upper half of the window
shows the entire chromatogram and the frame, the lower half displays
the zoomed section. Use the mouse to move and position the frame in
the upper window section. The shape of the mouse cursor ( )
indicates that this mode is enabled. Of course, you can draw a new
frame at any time.
•
Select Report on the View menu to display the integration report (see
The Integration Report).
Reports
•
Select Show Spectra on the View menu to display the peak spectrum
The Spectra Plot).
of the currently selected peak (see
•
Compare two chromatograms by enabling the display of an additional
Chromatogram Comparison).
chromatogram (see
Integration
133
Manual Re-Integration
Chromeleon largely automates sample integration. However, in special
cases, the user may prefer to perform the changes manually. Chromeleon
allows you to move peak delimiters manually, to insert and delete peaks, or
to modify the baseline, etc.
Select the Automatic Tool on the context menu to perform these changes
directly in the chromatogram. The shape of the mouse pointer indicates
which operation can be performed.
Move left or right peak delimiter
Change baseline point (left/right/center)
Move baseline point (left/right/center)
Move
Move baseline segment
Move ⇒Detection Parameter
Insert peak
Zoom out an area
Display UV spectrum
Operation not possible
Select the individual commands such as Baseline Tool, Insert Peak Tool,
or Zoom Tool, if you only need a specific scope of functions.
For more information about the operations that can be performed, refer to
Manual Re-Integration.
How to ...: Actions in the Chromatogram
Tip:
If manual modifications are performed, display the integration report to see
the numerical results (see Reports
The Integration Report).
134
Integration
Chromatogram Comparison
The most exact method to compare two or more samples or
chromatograms is to compare their numerical results. However, in many
cases, it may be sufficient to overlay the chromatograms. This is referred to
as a chromatogram comparison.
What can be compared?
Chromatogram comparison always compares single channels. It is
irrelevant whether these channels are from the same sample or from
different samples. Theoretically, an unlimited number of chromatograms
can be displayed simultaneously in Chromeleon.
However, the presentation becomes confusing when many chromatograms
are displayed. That is why Chromeleon prompts the user to confirm that
more than 20 chromatograms shall be displayed. This is to prevent that too
many chromatograms are displayed due to an operator error, which may
result in a decrease of system performance.
How are chromatograms compared and displayed?
To compare chromatograms, display them in the Integration window. One
chromatogram is the active chromatogram. To make a chromatogram the
active chromatogram, select it with the mouse. Only for the active
chromatogram, additional information, such as the decoration, etc. is
displayed. The names of all chromatograms displayed are indicated above
the Integration window. The name of the active chromatogram is written in
a different color. Click the name of any chromatogram to select it as the
active chromatogram.
Select Decoration on the context menu to edit the appearance of the
window and the active chromatogram.
How do I select the chromatograms or channels to be compared?
•
Select one or several samples in the Browser and drag them into the
open Integration window. Chromeleon automatically attempts to load
the ¾Channel of the current sample. If this is not possible, for example,
because the channel does not exist, the system loads the default
channel. The default channel is the first channel that appears in the list
when you open a sample. (Select Open on the context menu in the
Browser.)
Integration
135
•
Select Add Overlay ... on the File menu to display a specific channel
of any sample in an opened integration window.
•
Select Open > All Channels to compare all channels of a single
sample.
•
Select Compare to compare a specific channel in several selected
samples.
•
Perform a query to specifically compare samples with certain
properties.
•
Simultaneously click the Next Chromatogram icon ( ) and press the
CTRL key to additionally display the chromatogram of the next sample.
•
Simultaneously click the Previous Chromatogram icon ( ) and
press the CTRL key to additionally display the chromatogram of the
previous sample.
•
Simultaneously click the Next Channel icon ( ) and press the CTRL
key to additionally display the next channel of the same sample.
•
Simultaneously click the Previous Channel icon ( ) and press the
CTRL key to additionally display the previous channel of the same
sample.
For more information about how to select chromatograms and/or channels
to be compared, refer to How to ...: Actions in the Chromatogram
Performing a Chromatogram Comparison.
Placing chromatograms and channels in relation to each other
External factors such as the flow rate, solvent, column quality, detector
amplification, etc. considerably influence the appearance of the
chromatogram.
As you cannot modify these conditions later, other ways must be used to
perform the comparison as exact as possible. This is achieved by adapting
the position and the size of a chromatogram to match another. Chromeleon
provides several options. You can:
•
Assign chromatograms an offset in x- and y-direction.
•
Normalize the chromatogram time, i.e.
chromatograms at a specific retention time.
you can overlay
the
136
Integration
•
Normalize the peak height, i.e., you can adjust the height of a specific
peak.
•
Stretch and compress chromatograms.
•
Subtract chromatograms from each other.
Select Decoration on the context menu of the active chromatogram and
enter the settings in the Comparison tab page.
For more information, refer to How to ...: Actions in the Chromatogram
Performing a Chromatogram Comparison.
Data Smoothing
Data smoothing applies a digital filter to sample data to reduce signal noise
and helps improve chromatogram appearance and reproducibility of peak
baselines. Data smoothing is performed in the integration window. (For MS
chromatograms, data smoothing is defined in the ¾PGM File or during
¾Mass Trace extraction.) After smoothing, the smoothed chromatogram is
displayed overlaid over the original chromatogram. The original sample
data file is not altered and the smoothed data file is stored separately.
Filter Types
The Savitzky-Golay filter smoothes to least-squares fit, using a weighting
function based on second-degree and third-degree polynomials. SavitzkyGolay smoothing is useful for reducing high-frequency noise of a data set
that is continuous (such as a chromatogram) without significantly degrading
the underlying signal.
The Moving Average (= Boxcar) filter is a simple algorithm that produces
a set of output values in which each output value is equal to the average of
n points centered around the corresponding input value, where n
represents the filter size. Because the Moving Average filter equally
weights each point, its ability to discriminate between noise and signal is
limited.
The Olympic filter is very similar to the Moving Average filter, except that
the maximum and minimum points of each input data set are rejected
before the average is calculated. This provides better rejection of impulse
noise (spikes) than the moving average filter.
Integration
137
In addition, the Gaussian filter is available for acquiring MS
chromatograms and extracting a mass trace (in the Mass Spectra window).
This filter applies the Gaussian distribution for chromatogram smoothing.
Filter Size
Filter size is the number of input data points used to generate each output
data point. The filter size is an odd number between 5 and 999. Use a
narrow filter size if desired peaks are narrow, and a wider filter size for
wider peaks. As a rule of thumb, select a filter size that approximately
equals the peak's half width. Note that too narrow a filter results in
insufficient smoothing while too wide a filter can lead to distorted data.
Tip:
Distortion of data during data smoothing mainly affects the peak height.
Therefore, it is generally better to evaluate smoothed chromatograms by
area rather than height.
Iterations
If a filter is applied several times, by far the highest smoothing result
(>95%) is achieved when the filter is applied the first time. Thus, normally a
single smoothing step is sufficient. However, applying a narrower filter
multiple times often provides improved noise reduction without the signal
degradation that can occur when using a wider filter size. This requires
additional processing time, however, so a wider filter size may be
preferable if its results are acceptable.
For additional details, refer to How to ...: Actions in the Chromatogram
Performing Data Smoothing.
For MS Chromatograms, refer to How to ...: Actions Related to Mass
Extracting Mass Traces afterward.
Spectrometers
Combination of Channels
Chromeleon allows you to combine two 2D channels using arithmetic
operations. Each data point is created by combining the associated two
data points from the existing channels (i.e., the data points at the
corresponding time), using the desired operation. The resulting channel is a
2D channel, too.
138
Integration
Note:
If there is a data point for channel A at a specific time but not for channel B,
the missing data point is calculated by linear interpolation. That is why the
resulting chromatogram may be shorter than the two original
chromatograms.
You can combine channels:
•
In the chromatogram: The resulting channel is calculated immediately.
•
As ¾Post-Acquisition-Step in the PGM Editor: The resulting channel is
calculated immediately after data acquisition is complete.
Arithmetic combinations are not restricted to chromatograms in the strict
sense. They can be used for all 2D channels, except for temporary
channels.
Peak Ratio
If the baseline-corrected signals of two channels (of the same sample!) are
related to each other, a rectangular curve results. This curve is referred to
as peak ratio. It is based on the observation that the ratio between two
detector signals must be constant, as according to the Lambert-Beer law
the detector signal (S) is always be proportional (c1) to the concentration
(K) of a dissolved substance.
S = c1xK
If the quotient q is formed of the two channels, the substance concentration
K is reduced. The quotient now only depends on the ratio of the two
wavelengths, and not of the time.
q=
S1 c1(λ1)
=
S 2 c2(λ 2)
If q is entered against the time, the (theoretical) result for each peak of the
sample is a horizontal line of the height c1(λ1)/c1(λ2).
The baseline and the ⇒Peak Purity Threshold parameter determine the
width of the rectangle. The ratio is only formed where both (!) peaks have a
baseline and where the intensity of both (!) peaks is above the defined
peak purity threshold. Thus, the range to be actually overlaid (= width of the
rectangle) is the intersection of the baseline and Peak Purity Threshold
condition. The default Peak Purity Threshold value is 10% of the peak
maximum. You can change this value in the QNT Editor.
Integration
139
The rectangle heights of two adjacent peaks differ if the corresponding
peaks have different spectra and if the two wavelengths are selected so
that the absorption quotient is significantly different.
Consider the following limitations and requirements:
•
The detector may not drift.
•
The correlation only applies to the linear range of the Lambert-Beer law
(<2000 mAU).
•
The solvent composition may not be altered (isocratic conditions).
•
The solvent only slightly contributes to the absorption. Baseline
correction allows you to eliminate the solvent absorption.
Note:
A peak ratio can only be formed with the signals of the same detector.
Forming the Peak Ratio
•
In the Integration window, overlay two channels of the same sample,
for example, by simultaneously clicking the Next/previous channel
icon while pressing the CTRL key.
•
Select Decoration on the context menu to open the Chromatogram
Decoration dialog.
•
Under Peak Decoration, select the Peak Ratio check box.
In addition to the two chromatograms, a rectangle curve should appear in a
different color should appear in any place in the Integration window where
two peaks are overlaid
140
Integration
Result
A regular rectangle shape, as shown at 12.70min in the illustration, can
serve as a criterion for evaluating the peak purity. The more the curve
deviates from the rectangle shape, the higher is the probability that the
overlaid peaks do not originate from the same substance. If the rectangle
slightly overshoots on the right or on the left, this is tolerable due to the
lower signal intensity at the peak start and the peak end.
Select the Peak Ratio Mean Value and RSD Peak Ratio report variables
to numerically express the result of the peak ratio.
The Spectra Plot
Overview
The Spectra Plot enables the display of UV spectra. The prerequisite for
the spectra plot is that the corresponding raw data is available. Raw data is
generated by recording a 3D field, using a ¾Photodiode Array Detector.
Open the spectra plot from the Integration method.
•
To open the spectra plot window, select Show Spectra on the View
menu or click the following icon:
.
The representation of a spectrum in the Spectra Plot is usually (height)
normalized: The height of the spectrum is represented in percent. Thus, it is
independent of the concentration (also, refer to ¾Normalization). As a
default, normalization is by the greatest relative maximum within the
spectrum.
Normalization allows you to objectively compare two spectra of different
concentrations. If spectra of the same peak, but with different peak heights
are overlaid, these will generally coincide despite the differences in
concentration.
•
Select Decorations on the View or context menu. On the Frame &
Axes tab page, determine the type of normalization that shall be
applied to the spectrum.
Normalized spectra representation allows you to perform the following
tasks:
•
Comparing two spectra such as a standard and a sample
•
Determining the number and position of minima and maxima
•
Selecting an appropriate type of normalization
Integration
141
•
Verifying the linearity of the Lambert-Beer law
•
Deciding: Baseline correction Yes/No
•
Determining exact integration limits by checking peak purity at various
wavelengths and peak heights
•
Identifying components
Displaying Spectra of one Peak
•
In the chromatogram plot, select the peak for which to display the peak
spectrum (= spectrum in the peak maximum).
•
If several peak heights were enabled on the Peak Spectra tab page in
the Decoration dialog box of the spectra plot, the spectra of different
peak heights are simultaneously displayed when you click the peak.
Displaying any Spectra of a Chromatogram
To extract any spectra of a chromatogram via mouse-click, follow the steps
below:
•
Select Tools on the context menu and enable Spectra Tool option or
click the corresponding icon on the Integration toolbar. A spectra
symbol that is added to the mouse points indicates that the mode has
been changed.
•
Click anywhere in the chromatogram to display the corresponding
spectrum.
•
Repeat the operation while pressing the Shift key to overlay several
spectra.
Displaying Spectra of Different Samples
To objectively compare spectra of different samples:
•
Select Decoration on the context menu of the spectrum to open the
Decoration dialog box and select the Peak Spectra tab page.
•
Select the Retention time spectrum of a fixed sample option and
click Browse... to navigate to the desired sample.
•
Alternatively, you may also use the retention time spectrum of the last
standard (= Retention time spectrum of recent standard), the
reference spectrum of the peak table (= Reference spectrum in
corresponding peak table), or any spectra that was found during
library screening (Spectra library screening result).
142
Integration
Match Factor, Difference Spectra, and 1st and 2nd Derivative of Spectra
As soon as two or more spectra are represented on the spectra plot, a
frequent question is the similarity between the various spectra.
The similarity is indicated by the Match Factor, the formation of difference
spectra or by representing the first or second derivative of a spectrum.
•
Select Decorations on the View or context menu of the spectrum, and
then select the Show match check box on the Label tab page.
Chromeleon returns a value for each represented spectrum specifying
the match degree relative to the main spectrum (0 = no match; 1000 =
perfect match).
•
On Analysis tab page, select whether the difference spectrum or the
first or second derivative of a spectrum shall be displayed in a second
window in addition to the actual spectra.
In the case of the match factor and the difference spectrum, the question
which spectrum is considered a main spectrum is especially important, as
this is the basis for the comparison and for all calculations.
The main spectrum is usually the peak spectrum extracted at the retention
time. If there is no peak spectrum, distinguish the following cases: If you
used the Spectra Tool to extract the single spectra from the chromatogram,
the spectrum that was extracted first is the main spectrum. If the spectra
were automatically extracted at different peak heights (see Displaying
Spectra of one Peak), the spectrum with the "oldest" retention time is
considered the main spectrum. When representing difference spectra, the
Difference to ... entry indicates the basis for calculation.
Comparing a Spectrum with Spectra of an Existing Spectra Library
To clearly identify a spectrum, compare it to a reference spectrum stored in
a ¾Spectra Library.
•
On the context menu, select Library Search to start the comparison.
The
Spectra Library window lists all library spectra with a minimum
similarity to the (normalized) spectrum. For more information about how
to perform library screening, refer to How to ...: Actions in UV Spectra
Starting a UV Spectra Search.
and/or Mass Spectra
If the spectra plot contains more than one starting spectrum, Chromeleon
always uses the spectrum displayed first and then compares it to library
spectra.
Integration
143
The Mass Spectra Plot
General
The Mass Spectra Plot enables the display of ¾Mass Spectra. The
prerequisite for opening the mass spectra plot is that the corresponding raw
data recorded by a ¾Mass Spectrometer are available. Open the mass
spectra from the Integration method.
•
To open the spectra plot, select Show Mass Spectra on the View
menu or click the following icon:
.
The representation of a mass spectrum is height normalized: The height of
the spectrum is represented in percent and thus independent of the
concentration (also, refer to ¾Normalization). As a default, this
normalization is by the ¾Base Peak of the spectrum.
Normalization allows you to objectively compare two spectra of different
concentrations. If mass spectra of the same peak, but from different peak
heights are overlaid, these will generally coincide despite the differences in
concentration. Due to their higher information density, mass spectra are
displayed in Chromeleon below each other, which is contrary to the display
of UV spectra that are overlaid.
Displaying Mass Spectra of one Peak
•
In the chromatogram plot, select the peak for which to display the mass
spectrum (= spectrum in the peak maximum).
•
If several peak heights were enabled on the Peak Spectra tab page in
the Decoration dialog box of the mass spectra plot, the mass spectra
from different peak heights are displayed simultaneously when you
click a peak.
Displaying Mass Spectra of Different Samples
To objectively compare mass spectra of different samples:
•
Select Decoration on the context menu of the spectrum to open the
Decoration dialog box and select the Peak Spectra tab page.
•
Select the Retention time spectrum of fixed sample option and click
Browse... to navigate to the desired sample.
144
•
Integration
Alternatively, you may also use the retention time spectrum of the last
standard (= Retention time spectrum of recent standard), the
reference spectrum of the peak table (= Reference spectrum in
corresponding peak table), or any spectra that was found during
library screening (Spectra library screening result).
Reports
145
Reports
Reports (Overview)
The report (or on-screen report, to distinguish it from the ¾Printer Layout
that defines the printout) includes several graphics and tables for displaying
all relevant sample data on the screen. The report is saved in the ⇒Report
Definition File, together with the printer layout.
To open the report tables, either select Show Report on the View menu or
click the following button:
. The report is usually displayed in the
Integration window. However, you can also have the report tables
displayed in the ⇒QNT Editor to directly check the effects of the changes
made in the QNT Method.
You can add, edit, or extend the individual report tables as required.
Chromeleon provides the following tables when you select Insert Report
on the Table menu:
Result Tables
Peak Results
These reports combine data of the current sample.
Calibration
Report
Displays all variables required for creating a calibration report.
Integration
Report
Displays all variables required for creating an integration report.
Sample Results
These reports combine data of the entire sequence.
Calibration
History
Displays all variables documenting the course of the calibration.
Peak Summary
Displays those variables for the entire sequence that are required for
creating a peak summary.
SST Summary
Report
Documents the results of the ¾System Suitability Test for the entire
sequence.
146
Reports
Audit Trails
Audit Trail
(Commands, SST,
...)
Displays the Audit Trail of the current sample. (Also, see Data
MS Instrument Info
Report
Displays information about the ¾Mass Spectrometer.
MS Status Log
Report
Displays the mass spectrometer settings.
MS Tune Data
Report
Displays the tune data of the ¾Xcalibur raw data file.
MS Instr. Method
Report (XRaw)
Indicates the MS method.
Management
Audit Trails).
Spectra Results
MS Raw Report
Displays the raw data of the current ¾Mass Spectrum.
Fraction Collection Results
Fraction Report
Displays all variables required to create a fraction report.
Tube Report
Displays the different variables describing the single fraction
collection tubes. This also includes the fraction report variables.
However, please note that these variables are not selected by
default.
QNT Tables
These reports combine data of the current sample.
Detection
Parameter
Displays all ⇒Detection Parameters of the current sample.
Peak Table
Displays all variables that are required for displaying the peak
table of the ⇒QNT Editor.
SST Table
Lists variables of the ¾System Suitability Test for the current
sample.
PGM Tables
Commands
Displays the ¾Program for the current sample.
Post Acq.
Processing
Displays the ¾Post-Acquisition Steps for the current sample.
Miscellaneous Tables
Database Query
Inserts any kind of database queries into the report.
History Report
Displays all variables required for creating a ¾History report.
Reports
147
Tip:
Select Print on the File menu to print chromatograms, spectra, report
tables, etc. at any time. The pages defined in the Printer Layout will be
printed. This also applies when you print from an on-screen report.
For more information about some of the reports mentioned above, refer to:
The Integration Report
The Calibration Report
The Peak Summary Report
The History Report
Special Report Tables
For information about how to create a report, refer to How to …:
Actions in the Report Table.
The Integration Report
The Integration Report (or more precise, the Integration Report Table) is
usually the first table of a report. For example, the defltdad ⇒Report
Definition File includes the following integration report tables:
•
Integration
•
Peak Purity
•
Peak Analysis
Contents
•
The integration report contains all relevant numeric data for the active
sample, such as the retention time, peak area and height, amount,
peak type, and other ⇒Peak Variables.
•
The individual columns contain variables of the ⇒Peak Results report
category. Nevertheless, you can add more columns from other
⇒Report Categories.
•
If the chromatogram is modified, for example, by manual re-integration
of a peak, this is reflected immediately by the changed report data.
148
Reports
•
Via the Windows clipboard, data of the report table can be integrated
directly in other Windows applications.
•
In the report, the values of different cells are added, subtracted,
multiplied, or divided in the same way as in MS Excel. Enable Layout
Mode on the Table menu.
Formatting
You can adapt the integration report to your requirements by selecting the
corresponding format commands on the context or Table menu. (The
format commands are only available after you have enabled Layout Mode
on the Table menu.)
For more information, refer to How to ...:
Actions in the Report Table.
The Calibration Report
The Calibration Report (or more precise, the Calibration Report Table) is
Integration Report:
very similar to the
•
Similar to the integration report, the calibration report displays data of
the current sample. However, in the calibration report table, this data
refers to the calibration of the current sample.
•
The individual columns contain variables of the ⇒Peak Calibration
report category. Nevertheless, you can add more columns from other
⇒Report Categories.
•
The calibration report table allows you to understand changes made
during the calibration (in the QNT Method).
For more information, refer to How to ...:
Actions in the Report Table.
The Peak Summary Report
Commonly used reports such as the
Integration Report and the
Calibration Report contain data of one sample only. In case of a
chromatogram comparison, only the data of the selected (active)
chromatogram is displayed.
Comparing peak data from different samples is possible via the Peak
Summary report table.
Reports
149
Sample Selection
•
If one sample is selected and opened from the Browser, the Peak
Summary is based on the corresponding ⇒Sequence.
•
If several samples are selected in the Browser, these selected
samples only will be part of the Peak Summary report table.
•
If a ¾Query is started in the Browser, the search result forms the basis
for the Summary. In this way, it is possible to compare peaks from
different sequences.
Peak Selection
•
Normally, no peak is selected in the chromatogram of the sample. The
Peak Summary Report therefore contains no entries. All fields are
marked n.a. (not available) or Div/0 (Division/0). Only when a peak is
selected manually within a chromatogram, the Peak Summary Table
receives entries. As soon as this is performed, this peak is searched in
all previously selected samples. The search result is displayed in the
Summary Report.
•
For each Peak Summary column (!), it is possible to define whether the
values of the currently selected (Selected Peak) or one specific peak
(Fixed Peak) are displayed. Access these options by selecting
Column Properties on the context menu.
•
If the currently selected peak is an identified peak, that is, if the peak
is labeled with its name in the peak table, all involved samples are
searched for this peak name. The corresponding values of all found
peaks are displayed in the Summary.
•
If the currently selected peak is a non-identified peak, that is, if the
peak is not labeled with its name in the peak table, a "fixed time
window" (± 5% of the detected retention time) is calculated for this
peak. All peaks within this time window will be included in the
summary. If two or more peaks are detected in the time window, the
peak that is nearest to the specified retention time will be selected. If
the peak is an identified peak, this will be indicated by Ambiguous? in
the Summary header.
150
Reports
Creating a Summary
•
Double-click one of the samples to open the Integration window.
•
Display the Report and select the default Summary worksheet
available Chromeleon. It contains a selection of the most important
default ⇒Peak Variables. For each sample that is part of the Summary,
a separate line is reserved in the report.
•
If the default Summary Report is not available, a new worksheet can be
defined. Proceed as described in: How to ...: Actions in the Report
Adding and/or Renaming a Worksheet.
Table
As in all reports, the currently selected sample in the chromatogram
window is highlighted in the Summary Table.
The History Report
The standard ⇒Report Definition Files include both, an
Integration
Calibration Report but no history report table. However,
Report and a
you can easily include the History Report table (see How to ...: Actions in
Adding and/or Renaming a Worksheet).
the Report
Contents
•
By default, the history report displays the ¾History of the current
sample. In order to display the histories of other objects, select Table
Properties on the context menu and make your selection on the
Objects tab page.
•
In addition, you can limit the displayed history as to time and/or certain
actions and/or users.
Formatting
•
You can sort the history report entries according to specific details,
such as Time, Name, Version...
•
The history mode includes a special layout mode. Enable Layout
Mode on the Layout tab page.
Reports
151
Special Report Tables
The standard ⇒Report Definition Files include both, an
Integration
Calibration Report but none of these special reports.
Report and a
However, you can easily include them (see How to ... Actions in the
Adding and/or Renaming a Worksheet).
Report Table
MS Reports
MS Instrument Info Report
Information about the ¾Mass Spectrometer.
MS Instrument Method Report
MS method.
MS Raw Report
Raw data of the current ¾Mass Spectrum.
MS Status Log Report
Mass spectrometer settings.
MS Tune Data Report
Tune data of the ¾Xcalibur raw data file.
Note:
If you do not have the MS Control option enabled on your PC, MS reports
will not be displayed in the Insert Report dialog.
Other Special Report Tables
Database Query
Results of a database query.
Detection Parameter Report
Indicates the⇒ Detection Parameters.
Program Report
¾Program of the current sample.
¾System Suitability Test.
SST Report
Note:
All MS report tables as well as the Database Query, Detection
Parameter, and Program report tables comprise only the default columns.
It is not possible to add more columns.
152
Reports
The Calibration Curve
153
The Calibration Curve
The Calibration Curve
The Calibration Curve method window allows you to evaluate the
calibration. For more information about the calibration, refer to Theory of
Calibration (Overview).
Calibration
•
Open the window from a different method, for example, from the
Integration window, by selecting Show Calibration Curve on the
View menu.
•
Or else, click the corresponding icon on the toolbar.
As described for the QNT Editor and Integration methods, various window
sections can be displayed. The window arrangement usually comprises the
Chromatogram, Calibration curve, and Report windows.
154
The Calibration Curve
•
Press the F4 key or the Shift+F4 keys to change to the chromatogram
of a different sample. Generally, all chromatograms of a sequence can
be thus displayed on after the other.
•
In the chromatogram, select the peak for which you want to display the
calibration curve. The peak will be indicated by a different background
color.
•
The Calibration tab page of the Report window shows the most
important calibration data, such as the ⇒Calibration Type, the number
of calibration points, the ¾Relative Standard Deviation (Rel.Std.Dev),
the ¾Correlation Coefficients as well as the calibration coefficients
¾Offset, ¾Slope, and ¾Curve (offset c0, slope c1, curve c2). In
addition, you can display, for example, the ¾Variance or the ¾Standard
Deviation (Std.Dev).
•
Select Decoration on the context menu to change the colors and the
captions of various window elements.
•
Select Column Properties or Table Properties on the context menu
to change the contents and the layout of the table. For more
Actions in the Report Table.
information, refer to How to ...:
For more information about the calibration curve, refer to How to …:
Actions in the Calibration Curve.
Note:
We would like to point out that the default use of the report variables in the
Integration, Calibration, and Peak Summary reports is not binding in
Chromeleon. You can freely configure each of the three reports, use any
variables, or rename the report as desired.
The Printer Layout
155
The Printer Layout
The Printer Layout (Overview)
The increasing number of samples makes printing analytical results a
complex and time-consuming procedure especially because results are not
only required on hardcopy but in all types of presentations.
Chromeleon considers this and emphasizes flexible report generation that
meets all your requirements. The Printer Layout is the appropriate tool for
this.
With each Chromeleon installation, different ¾Report Definition Files
(RDFs) are stored in the Dionex Templates > Reports directory of the
client PC.
•
Click the
icon on the Method toolbar to open the Printer Layout.
Note:
For more information about the printing options, refer to Basic Operation
Printing.
The ¾Printer Layout represents a type of folder for various worksheets.
Each sheet describes one or several printed pages (size of the
chromatogram, where to position it, columns included in the numerical
report, results represented in a chart, what is included in headers and
footers).
The Printer Layout is saved together with the (on-screen) report of the
Integration Window in the ⇒Report Definition File (RDF). With an
increasing number of worksheets in the Printer Layout of a Report
Definition File, the printing possibilities increase as well. For example, if you
want to print all samples of one sequence, it may be a good idea to present
the results of a calibration sample differently from the unknown samples,
and the lines of a ¾PGM File or summary report in yet another way.
Specify which sample or file type should be printed and the worksheet to be
used.
156
The Printer Layout
The Printer Layout of the default.rdf report definition file contains, for
example, the Integration, Calibration (Curr.Peak), Calibration (Batch),
Peak Analysis, Summary, and Audit Trail worksheets. Thus, it covers all
default requirements.
For more information, refer to:
Appearance and Function
Creating Your Own Templates and Worksheets
Printing
For information about how to create a printer layout, refer to How to ...:
Actions in the Printer Layout. Also, refer to
Specifying the
Printout for printing your results.
Appearance and Function
Important features of the Printer Layout are:
•
The appearance and the structure are similar to Microsoft Excel. The
functional scope is similar to Excel as well. To enter formulas and to
create diagrams, use the ¾Report Publisher, which is a Chromeleon
add-on product.
•
Toggle between the individual sheets of a Printer Layout by clicking the
tabs provided on the bottom of the window bottom.
•
Double-click the name of a tab to change its name, for example, to
change Integration to Integration Special.
•
Each sheet consists of a large number of columns (256) and lines
(16000).
•
Select Insert Row(s)/Column(s) or Delete Row(s)/Column(s) to
insert columns or to delete columns or lines on the worksheet.
•
Single areas or cells of a worksheet can be "filled" using the Windows
clipboard (Copy and Paste commands) or via the Insert ... command
on the context menu. (For more information, refer to The Printer
Creating Templates and Worksheets.)
Layout
The Printer Layout
157
• Chromeleon objects are marked by a red triangle. Chromatograms,
tables, calibration curves, etc., have a red triangle in all four corners. For
cells with single variables, the red triangle appears only in the upper
right corner of the cell.
Tip:
Note that you can place several report tables on a worksheet. The tables,
however, must be positioned one below the other, not next to each other!
The worksheets that are virtually unrestricted in horizontal and vertical
direction, allow creating page layouts larger that one printed page.
Depending on the selected page size on the printer, one or several pages
are required for printing this type of page layout. The worksheet is divided
in many invisible horizontally and/or vertically positioned print pages.
If the user, for example, inserts a chromatogram that does not fit on one
page, the rest is printed on a new page.
158
•
The Printer Layout
Select Page Setup on the File menu to specify the order ("from left to
right" or "top to bottom") in which to print the printed pages of a
worksheet.
Creating Templates and Worksheets
In addition to the default report templates provided in the Dionex
Templates > REPORTS directory, Chromeleon allows you to create your
own templates:
•
Click the
icon on the Method toolbar to open the Printer Layout
from your currently selected view. Chromeleon automatically opens the
report template of the current sequence. Select the Load Report
Definition command on the context menu to open a specific report
template.
Note:
If only the default report templates are available in your system, save it
under a new name via the Save Report Definition command. Afterward,
you can edit the duplicate as follows:
•
Enable Layout Mode on the Edit menu to perform manual changes on
the report template.
•
Select the worksheet you wish to edit, or insert an additional (empty)
worksheet via the Insert Sheet command (Edit menu).
•
Select Delete Sheet on the Edit menu to delete a superfluous
worksheet.
•
Select Insert ... on the context menu to insert chromatograms, tables,
diagrams, report variables, or other elements.
The Printer Layout
159
Caution:
Note that you can place several report tables on a worksheet. The tables,
however, must be positioned one below the other, not next to each other!
Each element inserted in this way reserves a specific area on the
worksheet. If a report template or a single worksheet serves as a print
template, these areas are "filled" with the values or graphics of the current
sample. It is also possible to determine whether the printed output includes
data and chromatograms of all channels of a sample or only of one
channel.
Note:
In addition to report variables that can be combined to form new functions
via the four basic arithmetical operations (plus powers), it is possible to
calculate additional functions known from Microsoft Excel. For a list of
supported functions, refer to ¾Additional Functions in the Glossary.
For more information about how to create worksheets and report templates,
Actions in the Printer Layout.
refer to How to ...:
Printing
From the Printer Layout
The Printer Layout allows you to print the created report template at any
time.
•
Select Print on the File menu to start printing.
•
Click Preview in the Print dialog box to preview the layout of the
printed output.
The printed output uses the data of the current sample with the layout of
the currently opened sheet.
160
The Printer Layout
From the Browser
You can print larger quantities of data, either single samples or even entire
sample series, from the Browser:
•
Use the mouse to select the samples to be printed. If you want to print
all samples of one or several sequences, select the associated
sequence(s).
•
To print samples from several sequences, perform a ¾Query
•
Afterward, select Batch Report ... on the context menu and select the
Report Definition to be used for the printed output. In addition, select
the channel to be printed and specify the printer.
In addition to the results, it is possible to print sample data, that is, the
information in the sample list.
•
Open a sample list and select Print Table ... on the context menu.
Peak Purity Analysis
161
Peak Purity Analysis
PPA: Peak Purity Analysis
Caution:
The basic requirement for using the PPA method is the availability of a
Dionex ¾Photodiode Array Detector. In this case, the data supplied by a
detector can be "read" and viewed on any client PC. The most common
method to relate chromatograms to spectra is the representation of data in
a ¾3D Field in the method PPA. The 3D field is the default view in the isopixel plot representation.
Use one of the following methods to display a sample (for which you have
the corresponding 3d raw data) in the method PPA:
•
Select the sample in the Browser and then select the Open > 3DFIELD
on the context menu, or
•
Change from a different method to the PPA method. Click the icon or
select PPA on the View menu.
View and Operation
In the default view, the method window is divided in four sections. The 3D
field window (A3) is the most important section. Select either Iso Pixel Plot
or 3D plot (see ¾3D Field Presentation Modes).
The cross-wires that are freely positioned via the left mouse button or the
arrow keys "extract" a chromatogram at a specific wavelength in horizontal
orientation from the plot representation. In vertical orientation, a spectrum is
extracted at the time t and is displayed above (A1) or next (A2) to the 3D
field (A3). Select the ¾Zoom function for a more detailed view.
162
Peak Purity Analysis
The display of the window sections A1 and A2 can be enabled or disabled
via the Show Chromatogram or Show Spectra options. Optionally, an
additional numeric report (PPA report) can be displayed (Show Report).
The line of the currently selected peak is highlighted by a different color.
In addition to the context menu commands that allow undoing a Zoom
operation, restoring the original 3D field, selecting a different 3D
representation, or displaying additional information such as the ¾PPI Index,
¾Match Factor, etc., there are additional operations that can be executed
via the keyboard or a mouse-click.
•
Double-click the A3 windows to open the 3D field Decorations ...
dialog box.
•
Right-click while the 3D field is redrawn to stop redrawing. This option
is useful when the wrong zoom area was chosen.
•
Double-click the time axis of the window to execute the Full Size
command. In the wavelength scaling, the same operation performs the
Autoscale command.
•
Press the CTRL key to make the currently displayed spectrum in the
spectra window "permanent," so that it is still displayed even when
moving the cross-wires. The spectrum extracted via the cross-wires is
displayed in addition. If the operation is performed several times, any
number of individual spectra can be displayed in the spectra window.
Peak Purity Analysis
163
•
It is also possible to combine pressing the CTRL key and moving the
cross-wires: Position the x-axis of the cross-wires on the required
wavelength and press the CTRL key. Then move the y-axis over a
peak in the chromatogram. The spectra window now shows all spectra
within the covered range. This procedure can be performed for various
peaks in a chromatogram. Release the CTRL key between the
individual peaks to represent each peak in a different color. For each
peak, a number of spectra can thus be displayed.
•
If the y-axis of the cross-wires is located on a peak, the peak spectrum
(= spectrum in the peak maximum) can be copied to the Windows
clipboard via the Copy command. From there, the spectrum can be
included in the spectra library. For this operation, approximately
positioning the y-axis is sufficient.
•
Select one of the Extract: ... commands to extract the active
chromatogram, the optimum integration path, the current spectrum or
the 3D field data and save the item under a separate name. For a
description of the required steps, refer to How to ...: Actions in the
Extracting Spectra, Chromatograms, 3D Field
PPA Window
Selecting the Optimum Integration Path.
Data and
Note:
Copying or printing a 3D plot is more time-consuming than copying or
printing the Iso plot!
•
Select Library Search on the context menu to search the spectra
library for the displayed spectrum. For more information, refer to How
Starting a UV
to ...: Actions in UV Spectra and/or Mass Spectra
Spectra Search.
Function
Use the PPA method
•
To analyze the peak purity (see How to ...: Actions in the PPA
Checking Peak Purity).
Window
•
To assign peaks interactively via the spectrum.
•
To extract chromatograms, spectra, and the optimum integration path
Selecting the
(see How to ...: Actions in the PPA Window
Extracting
Spectra,
Optimum
Integration
Path
and
Chromatograms, and 3D Field Data).
164
Peak Purity Analysis
•
To visualize chromatograms for presentation and archiving purposes
•
To evaluate baseline effects
•
To check the Lambert-Beer linearity range
•
To perform quantitative analysis of overlapping peaks
These methods are completed by various procedures and calculations for
result interpretation and/or comparison. These include:
¾Baseline Correction
¾Blank Run Subtraction
¾Normalization
¾PPI: Peak Purity Index
¾PPI: Match Factor
Spectra Libraries
165
Spectra Libraries
Spectra Libraries (Overview)
The partial method Spectra library enables the comparison of
¾Normalized and ¾baseline-corrected individual spectra with spectra from
various libraries. There are three sections:
Spectra Table
The upper section indicates all spectra contained in a ¾Spectra Library as
well as their data. Double-click a column header to sort the list according to
the criteria of the selected column, such as the name, ID, or number of
extremes. Select the first column of the table to select the corresponding
spectrum.
•
As soon as you move the mouse cursor over the first column, the
cursor changes to a horizontal arrow. Left-click to select a spectrum.
•
Press and hold the CTRL key to select several spectra.
166
Spectra Libraries
Spectra Plot
The spectra plot is shown in the lower left window section. It shows the
spectra of all substances selected in the spectra table.
•
Double-click the window or select Decoration... on the context menu to
change the spectra representation. For more information, refer to
The Spectra Plot.
Integration
Data Window
The lower right window section of the Spectra Library is reserved for
representing the data. It shows important data regarding the spectrum
currently selected in the spectra table.
Working with the Spectra Library
The objective of spectra administration is the identification of an unknown
substance based on its UV spectrum. If you have a spectrum of a
previously unidentified substance, for example, in the spectra window of the
PPA method, you can start searching various libraries from there.
Chromeleon compares the curve form of the two spectra (comparison),
calculates a similarity value (evaluation), and displays similar spectra (hit
criterion). The distinction made by Chromeleon depends on whether a
single spectrum is identified or all peaks of a sample.
Hit List
The hit list lists all library spectra that have a certain similarity to the
extracted (and normalized) spectrum. For more information, refer to How
to ...: Actions in UV Spectra and/or Mass Spectra
Starting a UV
Spectra Search.
Peak Tracking
Assigning each peak in a chromatogram the spectrum of a library that
matches the spectrum extracted at the peak maximum best is referred to as
peak tracking. For more information about Spectra Libraries, refer to:
Creating a New Library
Comparing Spectra
Spectra Libraries
167
Creating a New Library
Generating
Select the File > New > Spectra Library to open a new and empty spectra
library.
Filling
There are two options for filling a spectra library:
•
From the PPA: Peak Purity Analysis or Spectra Plot partial methods,
copy a spectrum to the Windows clipboard using the Copy command,
and then insert it in the open library using the Paste Spectra
command. (Also, see Data Reprocessing
PPA: Peak Purity
The Spectra Plot).
Analysis and/or Integration
•
Or else, generate a subset of an existing selection based on specific
¾Hit Criteria.
Note:
To be able to compare UV spectra with each other, the spectrum and the
reference spectrum should be recorded under identical conditions. The best
search results are therefore obtained based on spectra that were recorded
and saved by the user. It is highly recommended to write down the
conditions under which the spectrum is recorded. The notes will be very
useful when there are several spectra for the same sample.
Saving
Select Save as to save an open library under a different name to a
directory of your choice. Libraries have the file extension LIB.
Comparing Spectra
To compare two spectra with each other, the curve of the normalized
spectrum can be compared to those of individual library spectra. In some
cases, better results are achieved when the first or second derivations are
compared instead of the spectra themselves.
Via the comparison function (see ⇒Check Derivative), the user determines
the curve form to be used for the comparison.
168
Spectra Libraries
In the next step, the user determines how the individual curves are
compared with each other. This is by selecting one of three mathematical
methods (⇒Match Criterion).
The combination of comparison function and standard of comparison
results in a "similarity value" between 0 and 1000 that expresses the match
degree between the search spectrum and various library spectra. The
similarity value is known as ¾Match Factor. A perfect match has the value
1000.
Depending on the method (hit list or peak tracking), a similarity list is
displayed or each peak is assigned the most similar spectrum. Entering a
minimum similarity value (threshold) displays only the hits above the
threshold.
For more information about how to search spectra, refer to How to ...:
Starting a UV Spectra
Actions in UV Spectra and/or Mass Spectra
Search.
Result Presentation in a Hit List
Using the hit list, a similarity list sorted by the match factor and a
representation of the original spectrum and the library spectrum with the
best match are displayed. Entering a minimum similarity value excludes
dissimilar spectra.
Due to the frequently insufficient characteristics of UV spectra, it may be
necessary to further limit these hit lists. That is why ¾Hit Criteria are
available as additional filters. Only the spectra fulfilling the selected criteria
will be displayed.
How to …:
How to …:
How to ...:
169
170
How to …:
Actions in the Browser
171
Actions in the Browser
The Browser serves to manage, copy, move, and delete chromatographic
Moving and Copying
data and files. For more information, refer to
Elements. For general information about the Browser, refer to Data
The Browser.
Management
Caution:
Browser functions and structure are similar to the Windows Explorer.
However, do not confuse the Browser with the Windows Explorer! Do not
use the Windows Explorer for operations within Chromeleon datasources.
In the Browser, you can also create a sample list, set up datasources, or
search and save data in a datasource.
For information about which actions you can perform in the Browser, refer
to:
Creating a Sample List (Sequence)
Handling Files and Datasources
Signing Sequences Electronically
Performing a Query
System Wellness
Importing PeakNet Method Files (Release 4.5 Through 5.2)
Importing Agilent/HP ChemStation Data Files
For information about any actions that administrators can perform in the
Browser, refer to the Administrator Manual: How to …:
Actions in the Browser and in the Operating System
172
Actions in the Browser
Moving and Copying Elements
Use the Drag & Drop function to move or copy various elements in the
Browser; for example, directories, ¾Sequences, samples, and individual
files, such as ¾PGM Files, ¾QNT Methods, ¾Audit Trails, etc.
Drag & Drop via the left mouse button
To move or copy an element, hold down the left mouse button and move
the element to the target directory. To define the Drag & Drop behavior,
select Preferences on the File menu, and then select the desired option
under When dragging item between folders:
Ask
For each Drag & Drop action within a datasource, you are
prompted to click the desired button. Click Copy or click Move.
Copy
For simple Drag & Drop actions, i.e., when you do not hold down
any other key, the selected element is copied to the new position
without being deleted at the original position.
Move
(As in the
Windows Explorer)
There are two different cases:
Within a datasource: For simple Drag & Drop actions, the
selected element is moved to the new position and deleted from
the original position.
To a different datasource: The selected element is copied to the
target position, but is not deleted from the original location.
Independent of the selected setting, you can force the desired behavior for
each Drag & Drop action by pressing an additional key:
<CTRL>
Copies the element.
Shift key
Moves the element.
Tips:
If network failure occurs while a Move action is in progress, the elements
either in the target directory or in the original directory will be incomplete.
After rebooting your computer, check whether the elements were moved
correctly. If they were not, complete the action as required.
If you cancel a Move action, all data copied so far will be deleted. This may
take some time.
Sequences that are locked and signed can only be moved as a whole.
Locked directories cannot be moved at all.
Actions in the Browser
173
Drag & Drop via the right mouse button
Hold down the right mouse button and move an element to the target
directory. When you release the mouse button, the context menu is
opened:
Select the desired action (Copy, Move, or Cancel).
Copy elements via Copy & Paste
To copy the desired element, you can also select the Copy and Paste
commands on the Edit or context menu or click the corresponding icons on
the Standard toolbar. First, copy the element, and then paste it at the
desired location. Or else, use the corresponding shortcuts:
<CTRL> <C> = Copy
<CTRL> <V> = Paste
Move elements via Cut & Paste
To move the selected element, you can also select the Cut and Paste
commands on the Edit or context menu or click the corresponding icons on
the Standard toolbar. First, cut the element and then paste it at the desired
location. Or else, use the corresponding shortcuts:
<CTRL> <X> = Cut
<CTRL> <V> = Paste
Behavior if a file already exists in the target directory
If you move a file to a target directory in which a file with the same name
already exists, a dialog appears. You are prompted to confirm overwriting
the existing file. Click either Yes or Yes to all to confirm the action. Click
either No or No to all to cancel the move.
174
Actions in the Browser
Tips:
If the Move action is later cancelled by the user or terminated due to an
unforeseeable error, all data overwritten so far will be lost.
To overwrite a file, you need the corresponding Delete ¾Privilege. For
example, to overwrite a sequence, you need the DeleteSEQ privilege. The
administrator can assign you this privilege in the ¾User Manager (CmUser
program). A Delete privilege is not required if you move a file (the file is no
longer available at the original location after the move, i.e., it is "deleted"
there).
Creating a Sample List (Sequence)
There are two basic ways how to create a sample list. (For more
information about the sample list, refer to Samples and Sequences
The Sample List (Sequence)):
1.
Manually edit an existing sequence and save it under a different name.
2.
Automatically create a sequence using the Sequence Wizard. (For
The
information about the wizard, refer to Samples and Sequences
Sequence Wizard).
Manually creating a sample list
•
The Browser), select the
In the Browser (see Data Management
sequence whose QNT and ¾PGM Files you want to use for the new
sequence, probably after having edited them. Select Save as on the
File menu to save the sequence under a different name. Edit the new
sequence as necessary.
•
In the lower right Browser section, enter the names and the properties
of the standard samples and the unknown samples to be analyzed:
•
Click a cell in the table and enter the new value or the name via the
keyboard. To open a dialog box for assistance, press the F8 key.
•
In the ⇒Name column, enter a name for each sample to be analyzed.
•
In the ⇒Type column, select the sample type from the drop-down list
(Unknown, Blank, Validate, Standard, Spiked, or Unspiked).
•
In the ⇒Pos. (sample position) and ⇒Inj. Vol. (injection volume)
columns, type the autosampler position from which to inject and the
substance volume.
Actions in the Browser
175
•
In the ⇒Program and ⇒Method columns, determine
¾Chromatographic Methods to be used for the analysis.
•
In the ⇒Status (sample status) column, define how often a sample
shall be processed. Select Single to process the sample only once.
Select Multiple to process the sample several times.
•
To analyze two unknown samples (Sample 1 and 2) on the basis of a
2-point calibration, the input can be as follows:
•
It is not imperative that you fill the other columns, e.g., ⇒Weight
(Sample Weight Factor), ⇒Dil. Factor (dilution factor) and ⇒Inj.
Date/Time. Either they contain special parameters or the system fills
them automatically after the analysis.
•
If you are not sure which values or names to enter, select the cell and
press the F8 key. An edit dialog box appears listing the allowed values
and/or options. Enter the desired value or select the desired option.
Clicking OK automatically updates the cell.
•
Press the F1 key to display more information about the individual
columns.
•
Press the F9 key to fill all subsequent cells of a column with the same
input.
•
To enter special values, create ¾User-defined Columns (see
Creating User-defined Columns).
•
To take an overview of the most important results in the sample list
after the samples have been analyzed, add ¾Sequence Report
Creating a Sequence Report Column).
Columns (see
•
Save the sequence under a new name, selecting Save on the File
menu.
•
Check the sequence properties by pressing <Alt> + <Enter>. Or else,
select the sequence, and then select Properties... on the context
menu.
the
176
Actions in the Browser
Note:
Of course, it is also possible to edit an existing sequence. Exception: It is
not possible to edit a sample that is currently running (the corresponding
line in the sample list is highlighted in a different color).
Automatically creating a sample list
•
In the Browser, select New on the File menu. Select Sequence (using
Wizard) from the list.
•
Click OK to open the Sequence Wizard. (For information about the
The Sequence Wizard.)
wizard, refer to Samples and Sequences
•
Follow the instructions to create a basic sequence structure.
•
Press the F1 key for more help.
For more information about the appearance of the sample list, refer to
Displaying Sequence Columns.
Creating User-defined Columns
New columns can be created either in the sample list of the Browser (see
The Browser) or in the peak table of the QNT
Data Management
Editor:
1. a) In the sample list of the Browser: Click the datasource for the
sequence for which you want to create a new column. Select
Properties... on the context menu, and then click the User-defined
Columns tab page.
b) In the peak table of the QNT Editor: On the context menu or Edit
menu, select Columns, and then select Display User-defined
Columns.
2. Select <New user-defined column> from the Columns list to enable
the Properties section.
3. Type a caption for the new column in the Name input field.
Actions in the Browser
177
Note:
When you enter the name, keep the following restrictions in mind:
•
Use only characters, numbers, and the underscore. The first
character must be a letter.
•
Do not use the German "Umlaute" or "ß."
•
Names starting with SEQ_ or SMP_ are not permitted.
•
If the desired name is also an SQL keyword, we recommend
preceding it with an abbreviation of your company name (or some
other agreed-upon text). For example, if the new caption is
INTEGER, you might enter it as DX_INTEGER, where DX stands
for Dionex. This will prevent collisions with SQL keywords and
help you instantly recognize user-defined columns.
•
Several protected names, such as Retention, Wavelength, Flow,
etc., cannot be used as names for user-defined columns unless a
prefix or suffix is added. Since it would be very time-consuming to
check long lists of protected names before assigning a name, it is
recommended in this case, also, that you use a company
abbreviation as a prefix. For example, instead of "Flow," assign
the name 'DX_Flow' to a user-defined column.
•
Names of columns that have been deleted in the current ‘dialog
session’ cannot be entered again. You must close and then reopen the dialog box before re-entering these names.
4. Specify the Value Type for the new column by selecting the
corresponding option from the drop-down box:
•
Integer (whole numbers)
•
Floating point (numbers with a defined number of decimal places)
•
Date
•
Time
•
Date and Time
•
Enumeration (open the combo box to select an item from the list)
•
String (any sequence of characters. Please note: Spaces at the
end of the string are deleted.)
178
Actions in the Browser
5. Type the Dimension for the values in the new column in the
corresponding input field.
6. More specification fields may be displayed, depending on the selected
value type.
Note:
The operating system restricts date input to the period of 1/2/1970 12/30/2037.
7. a) When creating a user-defined column in the sample list of the
Browser, click Append Column to add the new column to the
Columns list. Click OK to append the new column to the sample list.
b) When creating a user-defined column in the peak table of the QNT
Editor, click Append Column to append the new column to the peak
table.
Any new column created in the sample list of the Browser applies to the
entire ¾Datasource; i.e., it becomes part of all ¾Sequences in the
datasource.
Tips:
Be careful when creating user-defined columns in the sample list of the
Browser. Be sure not to assign the same column name twice in different
datasources or on different computers that may communicate with each
other. Otherwise, unless the column definitions are identical, problems may
occur when you copy sequences or when you
Restore backup files.
When you have entered or changed user-defined columns in the sample
list, shut down and restart the ¾Server to use the new and/or changed
columns in the ¾PGM File.
There are some restrictions on the deletion of user-defined columns.
Chromeleon cannot delete user-defined columns unless they are empty. In
addition, Chromeleon cannot delete user-defined columns in some
database configurations, e.g., this is not allowed in Oracle, version 8.0, or
MS SQL Server, version 6.5. In this case, use the Oracle or MS SQL
Server database tools to delete the column. For the corresponding
database columns, refer to the SAMPLES database table. The column
header corresponds to the name of the user-defined column in Chromeleon
(without the preceding asterisk). Please note: Deleting a user-defined
column in Oracle or MS SQL invalidates ¾Electronic Signature for all
sequences containing this column.
Actions in the Browser
179
Note:
In a typical HPLC or IC configuration, it is possible to process at least 16
user-defined sample list columns in the program.
For two examples of how to use user-defined columns, refer to How to ...:
Actions in the Printer Layout
Calculating the Concentration
Percentage (in Relation to the Total Concentration).
New columns are added on the right-hand side of the table. However, you
can also change the order of the columns afterward. For more information,
refer to
Displaying Sequence Columns.
If you still cannot display all the values you need using user-defined
Creating a Sequence Report Column.
columns, also see
Creating a Sequence Report Column
It often makes sense to have some sample results displayed in the sample
list of a sequence. To do so, add sequence report columns to the sample
list, in addition to the default columns and the ¾User-defined Columns.
Thus, the sequence report columns can provide an overview of the most
important results of the single samples, already in the Browser.
Note:
You need the CustomizeColLayoutSEQ privilege to add sequence report
columns while the ¾User Mode is enabled.
To add a sequence report column:
•
Click the sequence, and then select Properties on the context or File
menu. The Properties dialog box appears.
•
On the Report Columns tab page, the Report Columns list contains
all existing sequence report columns:
180
•
Actions in the Browser
Click New... to open the Create Sequence Report Columns dialog
box.
Tip:
You can also open this dialog box from the sample list. Select either
Report Columns on the context menu or Sequence Report Columns on
the View menu, and then select New Report Column.
Actions in the Browser
181
•
First, select the desired report variable. All report variables of the
different ⇒Report Categories are available for selection. Click the '...'
button, and then select the desired variable from the Categories and
Variables lists.
•
Change the column name (Identifier) and the column header. (It is
usually not necessary to change the unit (Dimension)).
•
Having selected a peak-specific variable, define the peak to which it
shall refer. Select the desired option under Peak.
•
In the same way, if you have selected a channel-specific variable,
define the channel under Channel.
•
In addition, you can display the statistical values for the corresponding
column. Select the desired option: Sum, Average Value, or Relative
Standard Deviation.
182
Actions in the Browser
In this way, you can create different sequence report columns and thus,
display all required values in the sample list:
New columns are added on the right-hand side of the sample list. However,
you can change the order of the columns afterward or hide any columns
that are currently not required.
For more information, refer to
Displaying Sequence Columns.
Editing Sequence Report Columns
To edit report columns afterward:
•
Select the column by clicking the column header.
•
Select Report Columns on the context menu or select Sequence
Report Columns on the View menu. Then, select Modify Report
Column....
•
The Modify Sequence Report Columns dialog box appears. The
dialog box corresponds to the Create Sequence Report Columns
(see above). Make your settings as required.
Tip:
You can also open this dialog box from the sample list by double-clicking
the column header of the selected column.
Actions in the Browser
183
Note:
Or else, you can also select the sequence, and then select Properties on
the context menu. Click Modify... on the Report Columns tab page or
double-click the desired entry on the list to open the Modify Sequence
Report Columns dialog box.
Deleting a Sequence Report Column
To delete a sequence report column:
•
Select the sequence by clicking the column header.
•
Select Report Columns on the context menu or select Sequence
Report Columns on the View menu, and then select Delete Report
Column.
Tip:
You can also select the entire column by clicking the column header and
then pressing <Del>.
Note:
Or else, you can select the sequence and then select Properties on the
context menu. On the Report Columns tab, select the column from the
Report Columns list, and then click Delete. Alternatively, select the desired
entry and press <Del>.
Undoing Changes
There are different ways to undo changes made for a sequence report
column:
•
In the sample list: Press <Ctrl> + <Z> or select Undo... on the Edit
menu.
•
On the Report Columns tab page of the Properties of Sequence...
dialog box: Click Undo Changes.
If the sequence report columns are not sufficient to display all the values
you need, also see
Creating User-defined Columns.
184
Actions in the Browser
Displaying Sequence Columns
Chromeleon provides different options for displaying the single columns in
the sample list. Use these options to create the sample list according to
your requirements.
Displaying or Hiding Columns
Often, it is often not necessary to display all columns in the sample list.
Therefore, you can hide any columns that are currently not required:
•
In the sample list, select Display Columns... on the context or View
menu. In the Display Columns dialog box, select the columns you
want to display and determine the order in which they shall appear:
Under Visible Columns, all columns are listed that are visible in the
sample list. All columns that are currently hidden are listed under Hidden
Columns.
Actions in the Browser
185
A '#' character in front of the column name, e.g., #Area_Pyrene indicates
that the column is a report column. If the name of the report column, i.e., its
identifier, is different from the column header, the column header appears
in parenthesis and brackets after the column name, e.g., #nPeaks
("#Peaks"). An asterisk (*) in front of the column name, e.g., *Temperature,
indicates that this column is a ¾User-defined Column.
•
Under Visible Columns, select the columns you want to hide.
•
Click <<Remove<< to remove these columns from the Visible Columns
list.
In the same way, you can later add them again to the sample list. Select
them in the Hidden Columns list, and then click >>Add>> or >>Add to
End>>.
Note:
It is also possible to hide columns interactively. Left-click the right column
separator. While clicking, push the column together completely. To display
the column again, move the mouse cursor to the right of the column
separator until the cursor changes it appearance. Left-click and draw the
column separator to the right.
Changing the Order
You can also change the order in which the columns appear in the sample
list:
•
Select the column(s) to want to move.
•
Move the column(s) to the desired position via the Move Up and Move
Down buttons.
Note:
It is also possible to change the order interactively. Left-click and draw the
column to the desired position.
186
Actions in the Browser
Displaying Columns by Default
If the sample list includes many columns, not all of them can be displayed
on the screen at the same time. Use the scroll bar to move to the left or
right. However, you might want to see at least the most important columns
by default. Under Frozen Columns, define the number of leftmost columns
to be displayed even if the scroll bar is at the utmost right.
Also, refer to
Creating a Sample List (Sequence)
Creating User-Defined Columns
Creating a Sequence Report Column
Handling Files and Datasources
Chromeleon provides several ways to handle, store, and save the data
collected during operation:
Datasources on Removable Media
Opening Audit Trails
Creating Backup Files
Restoring Backup Files
Exporting Files
Datasources on Removable Media
¾Datasources can be installed not only on the hard disk of the local PC or
a network PC, but also on removable media, such as a disk drive, a ZIP
drive, or a CD-ROM.
•
Select Mount Datasource on the File menu. Chromeleon
automatically displays the Windows drive letters of all removable media
that are currently available.
•
Select a drive. Or else, select Browse... to navigate to the desired
datasource.
•
If Chromeleon finds an existing datasource on the selected media, the
system automatically connects to this datasource, displaying it in the
Browser in the same way as any other datasource.
Actions in the Browser
•
187
If Chromeleon does not find a datasource on the selected media, the
user can install a new one on this drive. The datasource will receive the
name of the drive.
Note:
You can easily copy datasources in Microsoft Access database format to a
removable medium. Copy the DATA directory of the local datasource or the
desired subdirectory of a network datasource to the corresponding
removable medium.
Caution:
If you have connected to a datasource on a removable medium via the
Mount Datasource command, do not remove the medium (diskette, Zip
disk, CD-ROM) until you have correctly closed the connection, by selecting
Dismount Datasource on the context menu. If you do not close the
connection in this way, the operating system will crash!
Opening Audit Trails
In the Browser, you can open two types of Audit Trails (see Data
The Audit Trail):
Management
Daily Audit Trails
Sample Audit Trails
In the ¾Standard Datasource, the daily audit trails are listed in the AUDIT
directory for the corresponding ¾Timebase. Double-click to open a daily
audit trail.
To display a sample audit trail, select the corresponding sample. Select
Open on the context menu, and then select Audit Trail. You can also
display sample audit trails in the Report. Double-click to open the
corresponding sample, and then go to the Audit Trail tab page.
188
Actions in the Browser
Creating Backup Files
Chromeleon provides a backup program for data storage and data
exchange. The program allows you to create backup files that include all
linked objects.
Before starting the ¾Backup, select the objects to archive in the ¾Browser.
All selection options in the Browser are supported.
After selecting the desired objects, select Export/Backup on the File
menu. You can back up the following objects:
Select a
To create a backup of
Directory
All subdirectories and the included files, sequences, etc.
Sequence
All samples and the files included in the sequence (QNT File and
¾Program File (PGM File)).
Sample
The sequence information (name, directory, title, etc.), the
selected samples, and the ¾PGM and ¾QNT Files used by these
samples.
Other file
The corresponding files.
To save samples found in a ¾Query, the following options are available:
Select a
To save
Sample
The sequence information (name, directory, title, etc.), the
(from the sample list in
selected samples, and the PGM and QNT Files used by these
the lower part of the right samples.
Browser pane)
Sequence
(from the sequence list
in the upper part of the
right Browser pane)
All samples contained in the query of the corresponding sequence.
Query
(in the left Browser
pane)
All samples contained in the query.
The following linked objects are copied for the backup:
Object
Link
Sequence
Preferred ¾Report Definition file (RDF)
Peak table
Calibration list for the 'fixed' calibration mode;
Blank run sample for 'fixed sample' blank run subtract mode;
Spectra library for Spectra Library Screening.
Query
Preferred report definition file (RDF)
Samples
QNT File
PGM File
If a link refers to an object included in the selected objects list, it is treated
as a normal object.
Actions in the Browser
189
Select Backup to open the Backup dialog box:
For a description of the individual fields, refer to Backup (in the online
Help).
Before starting the backup, Chromeleon determines the amount of storage
space required. For hard disks and network drivers, Chromeleon checks
whether sufficient storage capacity is available. If there is not enough
space, an error message appears and you can cancel the backup
procedure.
If you use removable media, Chromeleon displays the expected number of
media. If you use data compression, the number of actually required media
may differ from the displayed number.
You can monitor the procedure on the screen via a status bar that indicates
the percentage of stored data already copied to the medium:
190
Actions in the Browser
If the destination is a removable medium, you will be prompted when to
change the medium.
To perform a backup, you must have the PRIV_Backup privilege for all
selected objects (see ¾Privilege groups). The backup is logged in the file
history. To stop the backup, click Cancel. The backup then contains all
objects stored up to this point.
You can also delete the original objects after the backup has been
performed. A dialog box appears and prompts you to confirm this action. If
History Mode is enabled, a dialog box appears in which you can enter a
comment about the history.
Backup files have the extension .cmb. For more information about how to
restore backup files, refer to How to ...: Actions in the Browser
Restoring Backup Files.
Actions in the Browser
191
Restoring Backup Files
To restore backup files created via the ¾Backup command, select
Import/Restore on the File menu in the Browser, and then select
Creating
¾Restore (also, refer to How to … Actions in the Browser
Backup Files).
Tip:
You can also select the backup file by double-clicking the file name in the
Windows Explorer. Backup files are identified by their extension (*.cmb).
In the Restore dialog box, select the backup file you want to restore.
Chromeleon recognizes whether the file is stored on a removable medium,
a hard disk, or a network drive.
If the backup file is stored on removable media, you must insert the first
medium now because it includes the list of contents. Chromeleon displays
the list in the following dialog box:
192
Actions in the Browser
For a description of the individual fields, refer to Restore (in the online
Help).
The ¾Channels and ¾Linked Objects that are common to all samples are
listed in separate groups. The linked objects are displayed under the
corresponding objects.
Select the channels and the links to be restored from these groups. The
default is that all channels are restored, but that no links are restored.
The selected objects are restored, together with the raw data files of the
selected channels, the audit trail files, and the history files. A dialog box
similar to the Backup dialog box appears. If the backup is located on
several removable media, you will be prompted when to insert the next
medium.
If the directory for an object to be restored is missing, it is created by the
system. Links are always restored to the original location. The
corresponding sequence and datasource must exist. Missing directories will
be created, if necessary.
If the destination object already exists, you are prompted whether to
overwrite it. The following options are available: Yes, Yes to all, No, No to
all, and Cancel. For each restored object, the corresponding entry is
written to history.
To restore an object, you must have the PRIV_Restore privilege for the
destination directory. To overwrite an existing object, you must have both
the PRIV_Restore and PRIV_Copy privileges for the destination directory.
To stop restoration, click Cancel.
Tip:
If you have created a backup file that contains a new feature, please keep
in mind that you cannot read this file with a Chromeleon version that does
not support this feature. For example, this refers to:
• ¾Sequence Report Columns (available since Chromeleon 6.50)
• ⇒Std. Add. Group and ⇒Ref. Amount Set sample columns (available
since Chromeleon 6.60)
• The ¾Trend Plot (available since Chromeleon 6.50)
Actions in the Browser
193
Exporting and Transferring Data
Chromeleon provides various options for exporting data (see Data
Data Export):
Management
1. Data can be exported during or after a ¾Batch, using the Export Wizard
of the Batch Report.
2. Sequences can be transferred at a specified time, using the ¾Online
Transfer Agent (OTA).
For more information, refer to:
Exporting Data During or After a Batch
Transferring Sequences Automatically
Exporting Data During or After a Batch
Select the sample(s) or sequence(s) to be exported:
•
Select Batch Report on the File menu to open the Batch Report
dialog box.
•
To open the Export Wizard, select the Export check box in the Export
options section. (If the check box is already selected, click the Export
Settings… button.)
194
Actions in the Browser
•
Click the "..." button in the Location field to specify where data is
stored.
•
Use the Directory formula field to automatically create additional
folders under the existing location folders in the Windows Explorer. To
facilitate finding your files, we recommend using the same structure as
in the Browser. Use the default {seq.path}\{seq.name}.seq formula to
create the same structure.
If you do not know the syntax, click the {...} button to enter the formula.
For the {seq.path}\{seq.name} formula, select Sequence from the
Categories list, and then select Directory from the Variables list. Click
OK to confirm your selection. Enter a backslash and return to the
previous dialog box by clicking the {...} button again. Select Sequence
from the Categories list and Name from the Variables list. Confirm
your input by clicking OK.
•
Use the File name formula field to enter a formula for the file name.
Individual files are usually created for the corresponding samples.
Therefore, {smp.name}, i.e., the sample name, is an appropriate entry
if every sample has a specific name.
If you do not know the syntax, click the {...} button to enter the formula.
For the {smp.name} formula, select Sample from the Categories list
and Sample Name from the Variables list, and then click OK to
confirm your selection.
The default entry is {smp.number;04}. The number of the
corresponding sample is used for the file name. 04 indicates a four-digit
number; i.e., this entry creates file names such as 0001.txt or 0053.txt.
•
Under Export format(s), select the desired export format.
•
Click Next to go to the next wizard page. Select the desired export
options. Please note: The selected export option determines which
wizard pages appear. For example, for the ASCII format, if you want to
export raw data with the report, select the channel(s) first and then
select the sheets to be exported.
Tip:
Before exporting a Summary page, verify (in the Printer Layout) that a
peak was selected in the individual columns. If the setting is Selected
Peak, no data will be exported because you did not select a peak in the
Browser.
Actions in the Browser
•
195
Click Finish to complete the Export Wizard, and then click OK to start
exporting.
Exporting Data from Different Samples
For more information, refer to
Transferring Sequences Automatically.
to a Single File and
Exporting Data from Different Samples to a Single File
Use one page of the ¾Printer Layout containing a peak summary table
(which is usually the Summary page) to export data for a specified peak
from different samples of a sequence to just one file. First, define the
columns of interest:
•
Enable the ¾Layout Mode on the Edit menu.
•
Double-click the header of the column of interest to open the report
column properties. Select the desired peak, and then select Save
Report Definition… on the Workspace menu to save the Report
Definition File (RDF).
Tip:
If the setting is Selected Peak, it is not possible to export any data.
Although data is always exported from the Browser, it is not possible to
select the peak there.
•
Change to the Browser and follow the description in
Data During or After a Batch. Use the following settings:
Exporting
•
In the File name formula field, enter {seq.name}, i.e., the formula,
for the sequence name; this generates a single file for the
sequence without a superior directory.
•
Select ASCII, Excel, or PDF as the export format.
•
Select Summary or the corresponding page of the peak summary
table as the report page to be exported.
•
Click Finish to complete the Export Wizard, and then click OK to
start exporting.
For more information, refer to
Transferring Sequences Automatically.
196
Actions in the Browser
Transferring Sequences Automatically
Sequences found with a ¾Query can be transferred at specified times,
using the ¾Online Transfer Agent (OTA).
Tip:
When the ¾User Mode is enabled, you need administrator rights to be able
to transfer sequences automatically.
In the Browser, select Preferences on the File menu. The Preferences
dialog box is opened. The Online Transfer Agent tab page shows a Log
File displaying errors, warnings, and information, as well as a list of the
tasks to be executed.
•
Double-click the New Job entry at the bottom of the list to enter a new
job. The Scheduler Job Type dialog box is opened.
Actions in the Browser
•
197
Select Transfer sequences selected by a query to open the New
Scheduler Job dialog box. The dialog box contains two tabs:
• On the Time Plan tab page, define when, and how often, data will
be transferred.
• On the Transfer tab page, select the source and target of the
transfer job to be executed by the OTA.
For more information, refer to
Exporting Data During or After a Batch
Exporting Data from Different Samples to a Single File
Signing Sequences Electronically
Electronic signature is an important tool for securing data within the scope
of quality assurance and ¾GLP. (For general information about electronic
Electronic Signature.)
signature, see Samples and Sequences
In order to sign sequences electronically, the ¾User Mode must be enabled
and the logged-on user must have the corresponding signature
¾Privileges; for example, SignResults.
For more information, refer to the Administrator Manual: Chromeleon
User Management
Signature Privileges
Tip:
¾Electronic Signature is available only for user databases that were
created with a User Manager (CmUser) program version 6.10 or higher. If
an error message notifies you that electronic signature is not possible,
update your database.
You can sign a specific sequence only if you are authorized to do so. To
check and/or edit authorization, open the Properties dialog box of the
sequence from the context menu, and then click the Signature tab page:
198
Actions in the Browser
If you have been authorized in the CmUser database to modify the
signature requirements (ModifySignRequirements privilege), click Edit...
to edit the list of users authorized for each signature level.
If these conditions are met, ¾Sequences can be signed in three steps:
•
Submit
•
Review
•
Approve
The sequence's signature status determines which options are available on
the context menu:
•
Submit Results... (for unsigned sequences)
•
Review Results... (for submitted sequences)
•
Approve Results... (for reviewed sequences)
Actions in the Browser
199
You can also access these options by selecting Electronic Signature on
the File menu or by clicking the following icon on the standard ¾Toolbar:
The function of the button depends on the signature status.
For more information, refer to
Individual Steps of Electronic Signature
Checking the Signature Status and Undoing the Signature
Individual Steps of Electronic Signature
The first step of electronic signature is the Submit process. (For general
information about electronic signature, see Samples and Sequences
Electronic Signature.) Click a ¾Sequence that has not yet been
Signing
signed, and then select Submit Results as described in
Sequences Electronically.
Select the ¾Report Definition File (RDF) to be used for displaying the
sequence, select the channel for which you want to show the results, and
select the report sheets to be signed.
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Actions in the Browser
Tip:
The settings saved in the Report Definition File are used as defaults for the
worksheets to be signed and for the respective conditions. For more
information, refer to How to …: Actions in the Printer Layout
Specifying the Pages to be Printed.
After you have selected the report definition file, the system writes the
results to an ¾SOR-File (Signed Off Results). You can monitor the
procedure on the screen via a status bar that indicates the percentage of
results already copied to the file. Simultaneously, the pages of the report for
the individual samples are frozen. Afterward, the Check Signed Results
dialog box appears:
Actions in the Browser
201
This dialog box allows you to check the report to be signed. Use the arrow
keys to toggle between different sequence samples. For each sample, the
tab pages for the selected report sheets are displayed. Click OK when you
have finished checking the report. The Submit Signature dialog box
appears. To sign the SOR file, enter your User ID and the signature
password:
In the Comment field, you can enter additional information, such as any
critical or doubtful points you noticed while creating the report.
When a sequence is open, the SOR file is displayed under the other
sequence files in the top right Browser section. Double-click to open the
SOR file:
To review sequences that have been signed and submitted, select Review
Results. You can monitor the process on the screen. The files and
samples of the sequence that have already been reviewed are listed, as
well as the result of the review. For the file or sample under review, the
status bar indicates the percentage of completion:
202
Actions in the Browser
(Reviewing the results is much faster than submitting the signature. Thus,
the above dialog box may be visible only for a few seconds, especially with
short sequences.) To approve a sequence, proceed in the same way.
Checking the Signature Status and Undoing the
Signature
To check the signature status of a ¾Sequence, select the sequence and
then, select Properties on the context menu. The Properties dialog box is
opened. Click the Signature tab page for information about the users
authorized to sign, review, and/or approve the sequence.
(For general information about electronic signature, refer to Samples and
Sequences
Electronic Signature.)
Actions in the Browser
203
To check the sequence signature, select Electronic Signature > Verify on
the File menu or click the following icon on the standard ¾Toolbar:
Tip:
If you use the sample ⇒Types Spiked and/or Unspiked, please keep in
mind that
• An ¾Electronic Signature created with Chromeleon 6.50 or earlier is
invalid in Chromeleon 6.60 or higher.
• An electronic signature created with Chromeleon 6.60 or later is invalid
in Chromeleon 6.50 or earlier.
204
Actions in the Browser
Manipulations attempted on the signed sequence or errors in the signature
are identified during verification. To check the reports once again in such a
case, you have to remove the signature first. To remove the signature you
must have the UndoSignResults¾ Privilege. If you have this privilege, you
can select Electronic Signature > Undo Signature on the File menu or
click the following icon:
The corresponding ¾SOR-File (Signed Off Results) is deleted as well.
Performing a Query
The term ¾Query refers to the search for data based on specific search
criteria. In Chromeleon, you can search for samples and the corresponding
sequences based on freely selectable parameters. Either you can use very
detailed criteria to search for one specific sample, or you can use criteria
that are more general to search for a specific series of samples with the
same properties.
How To
•
In the ¾Browser, select Query on the context menu. Or else, select
New > Query (using the Wizard) on the File menu. Either way, a
¾Wizard guides you through the process of entering the required
conditions.
•
On the first Wizard page, determine the ¾Datasource in which the
query is performed. Also, determine whether the query is performed for
¾Sequence properties (Sequences) and/or sample properties
(Samples), and/or other conditions (Results).
•
Clicking Next> takes you to the next Wizard page(s). The pages that
are opened depend on the properties selected on the first Wizard page.
Determine the desired search criteria.
Click Finish to open the New Query Properties dialog box, which provides
four tab pages:
•
Use the General tab page to select the datasource in which the query
is performed.
Actions in the Browser
205
•
Use the Native SQL tab page to edit the SQL statements directly in the
¾SQL syntax of the corresponding ¾ODBC driver (contains translated
Chromeleon statements).
•
Use the SQL tab page to edit the search statement in an entry dialog
box.
•
Use the Result Restrictions tab page to limit the resulting sample list
by more result queries.
For more information, refer to:
Entering the Sample Query Using the Wizard
Selecting Search Criteria for Samples and/or Sequences
Examples (Wizard)
Specifying the Sample Query Using the Dialog Box
Examples (Dialog Box)
Editing a Query in SQL Syntax
Saving and Performing a Query
Entering the Sample Query Using the Wizard
On the first Wizard page, determine the ¾Datasource in which the ¾Query
is performed. Either select the Selected datasource option (the name of
the currently selected datasource is indicated in brackets) or select Fixed
datasource and then, select the desired datasource from the drop-down
list box.
In addition, determine the Field Type for which the query is performed.
Select:
•
¾Sequences and/or
•
Samples and/or
•
Results (variables from the different ⇒Report Categories)
On the Samples and/or Sequences Wizard page, click the Data Field
input field and select the variable for which you want to perform the query
from list. On the Results Wizard page, either enter the desired formula
manually or click the "…" button and then, select the desired formula from
the Edit Result Formula dialog box.
206
Actions in the Browser
Tip:
On the Results Wizard page, you cannot access ¾User-defined Columns
directly by clicking the '...' button. For user-defined columns from the
sample list of the Browser, select the Samples field type and then, select
the desired entry from the Data Field field on the Samples Wizard page.
For user-defined columns from the peak table of the QNT Editor, select the
Results field type, and then, enter the formula directly in the Formula field
on the Results Wizard page. The syntax of the formula is as follows:
peak_tab.user_x
where x is the name of the user-defined column.
You can also search for samples with specified ¾Audit Trail properties. In
this case, enter the formula for the desired Audit Trail variable. For
example, to search samples that were recorded at a ⇒LampIntensity >
500.000 counts/s, use the formula below:
AUDIT.LampIntensity(0.0,"forward")>500000.
Select an operator from the Operator field and enter the desired value in
Selecting
the Value field. For information about the operators, refer to
Search Criteria for Samples and/or Sequences.
To connect one search criterion with another search criterion, select the
desired logic connective from the rightmost combo box. The next entry line
is enabled only after you selected either AND or OR.
Click Finish to complete the entries. The New Query Properties dialog
box is opened. Use this dialog box to specify your query further (see
Specifying the Sample Query Using the Dialog Box). For examples
Examples (Wizard).
of how to enter the query using the Wizard, refer to
Selecting Search Criteria for Samples and/or Sequences
A ¾Query allows you to search for samples and sequences, using a variety
of sample or sequence properties. Enter the search criteria (see table) on
the Query Wizard: Sequences and/or Samples pages. To further restrict
the query, select the SQL tab page of the New Query Properties dialog
box. This dialog box is opened automatically after you have clicked Finish.
(You can also use this dialog box to edit an existing query.
Actions in the Browser
207
To reopen the dialog box (then called Properties of Query "xyz"), select
the query in the Browser, and then select Properties on the context menu.)
Click Edit/Insert to open the Edit Conditions dialog box. Select the
desired criteria from the Field list box.
Name
Search Parameter
Sample Name
⇒Name
Sample Number
⇒No.
Sample Type
⇒Type
Sample Status
⇒Status
Sample Comment
⇒Comment
Sample Replicate ID
⇒Replicate ID
Sample Inject Volume
⇒Inj. Vol.
Sample Inject Time
⇒Inj. Date/Time
Sample Dilution Factor
⇒Dil. Factor
Sample Weight
⇒Weight (Sample Weight Factor)
Sample Amount
⇒ISTD Amount
Sample Raw Data ID
Chromeleon sample ID.
Sample ID
Sample ID assigned by the user
Sample Program Name
⇒Program used for sample processing
Sample Method Name
⇒Method used for sample evaluation
*Sample x
¾User-defined Column (column name)
Sequence Name
Sequence Title
⇒Sequence
Sequence Preferred Channel
Sequence Preferred Report
Sequence Directory
Sequence Timebase
¾Timebase
Sequence Creation Date
Sequence Creation Operator
Sequence Last Update
Sequence Last Update Operator
Sequence Sign Status
Sequence Authorized Submit Users
Users authorized to submit the signed
sequence
Sequence Submit User
User who submitted the signed sequence
Sequence Submit Date
Date when the signed sequence was
submitted.
Sequence Authorized Review Users
Users authorized to review the signed
sequence.
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Actions in the Browser
Name
Search Parameter
Sequence Review User
User who reviewed the signed sequence.
Sequence Review Date
Date when the signed sequence was
reviewed.
Sequence Authorized Approved Users
Users authorized to approve the signed
sequence.
Sequence Approve User
User who approved the signed sequence
Sequence Approve Date
Date when the signed sequence was
approved
There are four different groups of search criteria:
1. Text variables, such as Sample or Sequence Name, Sample ID, etc.
2. Numerical variables, such as Sample Number, Sample Position, etc.
3. Variables with specific values, such as Sample Type or Sample Status,
etc.
4. Time variables, such as Sample Inject Time, Sequence Creation Date,
etc.
These search parameters must be further specified. Different operators are
available to link the search criterion with a comparative value. It depends
on the search criterion which operators are available:
Operator
Searches all samples...
Available for
group no.
=
that are equal to the specified character string.
1, 2, 3, 4
<>
that are not equal to the specified character string.
1, 2, 3, 4
>
that are larger than the specified character string.
1, 2, 4
<
that are smaller than the specified character string.
1, 2, 4
>=
that are larger than or equal to the specified character
string.
1, 2, 4
<=
that are smaller than or equal to the specified
character string.
1, 2, 4
contains:
that contain the specified character string.
1
contains not:
that do not contain the specified character string.
1
starts with:
that start with the specified character string.
1
does not start with:
that do not start with the specified character string.
1
ends with:
that end with the specified character string.
1
does not end with:
that do not end with the specified character string.
1
is like:
that fulfill the specified wildcard condition.
1
is not like:
that do not fulfill the specified wildcard condition.
1
Actions in the Browser
209
Operator
Searches all samples...
is between:
that are between two values.
1, 2, 4
is not between:
that are not between two values.
1, 2, 4
during the previous: that were created during a specified time before the
query.
Available for
group no.
4
is null
in which the variable does not exist
1, 2, 3, 4
is not null
in which the variable exists.
1, 2, 3, 4
If the operators >, <, >=, or <= are used for text variables, the alphabetical
order is considered; for example: A<B.
Note:
All SQL-time queries containing a relative reference (= during the
previous operator) are recalculated for each query.
Wildcards represent character strings. The following wildcards can be used
for text variables when the operator is either "is like" or "is (not) like":
Wildcard
Description
%
Represents any character string with 0 or more characters.
_(underscore)
Represents any single character.
[]
Represents a single character in a specified range; for example, [a-f].
[^]
Represents any character except the specified range.
When you have completed your entries, click Apply to start the query.
To restrict the query further, you can enter the desired restrictions on the
Result Restrictions page of the Query Wizard. Else, you can select the
Result Restrictions tab page of the New Query Properties (or
Properties of Query "xyz") dialog box. Click Edit/Insert to open the Edit
Conditions dialog box. Click the "…" button to open the Edit Result
Formula dialog box and select the desired report variable (see ⇒Report
Categories). The available logical operations depend on the selected
sample and sequence properties.
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Actions in the Browser
Note:
Depending on the number of samples resulting from the SQL query, a
result-type query may take some time. After approximately 3 seconds, a
window is opened indicating the status of the result-type query (in percent
of the samples to be tested). The user can use this window to stop the
query. In this case, the result of the query includes only those samples of
the SQL query that have passed the result test until then.
For examples of how to enter the query using the Wizard, refer to
Examples (Wizard).
Examples (Wizard)
Use the following SQL query to search for all samples for which
•
The sample type is ¾Matrix Blank and
•
The sample comment starts with Charge 123456 or
•
The sample inject time is between 6/12/2003 and 7/12/2003:
.
Actions in the Browser
211
Use the following settings to restrict the ¾Query to samples that contain
•
an Anthracene peak or
•
more than 10 calibration points:
.
For more examples, refer to
Examples (Dialog Box).
Specifying the Sample Query Using the Dialog Box
After you specified the ¾Query in the Query ¾Wizard and completed your
entries by clicking Finish, the New Query Properties dialog box is
opened. You can use this dialog box to specify the query further. You can
also use this dialog box to edit an existing query later. In this case, the
dialog box is named Properties of Query "xyz." To open the dialog box,
select the query and then, select Properties on the context or File menu.
The dialog box provides the four tab pages:
•
General tab page: Enter or edit the query title in the Title field. From
the Datasource drop-down list box, select the datasource for which the
query is performed. If this field remains empty (default), the query is
performed for the datasource in which the query is saved. The
212
Actions in the Browser
Preferred RDF File and Preferred Channel settings are used when a
sample is opened or a batch report is started.
•
Native SQL tab page: This tab page is read-only by default, providing
information about the SQL statement sent to the ODBC driver. (Please
note that the SQL statement is translated into the ¾SQL syntax of the
corresponding ODBC driver.) To edit the SQL statement manually,
select the Always use native SQL check box.
Caution:
This edited SQL statement is used whenever the query is performed,
as long as the Always use native SQL check box remains selected.
•
SQL tab page: Use this tab page to edit the SQL condition(s) that the
samples must fulfill. To open the Edit Condition dialog box, click Edit
> Insert. Or else, place the mouse cursor in the list field and doubleclick the left mouse button or select Edit Condition on the context
menu. Use this dialog box to add SQL conditions (select Restrict
Condition (AND) or Expand Condition (OR)) or to change the
existing condition (select Change Condition). .
•
Result Restrictions tab page: Use this tab page to specify the query
further. You can change, restrict, or expand the query. Open the Edit
Condition dialog box by clicking Edit/Insert and select Change
Condition, Restrict Condition (AND) or Expand Condition (OR).
Enter a report variable in the Formula field. Click the '...' button to open
the Edit Result Formula dialog box and select the desired formula.
(For more information about the report variables and their categories,
refer to ⇒Report Categories). Enter the desired operator. (For
information about which SQL operators are supported, refer to How
to ...: Actions in the Browser
Selecting Search Criteria for
Samples and/or Sequences.) Finally, type the desired reference value
in the Value field.
Tip:
It is not possible to access ¾User-defined Columns by clicking the '...'
button. Enter the formula for the user-defined column directly in the
Formula field:
Actions in the Browser
213
For user-defined columns from the sample list of the Browser, enter:
smp.x
where x is the name of the user-defined column.
For user-defined columns from the peak table of the QNT Editor, enter:
peak_tab.user_x
where x is the name of the user-defined column.
•
After you have made the necessary entries, click Apply to start the
search.
For examples of how to enter the query using the dialog box, refer to
Examples (Dialog Box).
Examples (Dialog Box)
To search for all samples of the current day, set up the following ¾SQL
query in the Edit Condition dialog box of the SQL tab page. (For
Specifying the
information about how to open this dialog box, refer to
Sample Query Using the Dialog Box.)
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Actions in the Browser
With these settings, the following SQL statement is displayed on the SQL
tab page:
Samples.smp_inject_time >= CURRENT_INTERVAL '0' DAY.
To restrict the above search to the standard samples of the current day,
reopen the Edit Condition dialog box and make the following settings:
The following SQL statements are listed on the SQL tab page:
Samples.smp_inject_time >= CURRENT_INTERVAL '0' DAY
AND Samples.smp_type = 'S'
To search for all samples in a ⇒Sequence named S7709, for which the
Benzene peak or a peak of the PAK (PAH) peak group was identified, use
the following SQL query:
Open the Edit Condition dialog box of the SQL tab page and enter the
condition for the sequence name:
Actions in the Browser
215
Change to the Result Restrictions tab page and click the Edit/Insert
button to open the Edit Condition dialog box. Enter the condition for the
Benzene peak:
Return to the Edit Condition dialog box and enter the condition for the
peak of the PAK (PAH) group:
With these settings, the following SQL statements are displayed:
On the SQL tab page:
Sequences.seq_name = 'S7709'
On the Result Restrictions tab page:
peak.name = 'Benzene'
OR peak.group = 'PAK'.
For examples about how to enter the query using the Wizard, refer to
Examples (Wizard).
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Actions in the Browser
Editing a Query in SQL Syntax
On the Native SQL tab page, the query created by Chromeleon is
displayed in SQL syntax. (For information about how to open these tab
Specifying the Sample Query Using the Dialog Box.)
pages, refer to
If you have a good knowledge of SQL, you can use the Native SQL tab
page to specify your ¾Query further. Select the Always use native SQL
check box and modify the statement via the keyboard.
Tip:
If you have edited the SQL statement on the Native SQL tab page, this
edited SQL statement is used whenever the query is performed, as long as
the Always use native SQL check box remains selected. When you clear
the check box, the current entries from the SQL and Result Restrictions
tab pages are used, overwriting your SQL entries on the Native SQL tab
page.
Saving and Performing a Query
Queries can be saved as a file in the datasource, similar to ⇒Sequences.
To create a query, select New on the File menu. In the New dialog box,
select Query (using Wizard) from the list box to open the ¾Query Wizard.
After you have entered all conditions for the query click Finish. The New
Query Properties dialog box is opened. Click Save to save the conditions.
The Save as dialog box is opened, indicating that the Object of type is
Query. Enter the name under which the query is saved.
In the Browser, the ¾Query is indicated by the following symbol:
.
If you select a query with the mouse, Chromeleon behaves in the same
way as for a sequence: If you select the query in the left ¾Browser pane,
the query is performed immediately and the results are displayed in the
right Browser pane. If you select the query in the right Browser pane, click
Return or double-click the file to perform the query and to display the
results.
To edit an existing query, select Properties... on the File or context menu
and edit the query as desired (see
Specifying the Sample Query
Using the Dialog Box).
Actions in the Browser
217
Answering Frequently Asked Questions
Question: What can I do when I can no longer copy data to the network
datasource?
Answer: The reason is probably a communication error. Verify that the
network connection still available. In rare cases, there might be a problem
with the datasource. In this case, first disconnect the datasource using the
Disconnect command in Chromeleon and then, reconnect the datasource.
For more information, refer to the Administrator Manual: How to …:
Actions in the Browser and in the Operating System:
Disconnecting a Datasource
Connecting a Datasource
Question: I appended a sample to a running sequence. How can I make
sure that the last sample is processed, too?
Answer: After appending the sample save the sequence by selecting Save
on the File menu. Only then, the sample is part of the sequence and can
be processed.
Question: Can I start a single sample from the Browser or the Control
Panel?
Answer: No, you cannot. You have to start a batch. However, the batch
may contain only one sequence with only one sample.
Question: Can I change the injection volume after the analysis is finished?
Answer: Yes, you can change the injection volume if you are authorized to
do so. (You must have the ModifyFinishedSample ¾Privilege. The
system administrator assigns this privilege in the ¾User Manager (CmUser
program).)
Tip:
Usually, you should not change the injection volume after the analysis. This
would falsify the analysis result when an autosampler injected the
sample(s). You should only change the injection volume after the analysis
when you entered the wrong volume in case of manual injection of the
sample.
218
Actions in the Browser
Question: Where are programs (PGM Files), QNT Methods, and Report
Definition Files (RDF) saved? Are they copied automatically to the
sequence?
Answer: There is no default location where these files are saved. When
creating the files you have to determine where they should be saved.
Programs and QNT Methods must also be saved to the sequence in which
you want to use them. When saving an old sequence under a new name,
using the Save as… command on the File menu, all files saved in the old
sequence are also saved in the new sequence. The ¾Status of the single
samples is set to Single, i.e., the raw data is not saved in the new
sequence.
Notes:
It may make sense to additionally save all programs to a Programs folder.
Save the QNT Methods and RDF's in the same way.
When saving a file to more than one location, make sure that the content of
the file is identical, in both locations.
Question: What happens with the files saved in a sequence when I copy
the sequence using the Drag&Drop command?
Answer: The Drag&Drop operation corresponds to the Save as…
command on the File menu. All files are copied together with the sequence.
Question: Is an Audit Trail recoded even if I lock the AUDIT directory?
Answer: Select the directory and then, select Properties on the context
menu. On the Access Control tab page, select the Locked check box. In
this way, the Audit Trail is still saved and a new Audit Trail will be created
the next day, too. However, it is no longer possible to change the files in
this directory or to copy files into this directory.
Question: How can I reduce the number of ¾Workspaces displayed on the
Workspace menu?
Answer: This operation is quite complex. Contact your system administrator
for assistance. (Close the Chromeleon client and edit the information in the
registry, using the RegEdit program. The path for accessing the information
is: HKEY_CURRENT_USER\Software\Dionex\Chromeleon\Recent
Workspaces.)
For tips to solve similar questions, also refer to How to …:
the Browser and the corresponding subtopics.
Actions in
Actions in the Browser
219
System Wellness
System Wellness provides built-in diagnostic and calibration features that
help prevent unscheduled system shutdowns and assure reliable operation
of system devices. System Wellness features are available for IC devices
that have a version of ¾Moduleware installed that supports System
Wellness. Wellness features are available for devices in the Summit HPLC
product line, also.
For an overview of System Wellness features and a list of supported
devices, refer to:
System Wellness for IC Devices (Overview)
System Wellness for HPLC Devices (Overview)
System Wellness tasks for IC devices are performed from the Browser. For
more information, refer to:
Opening a Wellness Control Panel
Viewing and Restoring Calibration Data
Uploading ICS-1500/2000 Calibration Data
Performing Device Calibrations
Performing Device Diagnostics
Entering Device Parameters
Also, refer to the Administrator Manual: How to ...: Actions in the
Server Configuration
Enabling and Disabling System Wellness
Functions for IC Devices.
Opening a Wellness Control Panel
¾Control Panels for performing System Wellness functions are provided for
various system configurations.
1. In the ¾Browser under the local ¾Datasource, open the Panels folder
and then, open the Wellness sub-folder.
2. Double-click the panel name that corresponds to your ¾Timebase
configuration.
220
Actions in the Browser
3. The control panel is opened and then, Chromeleon attempts to connect
to the timebase assigned to the panel.
4. If an error message appears stating that the timebase was not found,
close the message, and then, select Connect to Timebase on the
Control menu. Select the timebase to be connected to the panel.
5. After communication is established with the timebase, the various
calibration and diagnostic controls on the panel are enabled.
Viewing and Restoring Calibration Data
Calibration data for a System Wellness supported IC device is displayed on
the Wellness ¾Control Panel (see How to ...: Actions in the Browser
Opening a Wellness Control Panel).
Chromeleon stores three sets of calibration data for each System Wellness
supported IC device.
•
Current is the data most recently sent (uploaded) from the device. It is
the data currently stored in the device memory.
•
Previous is the data from the previous time calibration data was
uploaded from the device.
•
Factory is the data obtained (uploaded) from the device when it was
initially configured in the System Configuration program (see the
Administrator Manual: How to ...: Actions in the Server
Enabling and Disabling System Wellness
Configuration
Functions).
The Wellness control panel displays the current data and the date the
current calibration function was performed. If the factory value is the current
data, the date field displays "---."
•
To display previous data or factory data or download calibration data
from Chromeleon to the device, click Detail.
A Calibration Detail dialog box appears that displays the three sets of
calibration data values and their corresponding dates. The dates
indicate when the values were uploaded from the device to
Chromeleon.
•
To download calibration data to the device, select Current, Previous,
or Factory from the list to the right of the Download button. Then,
select Download.
Actions in the Browser
221
Uploading ICS-1500/2000 Calibration Data
After performing a calibration procedure or changing a calibration variable
from the ICS-1500 or ICS-2000 touch screen, use the procedure below to
update the System Wellness database with the new calibration data.
Note:
Do not open a System Wellness ¾Control Panel if the name includes
"Service.pan." These Wellness panels are reserved for use by Dionex
Service Representatives.
1. Open the System Wellness Control Panel for the ICS-1500/2000 (see
How to ...: Actions in the Browser
Opening a Wellness Control
Panel).
2. Locate the Update Wellness Database controls. If desired, click
Instructions for an overview of the procedure.
3. Click Upload to begin uploading (sending) calibration data to
Chromeleon.
Performing Device Calibrations
Wellness ¾Control Panels, which are supplied by Chromeleon, display
calibration data and provide script buttons for performing IC device
calibrations. After a calibration is performed, the device uploads the new
calibration data to Chromeleon.
Tip:
Many calibrations require setup steps before the actual calibration
command is given. Before selecting a calibration script button on a control
panel, refer to the topics below for details about the particular calibration
task you are performing.
Devices
Pumps, Detectors, Autosamplers
Pumps
Calibration Procedures
Leak Detector
Flow Rate
Pressure Transducer Offset
Degas
222
Actions in the Browser
Devices
Calibration Procedures
Detectors
Wavelength (AD25/PDA-100)
Conductivity Cell
(CD20/25/25A, ED40/50/50A, IC20/25/25A)
pH Reference Electrode
(ED40/50/50A Amperometry Mode)
AS50 Autosampler
Inject Port Volume
ICS-90 system
Pressure Transducer
Conductivity Cell
ICS-1000/1500/2000 systems
Flow Rate
Degas Pump
Conductivity Cell
Calibrating the Leak Detector
When to Calibrate:
After installing a new leak detector
If the leak detector diagnostic test fails
Every 6 months
Many System Wellness supported devices are equipped with leak
detectors. The calibration procedure for all of the sensors of IC devices is
the same.
1. Thoroughly dry the sensor.
2. Open the System Wellness ¾Control Panel for the device (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel.)
3. Under Calibration, leak detector, click the internal script button to
calibrate the sensor installed in the device itself. Click the external
script button to calibrate the sensor on a controlled device. For example,
for a pump, internal refers to the sensor in the pump and external refers
to the sensor in a chromatography oven controlled by the pump.
4. The device calibrates the sensor and uploads the new value to
Chromeleon. Chromeleon stores this new calibration value as the
current value.
Actions in the Browser
223
Calibrating the Pump Flow Rate
When to Calibrate:
Every 6 months
Items Needed:
Backpressure tubing to create 14 MPa ± 2 MPa (2000 psi ± 300 psi).
Use 0.076 mm (0.003 in) ID yellow PEEK tubing (P/N 049715)
Deionized water
Tared beaker
1. Verify that there is about 14 MPa (2000 psi) of backpressure.
2 Pump deionized water at 1.0 ml/min.
3. Allow the pump to stabilize for at least 5 minutes.
4. Collect water into a tared beaker for exactly 5 minutes.
5. Open the System Wellness ¾Control Panel for the pump (see How
to ...: Actions in the Browser
Opening a Wellness Control
Panel).
6. Enter the weight of the water into the weight entry field under flow rate
and press <Enter>. Chromeleon downloads the value to the pump and
stores this calibration value as the current value.
Calibrating the ICS-1000/1500/2000 Flow Rate
When to Calibrate:
Every 6 months
If the OQ/PQ flow rate accuracy and precision test fails
Items Needed:
Backpressure tubing to create 14 ± 1.4 MPa (2000 ± 200 psi); use
0.076-mm (0.003-in) ID yellow PEEK tubing (P/N 049715)
Deionized water
Tared beaker
Note:
Do not open a System Wellness ¾Control Panel if the name includes
"Service.pan." These Wellness panels are reserved for use by Dionex
Service Representatives.
224
1.
Actions in the Browser
Open the System Wellness Control Panel for the ICS-1000/1500/2000
Opening a Wellness
(see How to ...: Actions in the Browser
Control Panel).
Locate the Pump Flow Rate Calibration controls. If desired, click
Instructions for an overview of the calibration procedure.
Click Reset Cal to reset the flow rate calibration value.
Click 1.00 ml/min to set the pump flow rate.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Allow the Current Pump Pressure reading to stabilize at 14 ± 1.4
MPa (2000 ± 200 psi) for 20 minutes.
Click 5.00 min to start the timer and immediately start pumping
deionized water at 1.00 ml/min.
Collect water into a tared beaker for exactly 5 minutes.
Enter the weight of the deionized water (in grams) in the Enter new
Flow Rate Cal field.
Click Calibrate to begin the calibration. When the calibration is
complete, Chromeleon will download the new calibration value to the
ICS-1000/1500/2000 and will store it as the current calibration value.
Click Log to record the new calibration value in the Audit Trail.
Chromeleon will automatically update the Current Calibration Date
and Last Calibration Date fields.
Wait at least 15 minutes, and then recheck the flow rate to verify that
the calibration was successful.
Calibrating the Pressure Transducer Offset
When to Calibrate:
Every 6 months
1. Turn off the pump flow.
2. Open the waste valve.
•
For GP40/50, IP20/25 pumps: The waste valve is on the pressure
transducer. To open the valve, turn the knob about two turns
counterclockwise.
•
For GS50/IS25 pumps: The waste valve is on the secondary pump
head. To open the valve, turn the knob one-quarter to one-half turn
counterclockwise.
Actions in the Browser
225
3. Open the System Wellness ¾Control Panel for the pump (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel).
4. Click the offset script button under pressure transducer. The pump
calibrates the offset and uploads the new value to Chromeleon.
Chromeleon stores this new offset as the current value.
5. Close the waste valve.
Calibrating the ICS-90 Pressure Transducer
When to Calibrate:
Every 6 months
About 10 minutes before starting the calibration, toggle the injection valve
position a few times by clicking the valve Load and Inject buttons on the
ICS-90 Control Panel. This removes any air or contaminant buildup in the
injection valve loop.
1. Turn off the pump flow.
2. Open the waste valve on the front of the pump head by turning the knob
counterclockwise two turns.
3. Open the ICS-90 System Wellness ¾Control Panel (see How
to ...: Actions in the Browser
Opening a Wellness Control
Panel).
4. Click the Calibrate Offset script button under pressure transducer.
The pump calibrates the transducer and uploads the new value to
Chromeleon. The new offset is stored as the current value.
5. Close the waste valve.
6. Connect a pressure gauge between the pump outlet and the pressure
transducer. Turn on the pump and let the system stabilize. Note the
average pressure reading on the gauge, and enter this reading in the
measured field.
7. Click the Calibrate Slope script button under pressure transducer.
The pump calibrates the slope and uploads the new value to
Chromeleon. The new slope is stored as the current value.
8. Turn off the pump.
9. Disconnect the pressure gauge. Reconnect the pressure transducer to
the pump.
226
Actions in the Browser
Degas Calibration
When to Calibrate:
Every 6 months
1. Open the System Wellness ¾Control Panel for the pump (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel).
2 Select the degas script button. The pump performs the degas
calibration and uploads the new calibration value to Chromeleon.
Chromeleon stores this new value as the current degas value.
Calibrating the ICS-1000/1500/2000 Degas Pump
When to Calibrate:
Every 6 months
Note:
Do not open a System Wellness ¾Control Panel if the name includes
"Service.pan." These Wellness panels are reserved for use by Dionex
Service Representatives.
1. Open the System Wellness Control Panel for the ICS-1000/1500/2000
(see How to ...: Actions in the Browser
Opening a Wellness
Control Panel).
2. Locate the Degas Calibration controls. If desired, click Instructions for
an overview of the calibration procedure.
3. Click Calibrate to begin the calibration. When the calibration is
complete, the ICS-1000/1500/2000 will upload the new calibration value
to Chromeleon. Chromeleon will store the new calibration value as the
current calibration value.
4. Click Log to record the new calibration value and degas pressure in the
Audit Trail. Chromeleon will automatically update the Current Cal. Date
and Last Calibration Date fields.
Actions in the Browser
227
Wavelength Calibration
Use this manual wavelength calibration procedure for the Dionex AD25 and
PDA-100 detectors.
When the AD25 power is turned on, the wavelength is automatically
calibrated. The PDA-100 wavelength is automatically checked under
certain conditions, but is not automatically calibrated.
Manually calibrate the wavelength at the following time:
When to Calibrate:
After a failed wavelength calibration check
After a failed wavelength calibration
1. Verify that there is solvent flowing through the cell, the background
absorbance is low, and there are no bubbles in the light path.
2. Open the System Wellness ¾Control Panel for the detector (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel).
3. Click the wavelength script button under Calibration. The detector
performs the wavelength calibration routine and uploads the results
(Pass or Fail) to Chromeleon.
If wavelength calibration fails, refer to the troubleshooting section of the
detector operator's manual.
Calibrating the Conductivity Cell
(CD20/25/25A, ED40/50/50A, IC20/25/25A)
When to Calibrate:
After installing a new cell (use Method A)
Every 6 months (use Method B)
Items Needed
(Method B Only):
1.0 mM KCl solution: Prepare by dissolving 0.07456 g of reagent grade
KCl in one liter of 18 megohm deionized water
Backpressure tubing to provide at least 7 MPa (1000 psi). Use
0.076 mm (0.003 in) ID yellow PEEK tubing (P/N 049715)
Method A: For Calibrating New or Replacement Cells
1. Open the System Wellness ¾Control Panel for the detector (see How
to ...: Actions in the Browser
Opening a Wellness Control
Panel).
228
Actions in the Browser
2. In the conductivity cell calibration entry field, type the cell calibration
constant (written on a tag on the conductivity cell's cable) and press
<Enter>. Chromeleon downloads the value to the detector and stores
the value as the current cell calibration constant.
Method B: For Calibrating After Every 6 Months of Use
1.
Disconnect the pump output line from the injection valve.
2.
Connect the pump output line directly to the inlet of the DS3 or cell.
3.
Verify that there is a minimum of 7 MPa (1000 psi) of backpressure.
4.
Pump 1.0 mM KCl through the cell at 1.0 ml/min.
5.
If using a DS3, set the DS3 temperature to the intended operating
point and allow it to reach this temperature.
6.
Allow the conductivity to stabilize for about 5 minutes.
7.
Open the System Wellness control panel for the detector (see How
to ...: Actions in the Browser
Opening a Wellness Control
Panel).
8.
Click the calibrate script button under conductivity cell. The detector
calibrates the cell and uploads a new cell calibration constant to
Chromeleon. Chromeleon stores this value as the current cell
calibration constant.
After calibration, the conductivity reading should be 147.00 ± 2 µS/cm
and the cell calibration constant should be between 130 and 190. If
this is not the case, refer to the troubleshooting section of your
detector operator's manual.
9.
Flush the KCl solution from the system by pumping deionized water
through the cell. When the conductivity drops to near zero, stop the
pump.
10. Reconnect the pump to the injection valve and reconnect the line from
the suppressor to the cell inlet.
Actions in the Browser
229
Calibrating the ICS-90 Conductivity Cell
When to Calibrate:
After installing a new cell
Every 6 months
Items Needed:
1.0 mM KCl solution: Prepare by dissolving 0.07456 g of reagent-grade
KCl in one liter of 18 megohm deionized water
Backpressure tubing to provide at least 7 MPa (1000 psi). Use
0.076 mm (0.003 in) ID yellow PEEK tubing (P/N 049715)
1.
Open the ICS-90 System Wellness ¾Control Panel (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel).
2.
Use an Allen wrench to remove the two screws securing the DS5
Detection Stabilizer (which holds the cell) to the ICS-90 component
mounting panel.
3.
Pull the DS5 straight out from the component mounting panel to
unplug the cell from its electronics. Let the DS5 hang by the tubing.
4.
Allow the conductivity to stabilize for 5 to 10 minutes.
5.
Click the Calibrate Offset script button under conductivity cell.
6.
Line up the 9-pin connectors on the DS5 and the component mounting
panel and plug the DS5 back into the electronics. Replace the screws
and tighten.
7.
Disconnect the pump outlet line from port P (2) on the injection valve;
connect the line directly to the cell inlet using the yellow PEEK
backpressure tubing.
8.
Fill an eluent bottle with the 1.0 mM KCl solution and connect it to the
eluent out line. Prime the pump and then turn on the pump.
9.
Verify that there is a minimum of 7 MPa (1000 psi) of backpressure.
10. Allow the conductivity to stabilize for 5 to 10 minutes.
11. Click the Calibrate Slope script button under conductivity cell. The
cell is calibrated and a new cell calibration constant is uploaded to
Chromeleon. The new value is stored as the current cell calibration
constant.
12. After calibration, the conductivity reading should be 147.00 ± 2 µS/cm.
If this is not the case, call Dionex for assistance.
230
Actions in the Browser
13. Flush the KCl solution from the system by pumping DI water through
the cell. When the conductivity drops to less than 1 µS/cm, stop the
pump flow.
14. Reconnect the pump to the injection valve and reconnect the line from
the suppressor to the cell inlet.
Calibrating the ICS-1000/1500/2000 Conductivity Cell
When to Calibrate:
(Optional) After installing a new cell (use Method A)
Every 6 months (use Method B)
Items Needed
(Method B Only):
1.0 mM KCl solution: Prepare by dissolving 0.07456 g of
reagent-grade KCl in one liter of 18 megohm deionized water.
Backpressure tubing to provide at least 7 MPa (1000 psi); use
0.076-mm (0.003-in) ID yellow PEEK tubing (P/N 049715).
Note:
Do not open a System Wellness ¾Control Panel if the name includes
"Service.pan." These Wellness panels are reserved for use by Dionex
Service Representatives.
Method A: For Calibrating New or Replacement Cells (Optional)
1. Open the System Wellness Control Panel for the ICS-1000/1500/2000
(see How to ...: Actions in the Browser
Opening a Wellness
Control Panel).
2. Locate the System Status controls.
3. Click the Details… button to open the Calibration Details dialog box.
4. Locate the Conductivity Cell Cal Details controls.
5. Enter the cell calibration constant (written on the front of the cell) in the
Conductivity Cell Constant field and press <Enter>. Chromeleon will
download the new value to the ICS-1000/1500/2000 and will store it as
the current cell calibration constant.
6. Click Close to exit the dialog box.
Actions in the Browser
231
Method B: For Calibrating After Every 6 Months of Use
1.
2.
3.
4.
5.
6.
7.
8.
9.
Open the System Wellness Control Panel for the ICS-1000/1500/2000
(see How to ...: Actions in the Browser
Opening a Wellness
Control Panel).
Locate the Electric Conductivity Cell Calibration controls. If desired,
click Instructions for an overview of the calibration procedure.
Click Offset Cal to begin the offset calibration.
When the offset calibration is complete, click Slope Cal to begin the
slope calibration. When the slope calibration is complete, Chromeleon
will retrieve the new calibration value from the ICS-1000/1500/2000
and will store it as the current calibration value. Chromeleon will
automatically update the Current Calibration Date and Last
Calibration Date fields.
Disconnect the pump output line from the injection valve.
Connect the pump output line directly to the inlet of the conductivity
cell.
Verify that there is a minimum of 7 MPa (1000 psi) of backpressure.
Locate the Conductivity Cell Calibration controls. If desired, click
Instructions for an overview of the calibration procedure.
Click Cell 35 C to select the cell heater temperature. Monitor the cell
during the warm-up period:
• If you have an ICS-1000, monitor the cell temperature on a
standard ICS-1000 control panel. When the temperature reaches
35 °C, wait an additional 5 minutes and then go on to Step 11.
• If you have an ICS-1500 or ICS-2000, monitor the cell
temperature on the HOME page on the instrument front panel.
When the "=" symbol is displayed next to the Cell Heater control,
go on to Step 11.
10. Click 1.00 ml/min to set the pump flow rate. Begin pumping 1.0 mM
KCl through the cell.
11. Wait until the Total Conductivity reading stabilizes (in approximately
15 minutes), and then click Calibrate. When the calibration procedure
is complete, Chromeleon will retrieve the new calibration value from
the ICS-1000/1500/2000 and store it as the current calibration value.
After calibration, the conductivity reading should be 147.00 ± 2 µS/cm
and the cell calibration constant should be between 130 and 190. If
this is not the case, refer to the troubleshooting section of your ICS1000/1500/2000 operator's manual.
232
Actions in the Browser
12. Click Log to record the new calibration value in the Audit Trail.
Chromeleon will automatically update the Current Calibration Date
and Last Calibration Date fields.
13. Flush the KCl solution from the system by pumping deionized water
through the cell. When the conductivity drops to less than 1 µS/cm,
stop the pump flow.
14. Reconnect the pump to the injection valve and reconnect the line from
the suppressor to the cell inlet.
Calibrating the pH Reference Electrode
When to Calibrate:
After installation of a new reference electrode
Items Needed:
pH 7 buffer
A second buffer of known pH (usually a calibration buffer that most
closely matches the pH of the eluent used in your application)
1.
Carefully remove the combination pH/Ag-Ag/Cl reference electrode
from the amperometry cell, making sure to leave the electrode leads
connected to the cell.
2.
Place the electrode into a pH 7 buffer.
3.
Wait for the pH reading to stabilize (about 1 minute).
4.
Open the System Wellness ¾Control Panel for the detector (see How
to ...: Actions in the Browser
Opening a Wellness Control
Panel).
5.
Click the pH 7 script button under pH electrode.
6.
Remove the electrode from the pH buffer, rinse, and then dry it.
7.
Place the electrode in the second buffer.
8.
Wait for the pH reading to stabilize.
9.
Enter the pH of the second buffer into the edit field above the 2nd
buffer script button.
10. Click the 2nd buffer script button.
Actions in the Browser
233
Entering the AS50 Inject Port Volume
The inject port volume is the volume of tubing between the AS50 inject port
and the injection valve. The AS50 uses this information to determine how
much fluid to push through the line in order to position the sample correctly
in the loop for precision injections.
When to enter a
new inject port
volume:
After recalibrating the inject port volume on a new or existing
needle seal assembly.
For detailed instructions on replacing a needle seal assembly or calibrating
the inject port, refer to the AS50 operator's manual.
1. Open the System Wellness ¾Control Panel for the autosampler (see
How to ...: Actions in the Browser
Opening a Wellness Control
Panel).
2. In the Inject Port Volume field, enter the volume determined during
recalibration of the inject port volume on a new or existing needle seal
assembly.
3. Press <Enter>. Chromeleon downloads the value to the autosampler
and stores this calibration value as the current value.
Performing Device Diagnostics
Wellness control panels, which are supplied with Chromeleon, display
diagnostic test results and provide script buttons for performing the tests.
The following tests are available - see How to ...: Actions in the Browser:
Leak Detector (for any IC device equipped with a leak detector)
Wavelength Verification (for AD25 and PDA-100 detectors)
Pressure and Conductivity Diagnostics
(for ICS-90 Ion Chromatography System)
Conductivity Diagnostics
(for ICS-1000/1500/2000 Ion Chromatography Systems)
234
Actions in the Browser
Testing the Leak Detector
Many System Wellness supported devices are equipped with leak
detectors. The test procedure for all of the sensors of IC devices is the
same.
1. Thoroughly dry the sensor.
2. Open the System Wellness ¾Control Panel for the device (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel).
3. Under Diagnostic Tests, leak detector, select the internal script
button to test the sensor installed in the device itself. Select external to
test the sensor on a controlled device. For example, for a pump, internal
refers to the sensor in the pump and external refers to the sensor in a
chromatography oven controlled by the pump.
4. The device tests the sensor and reports the results to Chromeleon.
Possible test results are:
•
Passed (Dry)
•
Failed (Wet)
•
Failed (Open circuit): The sensor may be disconnected. Check the
connection.
•
Failed (Short circuit): The sensor may need replacing. Contact
Dionex for assistance.
•
Failed (Out-of-calibration): Calibrate the sensor (see How to ...:
Calibrating the Leak Detector) and
Actions in the Browser
retest. If the test still fails, the sensor may need replacing. Contact
Dionex for assistance.
Wavelength Verification
Use this procedure for the Dionex AD25 and PDA-100 detectors. The
Wavelength Verification test verifies the wavelength accuracy of the
AD25 or PDA-100 detectors. When this test is run, a holmium oxide filter is
placed in the light path and measured wavelengths are compared to
theoretical wavelengths for holmium oxide.
Actions in the Browser
235
1. Verify that there is solvent flowing through the cell, the background
absorbance is low, and there are no bubbles in the light path.
2. Open the System Wellness ¾Control Panel for the detector (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel).
3. Under Diagnostic Tests, wavelength verification, click the Verify
script button. The detector runs the test and then reports the results to
Chromeleon. The overall results (Passed or Failed) are reported and
the theoretical and measured values for three peaks in the holmium
oxide spectrum.
If the test fails, run the wavelength calibration (see How to ...: Actions in
the Browser
Wavelength Calibration) and then rerun the verification
test.
ICS-90 Pressure and Conductivity Diagnostics
Use this procedure to test the variance in pressure and conductivity
readings for the Dionex ICS-90 Ion Chromatography System.
1. Verify that the ICS-90 pump is on.
2. Open the ICS-90 System Wellness ¾Control Panel (see How
to ...: Actions in the Browser
Opening a Wellness Control
Panel).
3. Under Diagnostic, click the Start script button. The ICS-90 begins
collecting pressure and conductivity readings. To stop the test, click the
Stop script button. The ICS-90 reports the minimum, maximum, and
variance results for both pressure and conductivity.
To test the conductivity variance on the electronics alone, select the
Dummy Cell check box before running the test.
236
Actions in the Browser
Conductivity Verification (ICS-1000/1500/2000)
Use this procedure to test the conductivity variance on the DS6 conductivity
cell electronics.
Note:
Do not open a System Wellness ¾Control Panel if the name includes
"Service.pan." These Wellness panels are reserved for use by Dionex
Service Representatives.
1. Open the System Wellness Control Panel for the ICS-1000/1500/2000
(see How to ...: Actions in the Browser
Opening a Wellness
Control Panel).
2. Locate the Dummy Cell Test controls. Click Instructions for an
overview of the test procedure.
3. Click On to begin the test.
4. When the test is complete, click Log to record the new dummy cell
value in the Audit Trail. The new dummy cell value should be 21 µS; if it
is not, the CPU card may be defective. Contact Dionex for assistance.
Entering Device Parameters
For some devices, System Wellness ¾Control Panels include fields that let
Applying a Sodium
you enter various device parameters. Also, see
Correction (for ED40/ED50/ED50A Amperometry detectors).
Applying a Sodium Correction
If you are using a NaCl reference electrode with an ED40, ED50, or ED50A
detector in amperometry mode, turn on the sodium correction parameter.
This adjusts the detector's signal response for the NaCl electrode, instead
of the default AgCl reference electrode.
1. Open the System Wellness ¾Control Panel for the detector (see How
Opening a Wellness Control
to ...: Actions in the Browser
Panel).
2. Under Device Parameters, sodium correction, select On.
Actions in the Browser
237
Importing PeakNet (Releases 4.5 Through
5.2) Method Files
Overview
In order to use Method files from PeakNet (releases 4.5 through 5.2) with
Chromeleon, they must first be imported into Chromeleon.
When imported, the PeakNet 5 Method file is converted into a Chromeleon
¾PGM File and/or ¾Quantification Method (QNT Method). Converted PGM
Files contain timed events and setup parameters for all modules included in
the original PeakNet 5 Method. Converted QNT Files contain component
names, retention times, reference peaks, tolerance, calibration options,
groups, and level amounts for each detector in the PeakNet 5 Method.
PeakNet 5 Methods from the following modules can be imported: AS50,
GP40/GP50, IP20/IP25, AD20, AD25, CD20, ED40, IC20, UI20, DX-120.
For Methods that were created with AI-450 software, only the QNT portion
of the Method is converted.
Because of differences in the way that PeakNet 5 and the Chromeleon
function, some features available in a PeakNet 5 Method file are not
imported into Chromeleon. These include:
•
Replicate calibration information
•
CE Method parameters
•
High/low limit values
•
Outlier rejection
•
Linear weighting options
In addition, Chromeleon allows only a single component table per injection,
whereas PeakNet 5 allows one table per detector. When the PeakNet 5
Method contains multiple detectors, the first detector is converted entirely,
that is, information about every component and unknown peak is added to
the peak table. Then, for the other detector(s), only unique components
and unique unknown groups, which were not in the first detector's data, are
added to the table. This means that any reference component information
for detectors other than the first may be lost.
238
Actions in the Browser
How To
1. In the ¾Browser, select a ¾Datasource folder (not a sequence name).
2. Select Import/Restore on the File menu and then select PeakNet 5
and Method Files.
3. In the Import PeakNet 5 Method Files dialog box, navigate to the folder
that contains the PeakNet 5 Method file(s) to be imported. These files
are named with the .MET extension.
4. Select one or more of the Method files and click the Add button. The
files appear in the Selected files list.
5. To convert the PeakNet 5 Method into a PGM File for Chromeleon,
select the Program box. The PGM File is created in the folder shown
next to the Program box. The default is the datasource folder currently
selected in the Browser. To choose a different location, click the
Browse button and select the desired location.
6. To convert the PeakNet 5 Method component table and calibration
parameters into a Chromeleon QNT Method, select the QNT box. The
QNT File is created in the folder shown next to the QNT box. The
default is the datasource folder currently selected in the Browser. To
choose a different location, click the Browse button and select the
desired location.
7. Select the Inverted Curve Fitting check box to use inverted calibration
(concentration or amount is plotted against measured values). Clear the
check box for "normal" curve fitting (measured values are plotted
against concentration or amount). See How to ...: Actions in the QNT
Editor
Inverting Dependent and Independent Variables for
details.
8. Click the Import button. The selected PGM and/or QNT Files are
created.
The new files have the same name as the PeakNet 5 Method file, but
with PN5 appended and with PGM and QNT extensions. Once a
Method file is converted, the file name is removed from the Selected
files list.
Tip:
If the QNT option is selected, but the PeakNet 5 Method does not contain a
detector module, an empty QNT Method file is created.
Also, refer to
Files
Importing PeakNet (Release 4.5 Through 5.2) Data
Actions in the Browser
239
Importing PeakNet (Releases 4.5 Through
5.2) Data Files
Overview
In order to use data files from PeakNet (releases 4.5 through 5.2) with
Chromeleon, the files must first be imported into Chromeleon. When the
PeakNet 5 data file is imported, a ¾Sequence for Chromeleon is created.
The sequence includes the raw data from the data file, the embedded
PeakNet 5 Method (converted into ¾PGM File and ¾Quantification Method
(QNT Method) of Chromeleon, and additional information required to
complete the sequence. If multiple data files are selected for import, they
are grouped into one sequence. If the PeakNet 5 data file contains multiple
detectors, a separate sample line in the sequence is created for each
detector (up to four).
How To
1. In the ¾Browser, select a ¾Datasource folder (not a sequence name).
2. Select Import/Restore on the File menu and then select PeakNet 5
and Data Files.
3. In the Import PeakNet 5 Data Files dialog box, navigate to the folder that
contains the PeakNet 5 data file(s) to be imported. These files are
named with a .DXD or .Dxx extension (where xx is 01 - 99).
4. Select one or more data files and click the Add button. The files appear
in the Selected files list.
5. The selected data files will be imported into the folder shown in the
PeakNet Folder box. The default is the datasource folder currently
selected in the Browser. To import the data file(s) into a different
location, click the Browse button and select the desired location.
6. In the Sequence Name box, enter a name for the sequence to be
created from the imported data files.
7. Select the Inverted Curve Fitting check box to use inverted calibration
(concentration or amount is plotted against measured values). Clear the
check box for "normal" curve fitting (measured values are plotted
against concentration or amount). See How to ...: Actions in the QNT
Editor
Inverting Dependent and Independent Variables for
details.
8. Click the Import button. The selected PeakNet 5 data files are imported
into Chromeleon.
240
Actions in the Browser
The following files are created:
•
One sequence, which contains a sample line for each PeakNet 5 data
file selected for import. If any of the data files contained data from
multiple detectors, separate sample lines are created for each
detector's data.
•
One PGM File and one QNT File for each sample in the sequence. The
PGM and QNT Files are created from the PeakNet 5 Method
embedded in the PeakNet 5 data file.
The PGM and QNT Files are named as follows: the Method file name from
the PeakNet 5 data file is used, followed by a three-digit identifier, which
corresponds to the detector's position in the sequence, and then a pgm or
qnt extension.
Example
In the Import PeakNet 5 Data Files dialog box, three data files are selected
for import.
After importing, the following sequence is created. Notice that because the
Bioamine.d04 data file contained data from two detectors, two sample lines
were created in the sequence:
Actions in the Browser
241
The following PGM and QNT Files were also created. These correspond to
the programs and methods listed in the sequence:
Also, refer to
Files
Importing PeakNet (Release 4.5 Through 5.2) Method
PeakNet (Release 4.5 Through 5.2)
Translation Tables
See the following topics for how PeakNet (releases 4.5 through 5.2) setup
and timed event Method parameters are translated to Chromeleon PGM
File commands.
AS50 Autosampler
Pump Modules and Eluent Generator
AD20 Detector
AD25 Detector
CD20 Detectors
ED40 Detectors
IC20 Systems
DX-120 System
UI20 Module
See
Detector Component Table Translation for how PeakNet
(release 4.5 through 5.2) component table parameters are translated to
Chromeleon QNT Method parameters.
242
Actions in the Browser
AS50 Autosampler Setup and Timed Event Parameter
Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
TTL 1 (checked/unchecked)
Sampler_TTL_1.State (=0v/5v)
Setup & timed
portions
TTL 2 (checked/unchecked)
Sampler_TTL_2.State (=0v/5v)
Setup & timed
portions
Relay 1 (checked/unchecked)
Sampler_Relay_1.State
(=Closed/Open)
Setup & timed
portions
Relay 2 (checked/unchecked)
Sampler_Relay_2.State
(=Closed/Open)
Setup & timed
portions
CSV (A/B)
ColumnValve.State(=Col_A/Col_B)
Setup & timed
portions
Sample NeedleHeight
NeedleHeight
Setup portion
ColumnTemperature
ColumnTemperature
Setup portion
TrayTemperature
TrayTemperature
Setup portion
Cycle Time
Cycle
Setup portion
Valve (Load/Inject)
Load/Inject
Setup & timed
portions
If Inject, command "Wait InjectState"
is added
Pipet function
(Source/Volume/Destination)
Pipet (SourceVial/Volume/
DestinationVial)
Setup portion
Mix function
Mix
Setup portion
(Vial/Volume/Cycles)
(SourceVial/Volume/NumberOfTime
s)
Flush function (Volume)
FlushSP (Volume)
Setup portion
Delay function (Delay Time)
DelaySP (Time)
Setup portion
Needle function (Height)
SetNeedleHeight (Height)
Setup portion
Dilute function
Dilute
Setup portion
(Concentrate Source/
Concentrate Volume/
(SourceVial/
Diluent Source/
Diluent Volume/
ConcentrateVolume
SourceReservoir/ DiluentVolume/
DestinationVial)
Destination)
Dispense function
Dispense (SourceReservoir/Volume/
DestinationVial)
Setup portion
(Source/Volume/Destination)
Wait function
Wait (SamplePrep)
Setup portion
Actions in the Browser
243
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Wait for stable temperature
(checked/unchecked)
WaitForTemperature (=True/False)
Setup portion
In any case 2 commands are added:
Setup portion
1."Flush Volume=100"
2."Wait
Output Labels
FlushState"
Syringe Speed always set to default
value = 3
Setup portion
CutSegmentVolume always set to
default value = 0
Setup portion
Not converted
Comments
Comments
Description
Comments
Pump and Eluent Generator Setup and Timed Event
Parameter Translation
Pump Modules Translation Table
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Oven Temperature
Temperature
Setup portion
High Pressure Limit
Pressure.UpperLimit
Setup portion
Low Pressure Limit
Pressure.LowerLimit
Setup portion
Eluent Label(A/B/C/D)
(%A/%B/%C/%D).Equate
Setup portion
Eluent percentage (B/C/D)
%B/%C/%D
Timed portion
Inject
InjectValve.State
Setup & timed portions
(checked/unchecked)
(=InjectPosition/LoadPosition)
Column
ColumnValve.State
(checked/unchecked)
(=Col_B/Col_A)
TTL 1 (checked/unchecked)
Pump_TTL_1.State (=0v/5v)
TTL 2 (checked/unchecked)
Pump_TTL_2.State (=0v/5v)
Setup & timed portions
Relay 1
(checked/unchecked)
Pump_Relay_1.State
(=Closed/Open)
Setup & timed portions
Relay 2
(checked/unchecked)
Pump_Relay_2.State
(=Closed/Open)
Setup & timed portions
Flow
Flow
Setup & timed portions
Eluent percentage (%A)
Not converted
Calculated from %B,
%C, and %D
Setup & timed portions
Setup & timed portions
244
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Actions in the Browser
Chromeleon PGM Command
Comments
Pump (On/Off)
Not converted
Curve
Curve
Comment
Comments
Piston Size
Not converted
Set in Server
Configuration instead
Pressure Units
Not converted
Set in Server
Configuration instead
Description
Comments
Eluent Generator Translation Table
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Eluent Concentration
Concentration
Timed portion
TTL 1(checked/unchecked)
EluentGenerator_TTL_1.State
(=0v/5v)
Timed portion
Offset Volume
Not converted
Set in Server
Configuration instead
Curve
Curve
Eluent Label
Not converted
TTL 1 Output
Not converted
AD20 Setup and Timed Event Parameter Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Rate
Data_Collection_Rate
Setup portion
UV_VIS_1.AcqOff
Timed portion
Time
(at time=time of AcqOn + Time)
TTL 1(checked/unchecked)
UV_TTL_1.State (=0v/5v)
TTL 2(checked/unchecked)
UV_TTL_2.State (=0v/5v)
Setup & timed portions
Setup & timed portions
Relay 1
(checked/unchecked)
UV_Relay_1.State
(=Closed/Open)
Setup & timed portions
Relay 2
(checked/unchecked)
UV_Relay_2.State
(=Closed/Open)
Setup & timed portions
Offset (checked)
Autozero
Setup & timed portions
Mark (checked)
UV_Analog_out.Mark
Setup & timed portions
Actions in the Browser
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
245
Comments
Range
UV_Analog_out.Recorder_Range
Setup & timed portions
Wavelength
Wavelength
Setup & timed portions
Collection Begin (checked)
UV_VIS_1.AcqOn
Setup or timed portion
UV Lamp
UV_Lamp
Setup portion
(Off/Low/High)
(=Off/Low/High)
If =Low/High, command
"Wait UV_Lamp_Ready" is added
Visible Lamp (Off/Low/High)
Visible_Lamp (=Off/Low/High)
Plot Scales
(Minimum/Maximum)
Not converted
Detector Units
Not converted
X-Y Data
Not converted
Setup portion
AD25 Setup and Timed Event Parameter Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Rate
Data_Collection_Rate
Setup portion
Time
UV_VIS_1.AcqOff
Timed portion
(at time=time of AcqOn + Time)
TTL 1(checked/unchecked)
UV_TTL_1.State (=0v/5v)
TTL 2(checked/unchecked)
UV_TTL_2.State (=0v/5v)
Setup & timed portions
Setup & timed portions
Relay 1
(checked/unchecked)
UV_Relay_1.State
(=Closed/Open)
Setup & timed portions
Relay 2
(checked/unchecked)
UV_Relay_2.State
(=Closed/Open)
Setup & timed portions
Offset (checked)
Autozero
Setup & timed portions
Mark (checked)
UV_Analog_out.Mark
Setup & timed portions
Range
UV_Analog_out.Recorder_Range
Setup & timed portions
Wavelength
Wavelength
Setup & timed portions
Collection Begin (checked)
UV_VIS_1.AcqOn
Setup or timed portion
UV Lamp
UV_Lamp
Setup portion
(On/Off)
(=On/Off)
If =On, command
"Wait UV_Lamp_Ready" is added
Visible Lamp (On/Off)
Visible_Lamp (=On/Off)
Setup portion
Offset Level
UV_Analog_out.Offset_Level
Setup portion
246
Actions in the Browser
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Calibration
Recorder_Calibration
(AU/Zero/Full_Scale)
Setup portion
(Off/Zero/Full Scale)
Polarity (Negative/Positive)
UV_Analog_Out.Polarity
Setup portion
Rise Time
Rise_Time
Comment
Comments
(=Negative/Positive)
Description
Comments
Labels
Not converted
Plot Scales
(Minimum/Maximum)
Not converted
Detector Units
Not converted
X-Y Data
Not converted
Setup portion
CD20 Setup and Timed Event Parameter Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Rate
Data_Collection_Rate
Setup portion
Time
AcqOff
Timed portion
(at time=time of AcqOn + Time)
Range (µS)
ECD_Analog_Out.Recorder_Rang
e
Setup & timed portions
TTL 1(checked/unchecked)
ECD_TTL_1.State (=0v/5v)
Setup & timed portions
TTL 2(checked/unchecked)
ECD_TTL_2.State (=0v/5v)
Setup & timed portions
Relay 1
(checked/unchecked)
ECD_Relay_1.State
(=Closed/Open)
Setup & timed portions
Relay 2
(checked/unchecked)
ECD_Relay_2.State
(=Closed/Open)
Setup & timed portions
Offset (checked)
Autozero
Setup & timed portions
Mark (checked)
ECD_Analog_Out.Mark
Setup & timed portions
Temp. Comp.
Temperature_Compensation
Setup portion
Cell Temp.
DS3_Temperature
Setup portion
SRS Current
(Off/50/100/300/500 Ma)
SRS_Current
Setup portion
Collection Begin (checked)
AcqOn
(=Off/50/100/300/500)
Setup or timed portion
Actions in the Browser
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Plot Scales
(Minimum/Maximum)
Not converted
Detector Units
Not converted
X-Y Data
Not converted
247
Comments
ED40 Setup and Timed Event Parameter Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command of
Comments
Rate
Data_Collection_Rate
Setup portion
Time
AcqOff
Timed portion
(at time=time of AcqOn + Time)
Range (µS)
ECD_Analog_Out.Recorder_Range
Setup & timed
portions
TTL 1(checked/unchecked)
ECD_TTL_1.State (=0v/5v)
Setup & timed
portions
TTL 2(checked/unchecked)
ECD_TTL_2.State (=0v/5v)
Setup & timed
portions
Relay 1
(checked/unchecked)
ECD_Relay_1.State (=Closed/Open)
Setup & timed
portions
Relay 2
(checked/unchecked)
ECD_Relay_2.State (=Closed/Open)
Setup & timed
portions
Offset (checked)
Autozero
Setup & timed
portions
Mark (checked)
ECD_Analog_Out.Mark
Setup & timed
portions
Temp. Comp.
Temperature_Compensation
Setup portion.
Only for
ED40/ED50c
(conductivity)
Cell Temp.
DS3_Temperature
Setup portion.
Only for
ED40/ED50c
SRS Current
SRS_Current
Setup portion.
(Off/50/100/300/500 mA)
(=Off/50/100/300/500)
Only for
ED40/ED50c
248
Actions in the Browser
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command of
Amperometry Cell (On/Off)
Cell (=On/Off)
Comments
Setup portion.
Only for
ED40/ED50d (DC
amperometry) &
ED40/ED50i
(integrated
amperometry)
Oven Temperature
(enabled)
Oven_Temperature
Setup portion.
Only for
ED40/ED50d &
ED40/ED50i
Voltage
DC_Voltage
Setup portion.
Only for
ED40/ED50d
Collection Begin (checked)
AcqOn
Plot Scales
(Minimum/Maximum)
Not converted
Detector Units
Not converted
X-Y Data
Not converted
Setup or timed
portion
IC20 Setup and Timed Event Parameter Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Rate
Data_Collection_Rate
Setup portion
Time
ECD_1.AcqOff
Timed portion
(at time=time of AcqOn + Time)
High Pressure Limit
Pressure.UpperLimit
Setup portion
Low Pressure Limit
Pressure.LowerLimit
Setup portion
SRS Current
(Off/50/100/300/500 Ma)
SRS_Current
Setup portion
(=Off/50/100/300/500)
Oven Temp.
DS3_Temperature
Setup portion
Temp. Comp.
Temperature_Compensation
Setup portion
Inject(checked/unchecked)
Pump_InjectValve.State
Setup & timed portions
(InjectPosition/LoadPosition)
TTL 1(checked/unchecked)
Pump_ECD_TTL_1.State (=0v/5v)
Setup & timed portions
TTL 2(checked/unchecked)
Pump_ECD_TTL_2.State (=0v/5v)
Setup & timed portions
Actions in the Browser
249
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Relay 1
(checked/unchecked)
Pump_ECD_Relay_1.State
(=Closed/Open)
Setup & timed portions
Relay 2
(checked/unchecked)
Pump_ECD_Relay_2.State
(=Closed/Open)
Setup & timed portions
Offset (checked)
Autozero
Setup & timed portions
Mark (checked)
Mark
Setup & timed portions
Flow
Pump_ECD.Flow
Timed portion
Range
Recorder_Range
Setup & timed portions
Eluent (A/B)
Pump_ColumnValve.State
Setup & timed portions
Collection Begin (checked)
ECD_1.AcqOn
Setup or timed portion
Pressure Units
Not converted
Set in Server
Configuration instead
Piston Size
Not converted
Set in Server
Configuration instead
Pump (On/Off)
Not converted
(=Col_A/Col_B)
Comment
Comments
Description
Comments
Labels
Not converted
Plot Scales
(Minimum/Maximum)
Not converted
Detector Units
Not converted
X-Y Data
Not converted
DX-120 Setup and Timed Event Parameter Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Rate
Data_Collection_Rate
Setup portion
Time
ECD_1.AcqOff
Timed portion
(at time=time of AcqOn + Time)
Pump (On/Off)
Pump (=On/Off)
Setup portion
Column (A/B)
Column (=A/B)
Setup portion
Wait RinseComplete command
added
Only if System Mode is
Column
PressureUnit (=psi/MPa)
Setup portion
Pressure (psi/MPa)
250
Actions in the Browser
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
SRS/Cell (On/Off)
SRS (=On/Off)
Setup portion
Eluent Pressure (On/Off)
EluentPressure (=On/Off)
Setup portion
Offset (checked)
Autozero
Setup & timed portions
Inject(checked/unchecked)
Pump_InjectValve.State
Setup & timed portions
(InjectPosition/LoadPosition)
TTL 1(checked/unchecked)
ECD_TTL_1.State (=0v/5v)
Setup & timed portions
TTL 2(checked/unchecked)
ECD_TTL_2.State (=0v/5v)
Setup & timed portions
Controlled AC
(checked/unchecked)
ControlledAC (=On/Off)
Setup & timed portions
Eluent (A/B)
Eluent (=A/B)
Setup & timed portions
Only if System Mode is
Eluent
Collection Begin (checked)
ECD_1.AcqOn
System Mode
Not converted
Comment
Comments
TTL Output Labels
Not converted
Plot Scales
(Minimum/Maximum)
Not converted
Detector Units
Not converted
X-Y Data
Not converted
Setup or timed portion
UI20 Setup and Timed Event Parameter Translation
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Rate (A&B)
Data_Collection_Rate
Setup portion
Time (A&B)
Channel_A.AcqOff for Channel A
Timed portion
Channel_B.AcqOff for Channel B
(at time=time of AcqOn + Time)
TTL 1(checked/unchecked)
Interface_TTL_1.State (=0v/5v)
Setup & timed portions
TTL 2(checked/unchecked)
Interface_TTL_2.State (=0v/5v)
Setup & timed portions
TTL 3(checked/unchecked)
Interface_TTL_3.State (=0v/5v)
Setup & timed portions
TTL 4(checked/unchecked)
Interface_TTL_4.State (=0v/5v)
Setup & timed portions
Relay 1
(checked/unchecked)
Interface_Relay_1.State
(=Closed/Open)
Setup & timed portions
Actions in the Browser
251
PeakNet (Release 4.5
Through 5.2) Method
Parameter
Chromeleon PGM Command
Comments
Relay 2
(checked/unchecked)
Interface_Relay_2.State
(=Closed/Open)
Setup & timed portions
Full Scale Voltage (mV)
Full_Scale_Voltage
Setup portion
(10/100/1000/10000)
(0.011/ 0.110/ 1.100/ 11.000)
Collection Begin (checked)
Channel_A.AcqOn for Channel A
Setup or timed portion
Channel_B.AcqOn for Channel B
Comment
Comments
Labels
Not converted
Plot Scales
(Minimum/Maximum)
Not converted
Units
Not converted
X-Y Data
Not converted
TTL Inputs
Not converted
Trigger
Not converted
Detector Component Table Parameter Translation
PeakNet Release 4.5
Through 5.2 (PN5)
Component Table
Parameter
Chromeleon QNT
Method
Component Name
Peak Name
Retention Time
Retention Time
Comments
If peak Reference Component
column = None then Retention Time
options are set to Absolute.
If peak Reference Component =
<Component> then Retention Time
options are set to Time Distance to
Reference Peak and Reference
Peak = <Component> is assigned
Tolerance
Window
If Tolerance = time then Window
options are set to Absolute and
Greatest.
If Tolerance = % then Window
options are set to Relative and
Greatest.
Reference Component
Retention Time Options
See Retention Time comments for
rules.
252
Actions in the Browser
PeakNet Release 4.5
Through 5.2 (PN5)
Component Table
Parameter
Chromeleon QNT
Method
Comments
Internal Standard
Component
Standard
If global PN5 calibration options =
External then all DDS Standard =
External
If global PN5 calibration options =
Internal then
- If Internal Standard Component =
Internal Standard then "Use this
peak as Internal Standard" option is
set.
- If Internal Standard Component =
<Component> then Internal Standard
is set and Associated ISTD Peak is
assigned.
Calibration Standards
Level [1...32] Amounts
Amount column for each
level
An amount column for each level
labeled Std1, Std2, … Stdn is
inserted where n = total levels. The
amounts for each peak in the table
are filled in.
Check Standards Level
[1...32] Amounts
Amount column for each
level
An amount column for each level
labeled CStd1, CStd2, … CStdn is
inserted where n = total levels. The
amounts for each peak in the table
are filled in.
Curve Fit Type
Calibration Type
If Cubic in PN5 then set to quadratic.
- PN5 Fit Linear/quadratic with Origin
= Force is Linear/quadratic in the
DDS.
- PN5 Fit Linear/quadratic with Origin
= Ignore is Linear/quadratic with
Offset in the DDS.
- PN5 Fit Linear/quadratic with Origin
= Include is Linear/quadratic with
Offset and Include point (0,0) options
in the DDS.
- All DDS are set to No Weights.
- No Average Response Factor in the
DDS so set to Linear.
Origin
Calibration Type
See Curve Fit Type for rules.
Calibrate By
Calibrate By
- Area or Height
- If Relative then set to Relative Area
for Identified Peaks Only.
Relative Response
Component
Response Factor
Relative to Peak is set and correct
peak is assigned.
Relative Factor
Response Factor
Value for factor is assigned.
Actions in the Browser
253
PeakNet Release 4.5
Through 5.2 (PN5)
Component Table
Parameter
Chromeleon QNT
Method
Comments
Groups
Groups
- For each peak in a group the DDS
group item is set to that name.
- For each time range in a group, the
range is added to Unidentified Peaks
list.
Standardization
External/Internal
Calibration Standard
Volume
Reference Inject
Volume
Amount Units
Dimension of Amounts
Replace Retention Time
Use recently detected
retention times check
box is checked
Low Limit Amount
Not Available (N.A.)
High Limit Amount
N.A.
Total Levels for
Calibration Standards
N.A.
Total Levels for Check
Standards
N.A.
Replicates Table
N.A.
Replicates
N.A.
Rejection of Outlier
N.A.
Linear Weighting
N.A.
Replace/Average
Response
N.A.
Sample Volume Default
N.A.
Sample Weight
N.A.
Dilution Factor
N.A.
Internal Standard
Amount
N.A.
Unknown Response
Factor
N.A.
Response for Unknowns
N.A.
CE Information
N.A.
See handling in Quantification
section above.
Last Standard Options = Last Value
No replicate information will be
converted from PN5.
If CE information is contained it will
not be converted to the DDS.
254
Actions in the Browser
Importing Agilent/HP ChemStation Data
Files
In order to use Agilent/HP ChemStation data files with Chromeleon, you
must first import the files into Chromeleon. However, keep in mind that the
structure of the two systems is different:
•
Agilent/HP ChemStation contains single files.
•
Chromeleon is a database-aided system.
Select the type of data you want to import in the Wizard that guides you
through the process (see below):
Agilent/HP ChemStation
Chromeleon
Batch
One ¾Sequence that has already been analyzed (not a
¾Batch!)
Sequence
Sequence
Method
Contains elements of the ¾PGM File, the ¾QNT
Method, and the ¾Report Definition File (RDF)
Tip:
Only the transferable QNT part of each file is imported.
Sample
Single sample
Spectra Library
¾Spectra Library
Depending on your selection, the following Agilent/HP ChemStation data
are imported together with the selected data type:
Agilent/HP ChemStation
Chromeleon
2D/3D raw data file
The corresponding channels; for example, UV and 3D
channels.
Instrument data such as
pressure, flow, temperature, and
gradient composition
Sample LOG File
¾Audit Trail
How To
•
In the Browser, select Import/Restore on the File menu and then,
select Agilent/HP ChemStation.... The Agilent/HP ChemStation
Data Import Wizard is opened.
•
On the first page, select the data type to be imported. Clicking Next>
takes you to the next Wizard page.
Actions in the Browser
255
•
Click the Add… button. In the Select Agilent/HP ChemStation …
dialog box, navigate to the folder in the Windows Explorer that contains
the file(s) to be imported. (The picture refers to importing a sample.)
•
Select the first file to be imported. Click OK to confirm your selection.
This action automatically returns you to the Wizard. (When you import a
batch, a sequence, or a spectra library, the appearance of the dialog
box is slightly different. In this case, click Open to confirm your
selection.)
•
Repeat these steps until all desired files appear in the selected files list.
Clicking Next> takes you to the next Wizard page.
•
Determine the destination directory. (Depending on the data type to be
imported, you may have to determine the destination sequence and the
method to be used):
256
Actions in the Browser
•
Select Import instrument signals by the check box if you want to
include the instrument signals into the import. Clicking Next> takes you
to the next Wizard page.
•
All files selected for import and the desired location are listed.
•
Click the Finish button. The selected files are imported into
Chromeleon.
For more information, refer to
ChemStation Translation Tables.
ChemStation Translation Tables
For information about how ChemStation data are translated to Chromeleon
data, refer to:
Translating ChemStation Sequences and Batches
Translating ChemStation Samples
Translating ChemStation Methods
Translating ChemStation Spectra Libraries
Actions in the Browser
257
Translating ChemStation Sequences and Batches
ChemStation (CS) sequences and batches correspond to the ¾Sequences
in Chromeleon. Chromeleon uses all information available in the
ChemStation sequence or in the ChemStation batch to create a new
sequence with the same name. A message appears if this sequence
already exists. You can either overwrite the old sequence or cancel the
import.
The table below lists the parameters that are available for a sequence
and/or batch in ChemStation and their equivalents in Chromeleon:
ChemStation (CS)
Chromeleon
Comment (of a CS sequence)
Title
Instrument
Timebase
Operator Name (of a CS sequence)
Created by
Last modification time (of a CS sequence)
Created
Current CM user (user who imported the data)
Last Update by
Date/time of import
Last Update
-
Locked
-
Preferred RDF file
-
Preferred channel
Some ChemStation sequence and/or batch settings cannot be mapped
directly to Chromeleon settings:
•
Cal Level: Used for ChemStation calibration; not used in Chromeleon.
In Chromeleon, the corresponding information is available in the
⇒Amount column of the ¾QNT Method.
•
Inj/Location: Indicates the number of replicates. The corresponding
number of samples will be generated for the Chromeleon sequence.
•
Interval: If the interval value for one of the standards is > 0, the
ChemStation sequence runs in a special mode: The standard samples
are injected first and then, the unknown samples are injected. After x
unknown samples have been injected (where x is defined by Interval),
one or more standards are injected again. After importing, a sequence
is created in Chromeleon in which standards are injected again after x
unknown samples.
258
Actions in the Browser
Translation table for generating a sample list:
ChemStation
Chromeleon
Sample Name
⇒Name (sample
name)
Sample Type
⇒Type (sample type)
Sample
Calibration
Control Sample
Unknown,
Standard,
Validation.
Location
⇒Pos. (sample position)
Inj Volume
⇒Inj.
Vol.
volume)
-
⇒Program (¾PGM File)
Method Name
⇒Method
method)
Date/Time of injection (evaluated from CS log file)
⇒Inj. Date/Time (injection
date and time)
Product of Sample Amount and Multiplier
⇒Weight (Sample Weight
Factor)
Dilution
⇒Dil. Factor
Factor)
ISTD Amount
⇒ISTD Amount (Amount
of the ¾Internal Standard)
(injection
(quantification
⇒Status (sample status)
Datafile (standards are named automatically - see below)
⇒Sample ID
-
⇒Replicate ID
Sample Info
⇒Comment
(Dilution
The ChemStation does not name the calibration standards; the names are
automatically generated with a prefix and counter. Chromeleon needs the
exact file name for data import and thus, uses the same algorithm.
Translating ChemStation Samples
Chromeleon uses all information available for the ChemStation (CS)
sample to create a new sample with the same name. If the new sample is
imported into an existing sequence, the sample is appended to the
sequence. Existing samples will not be overwritten.
Unlike Chromeleon, ChemStation does not provide a sequence context for
the single samples. That is why the sample information is gathered from
raw data, instrument diagnosis, macros, and the LOG files.
Actions in the Browser
259
The table below lists the properties that are available for a ChemStation
sample and their equivalents in Chromeleon:
ChemStation
Chromeleon
Sample name information from a 2D/3D raw data file
⇒Name (sample name)
Cannot be evaluated from the CS sample data;
set to "unknown"
⇒Type (sample type)
Vial position information from a 2D/3D raw data file
⇒Pos. (sample position)
Inj Volume from the Lcdiag.reg file
⇒Inj. Vol. (injection volume)
Not specified, no entry.
⇒Program (¾PGM File)
Method information from a 2D/3D raw data file
⇒Method (quantification
method)
Set to "Finished"
⇒Status (sample status)
Date/time of injection (from the LOG file)
⇒Inj. Date/Time (injection date
and time)
Cannot be evaluated from the CS sample data, set to 1.0
⇒Weight (Sample Weight
Factor)
Cannot be evaluated from the CS sample data, set to 1.0
⇒Dil. Factor (dilution factor)
Cannot be evaluated from the CS sample data, set to 1.0
⇒ISTD Amount (Amount of the
¾Internal Standard)
Sample name (*.d)
⇒Sample ID
-
⇒Replicate ID
Sample information from the "sample.mac" file
⇒Comment
If you did not specify the ChemStation method to be used, all methods
used for the imported samples will be imported. If one of these methods
does not exist, Chromeleon generates a standard QNT Method.
Translating ChemStation Methods
The ChemStation (CS) method contains parts of the Chromeleon ¾PGM
File, the ¾QNT Method, and the ¾Report Definition File (RDF).
Chromeleon uses only the relevant information of a ChemStation method
(i.e., Peak Table and Integration Events) to create a new QNT Method with
the same name. A message appears if this method already exists. You can
either overwrite the old method or cancel the import.
260
Actions in the Browser
The table below lists the Integration Events of a ChemStation method and
the equivalent detection parameters in Chromeleon:
ChemStation
Chromeleon
Comment
Slope Sensitivity
Sensitivity
Translation factor: 20
Peak Width
Peak Slice
Translation factor: 0.8
Area Reject
Minimum Area
Translation factor: 1/60
Height Reject
Minimum Height
Shoulders
Peak Shoulder Threshold
Off = 0ff, On = 0.1
Baseline Now
Lock Baseline
On
Baseline at Valleys
Valley to Valley
Off = Off, On = On
Baseline Hold
Lock Baseline
On = At Current Level, Off = Off
Tail Tangent Skim
-
Tangent Skim Mode
-
Area Sum
Peak Group Start
On
Peak Group End
Off
Integration
Inhibit Integration
Off = On, On = Off
Negative Peak
Detect Negative Peaks
Off = Off, On = On
Split Peak
-
Fixed Peak Width
Peak Slice
Auto Peak Width
-
Detect Shoulders
Peak Shoulder Threshold
Off = 0ff, On = 0.1
Shoulder Mode
-
-
Solvent Peak
-
Baseline Backwards
-
Baseline Next Valley
-
Unassigned Peaks
-
Chromeleon provides equivalents in the QNT Method for the following peak
table parameters of a ChemStation method:
ChemStation
Chromeleon
Compound
Peak Name
RT
Ret. Time
RT Window Low
Window
RT Window High
Comment
Calculated from the difference:
RT High - RT Low
Actions in the Browser
261
Translating ChemStation Spectra Libraries
When importing a ChemStation (CS) spectra library into Chromeleon, the
library name is the same in Chromeleon and in ChemStation. All spectra of
the ChemStation library are imported into the Chromeleon library. Only the
unmodified spectra are imported, all specific wavelength entries are
ignored.
The table below lists the information available in a ChemStation spectra
library and the equivalent parameters in Chromeleon:
ChemStation
Chromeleon
File name of the imported library + hint "Imported CS library"
Name
User who imported the spectra library
Created by
Creation date of the imported spectra library
Created
Current user
Last Update by
Date/time of the import
Last Update
Information on the imported library
Title
262
Actions in the Browser
Actions on the Control Panel
263
Actions on the Control Panel
For general information about control panels, refer to Control
Control Panel.
The
The topics below provide information about how to open and/or modify a
control panel and how to modify the control elements or change the
timebase assignment:
Opening a Control Panel
Connecting a Control Panel with a Timebase
Creating and Starting an Online Batch
Modifying a Control Panel
Modifying a Control
Linking a Control to a Device
Creating a Script Button
Creating a Command Button
Creating Hidden Windows
In addition, you can simulate data acquisition and display sample and
sequence information:
Using/Recording Demo Data
Displaying Sample and Sequence Information
Opening a Control Panel
To process unknown samples or to control an instrument by Chromeleon,
open a ¾Control Panel, either manually or automatically.
Chromeleon is shipped with more than 100 default control panels that
provide all standard functions required for operation. The default panels are
usually stored in the Dionex Templates > Panels directory of the local
¾Datasource.
264
Actions on the Control Panel
Open a control panel manually
•
Select Open... on the File menu. In the Open dialog box, select
Control Panel from the Object of type drop-down list and navigate to
the folder that contains the control panels. All panels available in the
selected folder are listed. Type the panel name in the Name field. Or
else, click the name in the list. In this case, the panel name is
automatically written into the Name field. Click Open to open the panel.
•
Alternatively, you can select the panel directly from the ¾Browser.
Double-click the panel name to open the panel.
Each control panel is connected with a ¾Timebase. When you open a
control panel, Chromeleon automatically connects to the timebase saved
last. When installing the software, Dionex Service usually specifies the
timebase to which Chromeleon connects automatically. Of course, you can
connect the control panel to any other timebase whenever you want (see
Connecting a Control
How to ...: Actions on the Control Panel
Panel with a Timebase).
Tip:
To change the automatic timebase assignment for a control panel:
Select Properties on the Edit menu. On the Timebase tab page, delete
the entry in the Timebase field and click OK. When you open the control
panel again, no timebase will be assigned. Return to the Timebase tab
page, and select the desired timebase. Click OK to save the new
assignment.
Open a control panel automatically
When started, Chromeleon opens the most recently used ¾Workspace. If
the workspace contains a control panel, the corresponding control panel is
opened as well.
If you cannot open a workspace, for example, because you have not yet
created such a view, open the control panel manually.
Actions on the Control Panel
265
Connecting a Control Panel with a Timebase
When you open a ¾Control Panel, it is automatically connected with the
assigned ¾Timebase. You can modify the timebase assignment any time:
•
Open a control panel. Select Connect to Timebase... on the Control
menu.
The Timebase field on the left indicates the name of the currently selected
timebase, the Computer field indicates the name of the computer on which
the corresponding ¾Server is running, and the Protocol field indicates the
currently used communication protocol. The list box on the right shows an
icon for the local computer (My Computer) and, if the computer is
connected to a network, also an icon for the network neighborhood (also,
see Chromatography Components: Hardware and Software
Network Installation.)
•
Click the + character beside an icon to display the items underneath.
•
If the server is running, all timebases configured for the local server are
listed under My Computer. If the server is not active, the entry is The
server is not running.
•
All PCs available on the network are listed under Network
Neighborhood. Click the + character beside a computer name to
display the Chromeleon servers underneath. If there is no active
Chromeleon server, the entry is Server not found.
•
Select the desired timebase and click OK to confirm your selection.
The name of the timebase is automatically written to the Timebase
field on the left.
•
If you want to connect to a different timebase whenever you open the
control panel, delete the entry in the Timebase field. The panel is no
longer assigned to a specific timebase and the message stating that
the former timebase is not found does not appear.
•
Click Ok to have Chromeleon connect the control panel with the
selected timebase. If the connection is successful, you can control the
connected instruments, using the control elements on the control panel.
Tip:
If you want to connect automatically to the currently timebase when you
reopen the control panel later, save the panel with the current timebase
assignment.
266
Actions on the Control Panel
Problems when Connecting to a Timebase
It depends on the selected network protocol whether a non-local server is
displayed or can be accessed. The system needs several seconds to check
whether the selected protocol can be used. If the connection to a specific
timebase is not successful, this can be due to several reasons.
•
First, check whether the corresponding server is running.
•
If the server is running, try a different network protocol. Chromeleon
can communicate via various network protocols, such as IPX, TCP/IP,
or NetBEUI.
Tip:
Communication between two stations is possible only if the same
network protocol is installed and selected on both stations. It is usually
sufficient if you have installed the corresponding network protocols,
e.g., IPX/SPX compatible protocol; NetBEUI Protocol, or Internet
Protocol (TCP/IP). (Click Settings > Control Panel > Network and
Dial-up Connections > Local Area Connection, and then, select
Properties on the context menu.) The current network installation
determines which protocol is actually used. Please contact your
network administrator. To connect to a Windows 2000 or Windows XP
computer, use the Named Pipes protocol. Usually, the connection is
successful when this protocol is used.
•
It may happen that the required timebase exists (Server is running)
but that it is not displayed. In this case, return to the Connect to
Timebase dialog box. The timebase is usually displayed then.
This may also happen when you connect two computers using an ISDN
connection. Usually, the reason is that the network installation is not
100 per cent correct.
•
If you use an IPX/SPX compatible protocol, click Settings > Control
Panel > Network and Dial-up Connections > Configuration. Select
Properties on the context menu and then, select the NWLink
IPS/SPX/NetBIOS Compatible Transport Protocols check box.
If the connection is still not successful, contact Dionex Service.
Actions on the Control Panel
267
Problems when Controlling Instruments
In addition to the control mode, Chromeleon also provides a Monitor Only
mode. If you connect to a timebase that is only available on a network, you
can only monitor this timebase.
The Monitor Only status is also assigned when you attempt to access a
local timebase that is currently controlled by an external user.
•
Deselect Monitor Only on the Control menu to enable complete
control over the timebase.
Creating and Starting an Online Batch
Chromatography systems are often operated day and night to ensure the
best possible rate of utilization. To facilitate processing, Chromeleon allows
you to group different sequences in a ¾Batch:
•
Select Edit on the Batch menu. The Batch dialog box is opened.
•
On the Batch List tab page, click Add... to add the sequences to be
processed. (In the Browse dialog box, select Sequence as Object of
type and navigate to the desired sequence(s). Click Open to confirm
your selection.) If necessary, click Remove to remove any unwanted
sequences. To delay the start of a sequence, select the sequence and
click Set Delay. A dialog box is opened. Enter the date and time to
start the sequence and click OK.
268
Actions on the Control Panel
•
Use the Reporting tab page to determine the print and report options.
Select the Print/Export Report check box to open the Batch Report
dialog box. Use this dialog box to specify which pages of the ¾Printer
Layout you want to print or export and for which samples you want to
print/export the selected pages. Confirm your settings by clicking OK.
On the Reporting tab page, also determine whether the selected
pages shall be printed or exported immediately after the corresponding
sample has been processed or when the entire batch has been
finished. (For more information, refer to How to ...: Actions in the
Printer Layout
Printing the Results of a Sequence or a Sample
Batch.)
•
On the Error Handling tab page, determine the behavior of
Chromeleon if errors occur during batch processing.
Under Emergency Program, select the program to be run if an Abort
error occurs. Select the program from the drop-down list box or click
Browse to navigate to the desired file. (For more information, refer to
Creating an Emergency
How to ...: Actions in the PGM Editor
Program).
Under Power Failure Handling, determine the program to be run when
the server restarts after a power failure. Select the program from the
drop-down list box or click Browse to navigate to the desired file. (For
more information, refer to How to ...: Actions in the PGM Editor
Creating a Power Failure Program). In addition, determine how
the system shall proceed after running the power failure program.
Select the desired action from the drop-down list box.
•
On the Transfer tab page, determine whether the data shall be copied
when the batch has been processed. Select the Copy data to the
following location check box and then, click Browse to navigate to
the desired location. The options provided in the Options section
depend on whether the ¾Online Transfer Agent (= OTA) is installed on
your computer. If the Online Transfer Agent is installed, the following
options are available:
Actions on the Control Panel
269
If the Online Transfer Agent is not installed on your computer, the
Chromeleon client transfers the data. If the OTA is installed, data
transfer is performed by the OTA service. In this case, it is possible to
start the batch even if the client is not running and to have data
transferred to network datasources that you are not allowed to access.
Tip:
To use the Online Transfer Agent, make sure that the Multiple
Network Control license is available on your PC (see
Chromeleon
Licenses).
For information about how to install the Online Transfer Agent, refer to the
Administrator Manual: How to ...: Actions in the Server Monitor
Program
Setting Up the Online Transfer Agent for Network Access.
270
Actions on the Control Panel
Modifying a Control Panel
Open the ¾Control Panel you intend to modify (see How to ...: Actions on
Opening a Control Panel).
the Control Panel
Saving the Panel under a Different Name
We recommend that you do not overwrite any panels from the Dionex
Templates > Panels directory. Also, before modifying an existing panel,
select Save as on the File menu and save the panel under a different
name.
Enabling Layout Mode
•
Enable ¾Layout Mode on the Edit or context menu.
Adding a Control
•
Move the mouse cursor over the ¾Layout Toolbar:
The quick info box provides a brief description of the ¾Control on
which the mouse cursor is placed.
•
Left-click to select and append the desired control to the cursor.
•
Move the mouse cursor to the desired position. Left-click to position the
new control.
Removing a Control
•
Left-click the control to be removed. A control frame marks the selected
control.
•
Press the Del key to remove the control. Or else, select Delete on the
Edit menu.
Modifying the Size and Position of Controls
•
To resize left-click the control and draw its ¾Control Frame to the
desired size.
•
To move a control left-click and drag the control to the desired position,
holding down the left mouse button. To select several controls
simultaneously, press the Shift key and left-click the desired controls.
Actions on the Control Panel
•
271
To align several controls, press the Shift key and left-click the desired
controls. Select ¾Align on the Edit menu and then, select the desired
option. The control frame of the control selected first is used as the
reference point and reference size for the other controls.
For information about how to change control properties, refer to How
to ...: Actions on the Control Panel
Modifying a Control.
Modifying a Control
In Online Mode
•
Right-click the ¾Control (= object) to be modified.
•
Select Properties... on the context menu to open the Properties dialog
box for the selected control.
In Layout Mode
•
Select the control to be modified.
•
Select Properties... on the context menu to open the Properties dialog
box.
Properties Dialog Box
The tab pages provided in the Properties dialog box vary, depending on
the selected control:
Name
Function
1.
General
Determine the caption, font, and text position and size for the control.
2.
Link
Link to the device for which the property is displayed.
3.
Autosize
Determine how the size of the control changes when the entire panel is
increased or reduced in size.
4.
Color
Select the colors for the individual components of the control.
5.
Style
Select the shape of the control.
6.
Button
Specify whether and which additional ¾Control Panel(s) are opened
when the corresponding Script button is clicked (also, see How to ...:
7.
Command
Type the command or program to be executed when the Script button is
clicked.
8.
Axis/Decoration Determine the scaling of the axes and the representation of the online
signals and 3D plot.
Actions on the Control Panel
Creating Hidden Windows).
272
Actions on the Control Panel
Name
9.
Function
Signals
Determine which signals are displayed and which offset is used.
10. Chrom.
Determine whether and how chromatograms overlap on the signal plot.
11. Device
Select the pump(s) for which gradient profiles are displayed.
12. Timebase
Connect with the selected ¾Timebase.
13. Default
Parameters
Specify the default parameters for a command that is linked to a
Command button.
14. Events
Select the events to be marked on the ¾Trend Plot.
15. Statistics
Select the statistics to be marked on the trend plot and define how they
are calculated.
16. Data Labels
Specify which data points on the trend plot are labeled, and the text of
the label.
17. Data
Select the trend variable to be plotted, the datasource from which trend
data is retrieved, and the time period of the trend data.
These tab pages are available for the following controls (the corresponding
links are available in the online Help):
1
2
3
Form
X
Check Box
X
X
X
Color Box
X
X
X
String
Display
X
X
X
Gauge
Indicator
X
X
X
4
5
X
X
7
8
9
10
11
12
13
14
15
16
17
X
X
X
X
X
X
X
X
X
X
X
X
Script Button X
X
Online
Signal Plot
X
X
Command
Button
X
Trend Plot
X
X
Group Box
X
X
Gauge/
Slider
X
X
X
X
X
Lamp
X
X
X
X
X
X
6
X
X
X
X
X
X
X
X
X
X
X
Actions on the Control Panel
1
2
3
Switch
X
X
X
Edit Field
X
X
X
Audit Trail
X
X
Gradient
Display
X
X
X
3D Plot
X
X
X
Rack
Display
X
Online Mass
Spectrum
Plot
X
X
4
5
6
7
8
9
10
273
11
12
X
X
13
14
15
16
17
X
X
X
X
X
X
X
For information about how to specify the property of a control, refer to How
Linking a Control to a Device.
to ...: Actions on the Control Panel
Linking a Control to a Device
To have a control display the status or the parameter of a device, you have
to link the element to the device:
•
Right-click the corresponding ¾Control and select Properties... on the
context menu.
•
Open the Link tab page.
•
From the Object list box, select the object to which you want to link a
property or parameter. For example, select an instrument, a function
(relay), a ¾Channel, or a system. Select the desired property from the
Object Property list box. Click OK to link the control to the object and
the property.
Example
To have a color box indicate whether the pump is connected to
Chromeleon, select Pump from the Object list box and Connected from
the Object Property list box:
274
Actions on the Control Panel
Tip:
Not every control is an ideal choice for representing a certain function or
parameter. The functions and properties that appear in the Object
Property list box depend on the selected control.
Note:
The Link tab page is not available for the signal plot, the gradient profile,
and the 3D plot. Use the Signals tab page for the signal plot and the
Device tab page for the gradient profile instead. You cannot change the
parameters for the 3D plot.
Creating a Script Button
Chromeleon lets you assign a command or an entire sequence of
commands to a Script Button. In this way, you only need to click the
corresponding button to have Chromeleon execute the command or
program. To create script buttons, you must have the corresponding
authorization.
Actions on the Control Panel
275
•
Right-click the script button, select Properties... on the context menu
and then, select the Command tab page.
•
Type all commands to be executed successively into the list box. The
syntax corresponds to the syntax used when creating a ¾Program.
When you have entered the desired commands, click Check... to check
the syntax. Click Try it now to test the operation before you complete
programming.
•
You can also use a script to start a previously written program: Copy
the entire program text and paste it into the list box on the Command
tab page. Or else, use the ⇒Branch command. Enter the Branch
command and then enter the path and name for the program to be
started, e.g.:
Branch „CM_Seminar\\Programs\\Equilibration"
This button allows you to start a column equilibration program that is
stored on the CM_Seminar datasource in the Programs directory.
For more information, refer to Control
The Program Syntax. For
Creating
practical tips, refer to How to …: Actions in the PGM Editor
Overview
a Program and Device Control
Linking a Control to a Command
To have a control execute a specific device command, you have to link the
control to both the device and the command:
•
Right-click the corresponding ¾Control and select Properties... on the
context menu.
•
Open the Link tab page.
•
From the Object list box, select the object to which you want to link a
property from the Object list box. For example, select an instrument, a
function (relay), a ¾Channel, or a system. Select the desired property
from the Object Property list box. Click OK to link the control to the
object and the property.
276
Actions on the Control Panel
Creating a Command Button
Chromeleon lets you assign a command to a command button so that you
can execute the command by clicking the button. To create a command
button, you must have the appropriate authorization.
•
Right-click the corresponding button and select Properties... on the
context menu.
•
Open the Link tab page.
•
From the Object list box, select the object to which you want to link the
command. From the Command list box, select the command to be
linked to the selected object.
•
Select the Default Parameters tab page.
•
Specify the default setting for the linked command and enter any
comment text you want to have associated with the command.
Creating Hidden Windows
In some cases, a ¾Control Panel has to fulfill so many different functions
that they do not fit in one window. For example, there is only few space left
for the signal plot. In this case, create a Script Button to open an
additional window for the remaining controls or, for example, a large signal
plot:
•
First, create an additional window: Enable Layout Mode on the Edit or
context menu.
•
Select New Window on the Window menu.
•
Click the Online Signal Plot icon on the toolbar. Add the signal plot to
the window by clicking inside the window and then, determine the size
of plot. Right-click the signal plot to open the Online Signal Plot
Properties dialog box. Use this dialog box to determine the properties
for the signal plot.
•
Right-click the background of the new window, i.e., right-click outside
beside the signal plot. Select Properties... on the context menu to
open the Form Properties dialog box.
•
On the General tab page, enter a name, e.g., Signal. On the Style tab
page, clear the Initially Visible check box.
•
Return to the original window by clicking the little cross on the top right
of the new window.
Actions on the Control Panel
277
•
Select the Script Button icon on the toolbar and add a new script
button to the original window. Right-click the button and select
Properties... on the context menu. The Script Button Properties
dialog box is opened.
•
The Button tab page indicates the names and captions of all windows
assigned to this panel. Select the new window and select the Show
Form check box.
If you now click this button, the signal plot is opened.
Using/Recording Demo Data
Simulating Data Acquisition
¾Demo (or Virtual) Mode lets you simulate data acquisition by loading a
pre-recorded demo file and displaying the data from the demo file on the
¾Control Panel's signal plot. The demo file is "read back" as though the
data were being acquired in real time.
1. To select Demo or Virtual Mode for a device, start the Server
Configuration program and select the desired device in the
corresponding ¾Timebase.
2. Select Properties on the Edit or context menu.
278
Actions on the Control Panel
3. For detectors that use demo mode, select Read under Demo Mode on
the General tab page and then, select an existing demo file from the
Demo File Name drop-down list.
For other detectors, select Virtual Mode on the General tab page.
Then, select the Demo Chromatogram tab, select the Read demo
chromatogram from: check box, and select a demo file from the dropdown list.
4. (Optional) Enable the Demo or Virtual Mode for all other devices of the
timebase on the corresponding General tab pages.
5. To run the demo file, open the control panel for the device, connect to
the timebase, and then enable data acquisition. The Data Acquisition
dialog box appears.
6. If the demo file contains more than one channel, select the desired
channel(s) and click OK. The demo file begins running. It runs
continuously (repeats) until data acquisition is disabled.
Recording Demo Data
1. To create a demo file for an installed detector, open the Properties
dialog box in the Server Configuration.
2. For detectors that use demo mode, select Write under Demo Mode on
the General tab page.
For other detectors, select Live Mode on the General tab page. Then,
select the Demo Chromatogram tab page and select the Write demo
chromatogram to: check box.
3. Enter a name for the demo file. If the device is connected via the Dionex
DX-LAN, select the Device ID under Communication.
4. To write the demo file, open the control panel for the device and enable
data acquisition. The data acquired from the detector is recorded in the
demo file.
5. To stop recording data, disable data acquisition. The demo file is then
complete. Alternatively, instead of starting and stopping data acquisition
manually, you can run a ¾PGM File that turns acquisition on and off.
Actions on the Control Panel
279
Displaying Sample and Sequence Information
In addition to the current analysis and status values, information specific to
sequences and/or samples can be displayed on a ¾Control Panel. Thus,
you can read from the control panel which sample is processed, to which
¾Sequence and ¾Datasource the sample belongs, which status the
sample has, and so on.
To display this type of information, link a ¾Control (alphanumeric display)
to one of the following functions (Object Properties):
Comment
Name
Number
Type
Datasource
Sequence
SequencePath
SequenceMoniker
Program
ProgramMoniker
Moniker
ID
For more information about controls, refer to the topics in How to ...:
Actions on the Control Panel.
Answering Frequently Asked Questions
Question: Why does an error message appear in Chromeleon when I shut
down a device?
Answer: The Chromeleon server reports an error because the device is no
longer connected. Stop the Chromeleon server. Right-click the
¾Chromeleon Monitor icon and click Stop Server.
Note:
Keep in mind that you have to restart the Chromeleon server before you
can record another chromatogram.
Question: How can I learn from a control panel which program is being sent
to the device and which commands are being executed?
Answer: The program used for analyzing the sample is displayed in the
Audit Trail section on the control panel. Under the program, the times at
which the different commands were executed by the device are listed.
280
Actions on the Control Panel
Question: What is the function of the Inject… command on the Control
menu? Does it also start data acquisition?
Answer: Only select this command to perform a manual injection. If you
select the command while a program is running, the program will be
interrupted. Data acquisition will not be started. To start data acquisition,
select Acquisition On on the Control menu.
Question: Several control panels are listed on the Window menu. How can
I delete an entry?
Answer: Select the control panel that is not used any longer. Close
panel by either clicking the left-most or right-most button on the top of
panel or by selecting Close on the File menu. If you have modified
panel, a dialog box appears in which you can confirm to save
modifications.
For tips to solve similar questions, also refer to How to …:
the Control Panel and the corresponding subtopics.
the
the
the
the
Actions on
Actions in the PGM Editor
281
Actions in the PGM Editor
Use the different views provided by the PGM Editor to edit your control
programs (¾PGM Files). To access a view, click the corresponding icon on
the shortcut bar:
•
Click Commands to edit a program if this is not entirely possible in the
device view. For more information, refer to How to …: Actions in the
PGM Editor
Creating a Program.
•
Click a device icon to edit the program for the device. (It depends on
the installed devices which device views are available in the PGM
Editing PGM Files in the Device Views of
Editor.) Also, refer to
the PGM Editor.
282
Actions in the PGM Editor
•
If installed, click Finnigan AQA (or MSQ) to create an MS method for
an MS method for the corresponding spectrometer. For more
information about the MS instrument method, refer to How to …:
Creating a PGM
Actions Related to the Mass Spectrometers
Creating a PGM File for the MSQ.
File for the aQa MS or
•
Click Post-acquisition steps to reprocess your data, e.g., to extract
channels and smooth data. For more information, refer to How to …:
Adding Post-Acquisition Steps.
Actions in the PGM Editor
For general information about the PGM Editor, refer to Control
PGM Editor.
The
Creating a Program
A ¾Program is used to define precisely timed start conditions and
⇒Control Commands. You usually create the program using the
Program Wizard and edit the program later in the PGM Editor (see
The PGM Editor). The minimum entries required for an
Control
operable program are as follows:
•
Signals to be recorded and their parameters
•
⇒Flow rate and solvent composition (⇒%B, %C, %D - for controlled
pumps, only)
•
⇒Inject command
•
Start of data acquisition (⇒AcqOn)
•
End of data acquisition (AcqOff)
•
End of the program (⇒End)
Verify that all relevant parameters are defined in the program. For
parameters that are not explicitly defined, Chromeleon uses the settings of
the last sequence.
We recommend to create the program automatically first (see
Automatically Creating a Program), and then edit the file via the PGM
Editing PGM Files in the Device Views of the PGM Editor
Editor (see
Manually Editing a PGM File in the Commands
or manually (see
View).
Actions in the PGM Editor
283
With respect to ¾GLP (Good Laboratory Praxis), we recommend adding
comments about all chromatographic settings (e.g., for the column,
detector, pump, and the sample components) as well as explanatory notes
at the beginning of the program. However, this is possible only in the
Commands view.
To see the structure of a simple program, refer to How to ...: Actions in
the PGM Editor
Program Example.
Automatically Creating a Program
Create the basic structure of a ¾Program using the Program Wizard. (For
The Program
general information about the Wizard, refer to Control
Wizard.)
•
Select New on the File menu, and then select Program File from the
list box. The Program Wizard guides you through program creation.
•
Enter the required information in the input fields on the corresponding
Wizard pages. Clicking Next> takes you to the next Wizard page.
•
If you need help, press the F1 key.
When input is complete, click Finish. Based on your entries, Chromeleon
creates an operable program. You are not required to pay attention to the
syntax or other programming details.
You can then modify and/or extend this basic program later as described in
Manually Editing a PGM File in the Commands View.
For a simple example, refer to How to ...: Actions in the PGM Editor
Program Example.
For examples for special programs, refer to How to ...: Actions in the
PGM Editor
Creating an Emergency Program
Creating a Power Failure Program
For an overview of the numerous programming capabilities, refer to How
to …: Device Control
Overview.
284
Actions in the PGM Editor
Editing PGM Files in the Device Views of the PGM Editor
The different device views in the PGM Editor offer a user-friendly way to
edit an existing program at any time according to your requirements.
•
Click a device icon on the left pane in the PGM Editor to change the
parameters and/or to enter new parameters for the associated device.
•
The corresponding page(s) of the
•
Enter the desired parameters or changes.
•
If you want to change and/or enter parameters for other devices, click
the respective icon and follow the description above.
Program Wizard are opened.
The pages of the Wizard or the PGM Editor provide only the most important
commands for the devices. The Commands view allows you to enter
Manually Editing a PGM File in
additional commands manually (see
the Commands View).
Note:
If your program already contains many comments, it may make sense to
edit the PGM File in the Commands view and not in the Device Views. If
you edit such a program in the Device Views, it may happen in some
configurations that the single commands appear at the "wrong" position.
Please note that comments that were entered at the end of a line are
removed. Therefore, please check the program before saving the changes.
Manually Editing a PGM File in the Commands View
If you want to modify and save an existing ¾Program, follow the steps
below. (For information about how to create a new program, refer to
Automatically Creating a Program. Modify the program in the
Editing PGM Files in the
corresponding device view as described in
Device Views of the PGM Editor.)
General Information
•
Select Open… on the File menu, and then select an existing PGM File
from the Open dialog box.
•
Verify that the server is running. If the server is not yet running, start
the server in the ¾Server Monitor Program.
Actions in the PGM Editor
•
285
Select Connect to Timebase on the Control menu and then, select
the timebase in which you want to use the modified PGM File.
• To open the Commands view, click the following icon on the icon bar:
•
Place the cursor on the position you wish to modify or press the Enter
key to insert a new program line.
Entering commands or properties
•
Press the F8 key to open the Commands dialog box.
•
Select the instrument (below called Device) for which you want to edit
an instruction. A device can be any instrument of a timebase, an
installed channel, a relay, a remote input, or the system itself. Devices
. Click the "+" character beside
are marked by the following icon
the device name to display the items underneath.
•
Each device has its own commands (
, ).
•
When you select a command or a property, additional input boxes
appear under the Retention Time input box. A short help text is
displayed in the Help section.
•
Enter the retention time when the command shall be executed or when
the status of a property shall be changed.
•
Assign either the required value (e.g., a number) or a new status (e.g.,
On) to the command or property.
•
Click OK to complete the input. Click OK & Prev or click OK & Next to
change to the previous or to the next program line.
•
Repeat the input procedure until you have changed all commands
and/or properties of interest.
) and/or properties (
,
,
Adding command/property values to the audit trail
• Press the F8 key to open the Commands dialog box.
• Select the ⇒Log command from the commands list. The devices listed
in the left window are displayed a second time, in the Property box on
the right. The structure in which the devices are displayed is the same
as described above. The Property box provides the commands and
properties from the left window and in addition several read-only
variables.
286
Actions in the PGM Editor
• Select the command or the property for which you want to write the
value at the time specified in the Retention Time field to the Audit Trail.
The Help section provides a short description of the selected command
or property.
• Click OK to enter the selected Log command into the program.
Adding comments to the audit trail
• Press the F8 key to open the Commands dialog box.
• Select the ⇒Protocol command from the commands list. Enter the
desired comment into the Text input box that appears on the right.
• Click OK to enter the Protocol command into the program.
Checks during command input
•
When you enter commands, Chromeleon performs different checks
(see ¾Check Command):
Syntax check: Does the entry correspond to the formal rules of the
program language?
Semantics check: Does the program make sense with regard to
chromatography?
•
If an error is detected, the corresponding line is displayed in red color.
Check and correct the entry. If necessary, repeat this procedure, using
the F8 box.
Adding comments
To enter a comment, place the cursor at desired position. Type the desired
the text, starting with a semicolon. Or else, press the F8 key to open the
Commands dialog box and enter the desired comment in Comment field.
Click OK to add the comment to the program.
Comments are displayed in green color, indicating that Chromeleon
considers the text as a comment. Any comment entered in this way does
not appear in the audit trail. It only serves to comment the program so that
you can, for example, easily recall and understand the various steps later.
Actions in the PGM Editor
287
Note:
If you want to edit your program later in the Device views (see Control/The
PGM Editor
The Device Views), we recommend entering all comments
right at the beginning of the program. In some cases, Chromeleon cannot
assign comments to the associated commands when re-sorting. Thus, it
may happen that some comments appear at the wrong position after you
have edited the program in the Device views.
For information about how to perform calculations in a program, refer to
Calculating in a Program.
Save the result as a new PGM File, using the Save as command.
Note:
Experienced users can enter these commands directly, that is, without
opening the F8 box. However, this requires profound knowledge of the
program syntax (see Control
The Program Syntax).
For a simple program example, refer to
Program Example.
For special program examples, refer to How to ...: Actions in the PGM
Editor
Creating a Shutdown Program
Creating an Emergency Program
Creating a Power Failure Program
Also refer How to ...: Actions in the PGM Editor
Columns in a Program.
Using User-defined
For an overview of the numerous programming capabilities, refer to How
Overview.
to ...: Device Control
288
Actions in the PGM Editor
Calculating in a Program
To use different values and signals in one program, Chromeleon lets you
use the four fundamental operations of arithmetic and powers. See below
for some examples:
Addition or Subtraction: Relative Sample Position
The two examples below refer to the ASI-100 autosampler. (For general
information about this autosampler, refer to the Administrator Manual:
Installation Instructions
ASI-100 HPLC Autosamplers.)
1. To determine that the vial that is five incremented positions away from
the current position is always used as the sample preparation vial, use the
following command line:
0.000 Sampler.PrepVial
Position + 5
For example, if you are currently processing the sample from position RB3,
the vial at position RB8 is used for sample preparation.
2. If the autosampler is currently at a position in the RA ring, the following
line takes you to the corresponding position in the RB ring:
0.000 Position Position + RB1 - RA1
Multiplication: Calculating the Volume
3. To record the solvent volume, which is transported through the column
while the current sample is processed, as a virtual channel use the
following line:
VirtualChannel_01.Formula Formula=pump.flow*system.retention
In addition, you have to define: VirtualChannel_01.FormulaMin and
VirtualChannel_01.FormulaMax.
Division: Ratio
4. To determine the ratio of two channels, record the quotient of the two
channels as virtual channel:
VirtualChannel_01.Formula Formula=UV_VIS_1/UV_VIS_2
Powers: Calculating the radioactive decay
5. Use the following formula to record a channel for a radioactive substance
as if this substance would not decay:
VirtualChannel_01.Formula
UV_VIS_1 / 2.718**
(-0.69314718*System.Retention/t½)
Actions in the PGM Editor
289
Note:
For detailed examples of the ¾Virtual Channel Driver, refer to How to ...:
Program Examples for Virtual Channels.
Device Control
Relational and logical operators: ¾Trigger Commands
6. Use the following condition to combine, for example, fraction collection:
Trigger FRACTION UV_VIS_1 OR UV_VIS_2 > 100
In the above example, fraction collection could start if a signal with a peak
area larger than 100 mAU is recorded on either the UV_VIS_1 or the
UV_VIS_2 channel.
The following operators are available for trigger commands:
Operator
>
<
=
AND
OR
NOT
XOR (=^)
Description
Greater than
Less than
Equals
All parameters must fulfill a condition.
Only one parameter must fulfill a condition.
Verifies that a parameter has no specified value.
Verifies that the values are different.
We recommend using the Fraction Collection device driver to control
fraction collection. For more information, refer to How to …:
Actions
Related to Fraction Collection.
290
Actions in the PGM Editor
Program Example
A ¾Program for a 20-minute chromatogram (flow rate: 1 ml/min,
components: A (60%) and B (40%), signals: UV_VIS_1 (256 nm),
UV_VIS_2 (300 nm), and 3D field: recorded in the 200 to 360 nm range at
a step of 0.5 seconds) could have the following appearance:
Pressure.LowerLimit =
Pressure.UpperLimit =
%A.Equate =
%B.Equate =
%C.Equate =
5
250
"%A"
"%B"
"%C"
3DFIELD.MaxWavelength =
3DFIELD.MinWavelength =
3DFIELD.BunchWidth =
3DFIELD.Step =
3DFIELD.RefWavelength =
3DFIELD.RefBandwidth =
360.0
200.0
1.9
0.5
600.0
1.9
UV_VIS_1.Wavelength =
UV_VIS_1.Bandwidth =
UV_VIS_1.Step =
UV_VIS_1.Average =
UV_VIS_1.RefWavelength =
UV_VIS_1.RefBandwidth =
256
1
2
On
600
1
UV_VIS_2.Wavelength =
UV_VIS_2.Bandwidth =
UV_VIS_2.Step =
UV_VIS_2.Average =
UV_VIS_2.RefWavelength =
UV_VIS_2.RefBandwidth =
300
1
2
On
600
1
Flow =
%B =
1.000
60
Actions in the PGM Editor
291
0.000 UV.Autozero
Inject
3DFIELD.AcqOn
UV_VIS_1.AcqOn
UV_VIS_2.AcqOn
20.000 3DFIELD.AcqOff
UV_VIS_1.AcqOff
UV_VIS_2.AcqOff
End
Notes:
•
A program for processing a sample batch must contain the ⇒Inject,
⇒AcqOn/Off, and ⇒End commands.
•
It is possible but it is not necessary to include the commands for
controlling the pump and the detector. Instead of entering the ⇒Flow
rate, eluent composition (⇒%B, %C, %D), and signal parameters
directly on the instruments, you can set these values in the program, as
shown in the example. This is only possible for controllable
chromatography instruments connected to Chromeleon via an RS-232
interface.
•
By definition, the injection time is t = 0.000. For all commands that are
to be executed before the injection time, the time entry is t < =0.000; for
example, the detector lamp on command, here -10.000 min.
•
You can omit the ⇒Lamp=On command if you allow sufficient time for
the lamp to reach the operating temperature.
•
Any PDA-100 Photodiode Detector command that triggers an Autozero
or Auto Offset command must be followed by either a Wait_AZ
command or a delay of at least 30 seconds until the next command.
The following commands trigger Autozero or Auto Offset: UV Lamp,
Visible Lamp, Wavelength, Bandwidth, RefWavelength, RefBandwidth,
Reference Mode, Rise Time, MinWavelength, and MaxWavelength.
•
For commands that are not listed but are automatically considered,
such as, the ⇒Step parameters of the UV_VIS_1 channel, either the
default values or the values that were last used are used; for example,
Step = 0.25. The decision which value is considered depends on the
respective device driver.
292
•
Actions in the PGM Editor
In your programs, keep in mind that the instruments need a certain time
to execute the different processes. If you use an HP autosampler, for
example, the program files must be at least 2 min long to allow the
¾Autosampler sufficient time until the next injection.
For special program examples, refer to How to ...: Actions in the PGM
Editor
Creating a Shutdown Program
Creating an Emergency Program
Creating a Power Failure Program
Program with Variable Run Time
Creating a Shutdown Program
When creating a shutdown program, keep the following aspects in mind:
•
Reduce the flow to make sure that as little solvent as possible is used.
However, do not reduce the flow to zero because this may result in
crystallization of salts in ion chromatography, for example.
•
Therefore, set the lower pressure limit (Pressure.LowerLimit) to 1. This
setting avoids that the pressure falls below the lower pressure limit
when the flow is reduced and that the pump is shut down.
•
In addition, the detector lamp(s) should be turned off.
Tip:
We do not recommend performing data acquisition in the shutdown
program, i.e., do not use an AcqOn command.
An example program could look as follows:
Pressure.LowerLimit = 1
Pressure.UpperLimit = 350
0.000 Flow = 0.500
0.500 UV_Lamp = Off
Flow = 0.100
End
Creating an Emergency Program and
Also, refer to
Power Failure Program.
Creating a
Actions in the PGM Editor
293
Creating an Emergency Program
If a ¾Batch was automatically aborted due to a severe or serious error, an
¾Emergency Program can be started. Determine which ¾Program is
executed by default.
Select Error Handling… on the Batch menu to open the Batch <Timebase> dialog box. On the Error Handling tab page, click Browse in
the Emergency Program section and navigate to the desired emergency
program.
Example 1:
Failure of the detector lamp is a severe error because data acquisition is no
longer possible. However, it is not necessary to stop the flow immediately.
Instead, the column should be reconditioned after the batch is aborted. You
may use, for example, the following emergency program:
;
0.000
10.000
10.500
Program: Emergency.PGM
Recondition column after
Flow
%A.Value
%B.Value
%C.Value
Flow
End
abort.
= 1
= 0
= 100
= 0
= 1
294
Actions in the PGM Editor
Example 2:
Power failure of an ¾Autosampler is a serious error. In this case, the entire
system should be stopped immediately to prevent more damage. The
Abort.PGM emergency program stops the pump flow and turns off the
detector lamp.
;
0.000
0.500
Program: Abort.PGM
The detector lamp and the pump flow are turned off.
Flow
= 0
Lamp
= Off
End
For a program example for the 1100 HPLC System, refer to the
Agilent 1100 HPLC
Administrator Manual: Installation Instructions
System: Program Tips.
Creating a Shutdown Program and
Also, refer to
Power Failure Program.
Creating a
Creating a Power Failure Program
You can determine how Chromeleon continues operation after a power
failure (see ¾Power Failure Protection). After booting the server, you may
run a power failure program first.
Select Error Handling… on the Batch menu to open the Batch
<Timebase> dialog box. On the Error Handling tab page, click the
Browse button in the Power Failure Handling section and navigate to the
desired power failure program.
If a power failure program is not yet available, create a program for the
timebase on which you want to use it. Make sure that all instruments are
reconnected to Chromeleon first. Very often self-tests have to be performed
during which the devices are not ready for access. Therefore, issue the
⇒Connect commands 1-2 minutes before the first ⇒Control Commands
are sent to the instruments.
Example:
Use the following program example, for example, for a ¾Timebase that
contains an ASI-100 ¾Autosampler, a UVD 340U ¾Photodiode Array
Detector, and a P580 pump:
Actions in the PGM Editor
-2.000
UV.Connect
Sampler.Connect
Pump.Connect
-1.500 Lamp =
295
On
; After the UVD 340U has been turned on, the detector needs some time
; for spectra; calibration. That is why you have to issue this
; command, too, some minutes before data acquisition is started.
0.000 Flow =
0.300
1.000 Flow =
1.000
End
Creating a Shutdown Program and
Also, refer to
Emergency Program.
Creating an
Program with Variable Run Time
To be able to create a program with variable run time for method
development, you need a generic device driver. This driver is not included
on the Chromeleon software CD. However, you may use the example
driver described in the Administrator Manual (see Installation
Example)
Instructions/Special Drivers/The Generic Device Driver
and then, generate the following program for this driver:
(…)
Flow = 1.000
%B = 60
0.000
UV.Autozero
Inject
3DFIELD.AcqOn
UV_VIS_1.AcqOn
UV_VIS_2.AcqOn
Stop_Device.Time = 15.000
Trigger EndTime System.Retention>Stop_Device.Time
3DFIELD.AcqOff
UV_VIS_1.AcqOff
UV_VIS_2.AcqOff
End
EndTrigger
999.000
3DFIELD.AcqOff
UV_VIS_1.AcqOff
UV_VIS_2.AcqOff
End
296
Actions in the PGM Editor
The program ends when the time defined for the stop device (here: 15.000
minutes) has been reached. You can change this time from the panel
during the program run. If the Stop-Device.Time is not reached, the above
program runs 999 minutes.
To change the run time quickly, create, e.g., an edit field on the control
panel, following the instructions in How to ...: Actions on the Control
Panel
Modifying a Control Panel. On the Link tab page, link the edit
field to the Time object property of the Stop_Device. You can then use this
edit field to set the run time to the desired value.
In addition, to identify the edit field, create a Color box above the field:
Converting Units
Chromeleon allows you to convert the registered units. Use the following
command, observing the correct syntax:
<Parameter> = <Value> [<Unit>]
Tip:
Chromeleon can only convert registered units, e.g., the pressure units bar,
psi, and MPa. Conversion is case-sensitive, i.e., Bar is not registered, and
thus, it would not be converted.
Example
If the pressure unit set on a GP50 pump is psi, you can nevertheless enter
the pressure limits as follows:
Pressure.LowerLimit =
Pressure.UpperLimit =
15 [bar]
200 [bar]
When the program is started, the pressure limits are converted into psi.
Chromeleon automatically converts the values and communicates the
following commands to the pump:
Pressure.LowerLimit =
Pressure.UpperLimit =
218 [psi]
2901 [psi]
Actions in the PGM Editor
297
This feature is important for programs that run on different systems. Even if
the pressure units are different for these systems, you can use the same
PGM File without adapting the values.
Chromeleon can convert the following units:
Value
Registered Units
Absorption
AU
mAU
µAU
Charge
C
µC
nC
Conductivity
S
mS
µS
nS
Current
A
mA
µA
nA
Density
kg/m³
g/ml
µl/min
µl/s
Flow rate
m³/s
ml/min
Flow acceleration
m³/s²
ml/min²
Fraction
None
%
Frequency
Hz
kHz
Inverse pressure
1/Pa
1/Mbar
Inverse temperature
1/K
%/K
Length
m
mm
µm
Mass
kg
g
amu
Molar concentration
mol/m³
mM
Pressure
Pa
bar
psi
Refractive index
RIU
µRIU
nRIU
pA
MHz
Refractive index/voltage
RIU/V
µRIU/V
Refractive index/time
RIU/s
nRIU/h
Temperature
K
°C
°F
min
Time
s
h
Velocity
m/s
mm/s
Voltage
V
mV
µV
Volume
m³
l
ml
nm
Å
MPa
kPa
ms
µs
µl
Note:
Concentration units, such as mg/ml and µg/ml, are not available in a PGM
File.
298
Actions in the PGM Editor
Using User-defined Columns in a Program
You may include ¾User-defined Columns from the sample list of the
Browser in a program, e.g., for special applications. However, you can only
use user-defined columns of the server's ¾Standard Datasource for which
the Value type is neither Time nor Date and time.
Caution:
Only experienced users should include and program user-defined columns
in a program. Errors in the control program may abort processing of the
single program steps, and thus the control program.
If you use user-defined columns in PGM files, verify that the column does
not contain empty fields. Otherwise, it may happen that the program is not
executed.
Tip:
You cannot use user-defined columns with a date or time format in PGM
files.
When user-defined columns are used in a program, the entry of the current
sample is usually used. The syntax is as follows:
<Command>
Sample.<UDC Name>
Tip:
You can also use the values of the user-defined column of the next or
previous sample (NextSample / PrevSample.<UDC-Name>) or of the
previous standard (PrevStand.<UDC-Name>).
Example 1: Defining the wavelength
In a control program, you can use user-defined columns, e.g., to define the
wavelength of a certain channel separately in the sample list for each
sample:
•
Create a user-defined column in the ¾Standard Datasource of the
server (see How to ...: Actions in the Browser
Creating Userdefined Columns).
Actions in the PGM Editor
•
299
Name the column, e.g., WL (for wavelength). To avoid confusion, do
not use a property that is already available in Chromeleon. That is why
you should not use, e.g., Wavelength. (Instead of WL, any other
agreed-upon abbreviation is acceptable.)
Tip:
Do not use a German "Umlaut" or "ß" in the name.
Restart the server.
•
In the program, enter, e.g., UV_VIS_1 for the channel:
UV_VIS_1.Wavelength =
Sample.WL
This entry means: For each sample, the wavelength used for the UV_VIS_1
channel is the wavelength defined in the corresponding cell of the userdefined column called WL.
Example 2: Starting and stopping fraction collection
You can use the Program Wizard to create user-defined columns to define
the fraction collection start and the end. (For information about the wizard,
refer to Control
The Program Wizard). For more information, refer to
PGM Wizard:
How to ...: Actions Related to Fraction Collection
Fraction Collection - General Options.
300
Actions in the PGM Editor
Restrictions when using user-defined columns in a program
You cannot use user-defined columns if the value that the parameter shall
reach must be known in advance. This applies to all gradient commands,
for example:
•
⇒Flow
•
⇒%B, %C, %D in % gradients
•
GC.Temperature in temperature gradients
•
Concentration
•
Pump.Curve and EluentGenerator.Curve (see ¾Gradient Curves)
Note:
Only if you issue these commands before you enter the retention time, you
can determine the corresponding values using user-defined columns.
For some instruments, the entire control program is downloaded before
sample processing, e.g., for the 1100 HPLC System. In this case, you
cannot make changes during the analysis.
Adding Post-Acquisition Steps
Use the Post-acquisition steps view of the PGM Editor to define
extraction and data smoothing steps to be performed after data acquisition.
The
(For general information about the PGM Editor, refer to Control
PGM Editor.) In addition, you can copy existing channels or combine them,
using arithmetic operations. A new data channel is created for each of
these steps. You can have these data reprocessing steps performed either
online after data acquisition or offline of chromatograms, UV spectra, or
¾Mass Spectra.
To open the Post-acquisition steps view, click the following icon on the
shortcut bar:
Actions in the PGM Editor
301
Click the gray line to add a new post-acquisition step. A dialog box
appears. Select the desired step. The following options are available:
•
Arithmetic combination of channels (2D channels
Combining Channels via Arithmetic Operations)
•
Copy Channel (see
•
Extract MS channel (for extracting a ¾Mass Trace - only if MS data
are available, see How to …: Actions Related to Mass
Extracting Mass Traces Afterward).
Spectrometers
•
Extract optimum integration path (for extracting the ¾Optimum
Integration Path - only possible if a 3D field is available, see How to …:
Selecting
Actions in the Peak Purity Analysis (PPA) Window
the Optimum Integration Path)
•
Extract UV channel (for extracting a UV channel extraction - only
possible if a 3D field is available; the procedure is similar to selecting
the optimum integration path)
•
Smooth data (for chromatogram ¾Smoothing - see How to …:
Performing Data Smoothing)
Actions in the Chromatogram
-
see
Copying a Channel)
For example, the following steps can be added:
In step 1 and step 6, two UV channels are extracted at 254 nm and
220 nm, respectively. The EXT254NM channel does not use compression;
only data that were acquired at 254 nm are used. For the EXT220NM, a
range of 10 nm was selected, i.e., the wavelengths from 215 to 225 nm are
used. In addition, compression is used during channel extraction.
Compression = 3 means that only every third data point is saved.
302
Actions in the PGM Editor
In step 3 and step 4, two MS channels (MS_1 and MS_2) are extracted.
The first mass trace covers the mass range 49.0 to 51.0 amu, while the
second trace covers masses from 137.0 to 145.0 amu. For both channels,
the Gaussian algorithm is used for data smoothing. Seven points are used
for data smoothing when the MS_N1 channel is extracted; five points are
used when the MS_N2 channel is extracted.
In step 2, the ¾Optimum Integration Path is saved as OPTINT channel. In
step 5, data smoothing is performed for this new channel using the
Savitzky-Golay ¾Filter. The channel is then saved again as
OPTINT_SG_009_001 channel. Filter size: 9 indicates that 9 input data
points each are used for creating one output data point.
To add additional steps, go to the bottom line. Or else, select Insert Line or
Append Line on the context menu.
The server performs the post-acquisition steps when data acquisition of the
last channel is completed and before the ¾System Suitability Test (SST) is
started. If there is a network failure (see ¾Network Failure Protection),
execution of the post-acquisition steps is delayed until the network source
is available again.
Manual Injections with Application
Templates
If no autosampler is found in the server configuration, Chromeleon
assumes that an AS40 Autosampler is connected to the Pump's Relay 1. If,
however, sample is being loaded and injected manually instead of via an
AS40, and an application template is being used to create the program, you
must modify the ¾PGM File to support this. The PGM File created by the
application template is saved in the sequence created by the Sequence
Wizard.
Open the PGM File and locate the AS40 load command. The command
typically occurs at the beginning of the timed events, at time -2.300. Below
is an example AS40 load command:
-2.300
Pump_Relay_1.Closed Duration=130.00
; For AS40 injection.
Replace this line with the following for manual injection
Actions in the PGM Editor
303
A manual injection line has been added to the PGM File immediately after
the load command:
;-0.100
Message "Load the sample into the injection loop, press OK
to continue"
The semicolon at the beginning of the line deactivates (comments out) the
manual injection ⇒Message. To reactivate it, remove the semicolon at the
beginning of the manual injection message and add one to the beginning of
the AS40 load command. Do not change the times preceding the
commands, even if they differ from the above examples.
;-2.300
Pump_Relay_1.Closed Duration=130.00
; For AS40 injection.
Replace this line with the following for manual injection
-0.100
Message "Load the sample into the injection loop, press OK
to continue"
Save and close the PGM File.
304
Actions in the PGM Editor
Actions in the QNT Editor
305
Actions in the QNT Editor
The settings selected in the quantification method determine which
calibration, peak table, and ⇒Detection Parameters are used to evaluate a
peak or an entire chromatogram (see ⇒QNT Parameters).
The peak table contains more columns than can be displayed clearly on the
screen. Thus, the columns are distributed on the following tabs:
•
Peak Table (general peak table)
•
Amount Table (parameters to determine the amount)
•
Peak Tracking (parameters to assign peaks using reference spectra)
•
MS Tracking (parameters to assign peaks using ¾Mass Spectra)
To determine which columns are displayed, select Display Columns on
the View menu and determine the columns to be displayed in the Display
Columns dialog box.
In addition to the tabs mentioned above, the QNT Editor provides the
following tabs:
•
General (general settings)
•
Detection (detection parameters)
•
Calibration (calibration parameters)
•
Spectra Library Screening (spectra search parameters)
•
SST (¾System Suitability Test parameters)
•
MS (¾Mass Spectrometry parameters)
•
UV (UV background subtraction)
To hide a sheet, select Select Sheets on the View menu and deselect the
desired sheet in the Select Sheets dialog box. To rename a sheet, doubleclick its tab in the QNT Editor and enter the new name.
306
Actions in the QNT Editor
For more information, refer to:
Creating a Peak Table
Defining Detection Parameters
Combining Peaks
Subtracting a Blank Run Sample
Disabling Calibration Samples
Calibration
Spectra Library Screening
Defining the System Suitability Test
Processing Mass Traces
Processing UV Channels
Creating a Peak Table
The peak table contains data for
•
Peak identification
•
Amount calculation
Specific peaks of a chromatogram are identified by a name. The
determined peak areas are converted into amount values (= ¾Formula for
Amount Calculation). The peak table contains the amount values for all
standard samples. Hence, it is the basis for each calibration. Usually, all the
information is entered into the peak table before the analysis is started.
To determine which columns are displayed on the Peak Table, Amount
Table, Peak Tracking, and MS Tracking tab pages, either select Display
Columns on the View menu or select Columns > Display Column on the
context menu. In this way, you can adjust the tab pages to your individual
requirements.
For more information, refer to:
Identifying Peaks
Identifying Peaks via Their UV Spectra (Peak Tracking)
Identifying Peaks via Their Mass Spectra (MS Tracking)
Defining Retention Times and Reference Peaks
Actions in the QNT Editor
307
Defining the Retention Index and the Kovats Index
Entering Amount Values
Selecting the Standard Method
Selecting the Calibration Function
(Columns: Cal. Type and Int. Type)
Weighting and Averaging Calibration Points
Defining the QNT Method for Several Detectors
Entering Reference Spectra
Autogenerating the Peak Table
Also, refer to How to ...: Actions in the Browser
defined Columns
Creating User-
Identifying Peaks
(Peak Name, Retention Time, and Window columns)
Most frequently, peaks are identified by their retention time.
•
Enter the names of all peaks to be identified in the ⇒Peak Name
column, line by line. Assign a nominal retention time to each peak by
entering a retention time value in the Ret. Time peak table column
(= manually creating a peak table). Or else:
•
Autogenerate Peaktable on the Edit menu to generate the
Select
peak table automatically, based on the current sample. In this case,
Chromeleon includes all integrated peaks of the current sample into the
peak table. The assigned peak names consist of the sequence name
and a consecutive number. Chromeleon uses the maximum values of
the single peaks to recalculate the values to be entered in the
⇒Retention Time and ⇒Window columns. All other entries are
replaced by the default values (= automatically creating a peak table).
If a peak is detected at the specified time in an unknown sample, the peak
is automatically assigned a name (fig. a).
Identification is possible even if the retention times deviate or if neighboring
peaks are very close. To allow this, use the Window (fig. b) peak table
parameter to define a tolerance range. If a peak is detected in this range, it
308
Actions in the QNT Editor
is identified even if the nominal and the actual retention times do not
coincide exactly (fig. c). If several peaks are detected in this range,
Chromeleon identifies the greatest peak, the first peak, or the peak nearest
to the retention time (fig. d). Which peak is identified depends on the option
selected in the Peak Window dialog box. In the Window column cell, the
two letters after the time range indicated the selected option.
set
peak name
actual
window
Tret (f)
Tret (n)
Tret (g)
Tret
a)
b)
c)
d)
To open the Peak Window dialog box, mark the Window column cell of
the peak of interest and then, either press the F8 key or double-click the
cell. Determine the Window Interpretation: Select Absolute to enter the
window width in minutes. Select Relative to define the window width in
percent.
In the Peak Match section, select the criterion for peak identification. If you
use a ¾Photodiode Array Detector, you can also identify the peaks by the
spectrum or by the spectrum and the retention time. For more information,
refer to
Identifying Peaks via Their UV Spectra (Peak Tracking). If
you use a ¾Mass Spectrometer for data acquisition, you can identify the
Identifying
peaks by their mass spectra. (For more information, refer to
Peaks via Their Mass Spectra (MS Tracking)).
The Window column in the peak table indicates the window width in
¾Decimal Minutes; for example, 0.25, followed by the abbreviation for the
selected window interpretation and the peak match criterion. To identify the
greatest peak in a 30 second window, the entry in the column must be as
follows: 0.25 AG (0.25 min or 15 seconds to the left and right of the
retention time). If the entry is 0.25 AN, the peak that is nearest to the
nominal retention time is identified.
Actions in the QNT Editor
309
Notes:
The retention times and window values stated in the peak table only serve
to identify a peak. The retention times indicated in a ¾Report are always
the actual retention times (= retention time in the peak maximum).
If the retention time of the same peak shifts from sample to sample due to a
column trend, it may happen that the peak leaves the retention time window
at some point. In this case, peak identification is no longer possible.
However, Chromeleon provides a method to reliably identify peaks even
then: Select the ⇒Use Recently Detected Retention Time check box on
the General tab page of the QNT Editor.
In addition to peak identification by the nominal retention time, it is also
possible to identify compounds by their substance spectrum.
Identifying Peaks via Their UV Spectra (Peak Tracking)
Chromeleon allows you to perform peak tracking. Peak tracking means
identifying peaks by comparing spectra. In addition to the Reference
Spectrum column, other peak table columns also allow you to influence
spectra comparison: Match Criterion, Check Derivative, Min. WL, Max.
WL, Threshold, Rel. Max. Deviation, and Check Extrema.
The values entered in these columns have the same meaning as described
in How to ...: Actions in the QNT Editor
Entering Criteria for the
Spectra Library Screening.
Tip:
If neither the minimum (Min. WL) nor the maximum wavelength (Max. WL)
are set, the comparison is performed for the entire wavelength range of the
reference spectrum.
Enable peak tracking in the Window column. In the corresponding F8
dialog box (press the F8 key in any cell in the Window column or doubleclick), select Spectrum or Spectrum and time under Peak Match.
310
Actions in the QNT Editor
The algorithm for peak identification by comparing spectra can be
described as follows: If, in the Window column of the peak table, the Peak
Match criterion for a peak is Spectrum or Spectrum and time, peak
tracking uses the reference spectra in the corresponding column. If
Spectrum and time is selected, the spectra comparison is limited to the
specified time window. Peak tracking generates a list of spectra sorted by
the match factor; a peak hit list is not displayed.
Tips:
Peak tracking is performed using the parameters (match criteria, etc.)
specified for the peak in the peak table.
The peak hit list includes only peaks with a match factor above the
threshold specified for this peak.
An empty peak hit list indicates that no spectrum fulfilling the match criteria
was found. This could be due to a very high threshold.
The peak hit list is calculated for all peaks in the peak table, for which peak
tracking was enabled. After calculation, the peak hit lists are checked for
multiple hits. If the Check the best hits only option is enabled, only the
best hits are compared for the individual peaks. In case of multiple hits, the
peak with the best match factor receives the name of the reference
substance.
Usually, multiple hits are not found if you select Spectrum and time as
match criterion.
Actions in the QNT Editor
311
All other peaks in the peak table, i.e., peaks for which the peak tracking
function is disabled, are identified via the window assignment (First,
Identifying Peaks.
Nearest, Greatest) as described in
If you acquire data with a ¾Mass Spectrometer, you can identify peaks via
Identifying Peaks
their mass spectra. (For more information, refer to
via Their Mass Spectra (MS Tracking)).
Identifying Peaks via Their Mass Spectra (MS Tracking)
(Mass peak x, MS threshold, MS filter conditions, and Check MS ret.
times columns)
¾Mass Spectra present a very reliable method for peak identification. For
the available parameters, refer to the MS Tracking tab page.
Double-click the respective cell or press the F8 key to open the Mass
spectrometry conditions for ... dialog box:
312
Actions in the QNT Editor
If you have entered at least the mass of one mass peak, you can enable
and disable peak identification via mass spectra by checking the Check
mass ratios check box.
Under Peak 1, enter the mass of the substance (range: 1.0 to 2000.0 amu),
which is expected at this peak. If you expect fragmentation and can
estimate the fragments, which are expected, you can enter two of them with
their expected intensities under Peak 2 and Peak 3.
Select the Threshold option to filter the noise. A mass is detected in the
mass spectrum only if its relative intensity compared to the ¾Base Peak
(largest peak) is higher than the threshold value.
The filter settings limit the mass spectra used for peak identification. This
option is required if you modify the polarity and/or use a different maximum
voltage for ionization of the sample.
Select the Check retention time of mass spectra option to use also the
retention times of all masses for peak assignment. All retention times must
be within one scan.
The settings made in the Mass spectrometry conditions for... dialog box
are not active immediately. You can activate them later from the Peak
Table tab page: Press the F8 key in the Window column or double-click to
open the Peak Window for... dialog box. Click the Check mass ratios
check box.
If you acquire data with a ¾Photodiode Array Detector, you can identify
peaks via their mass spectra. (For more information, refer to
Identifying
Peaks via Their UV Spectra (Peak Tracking)).
Defining Retention Times and Reference Peaks
(Retention Time column)
The Retention Time column allows you to select reference peaks for
relative retention times and to determine how the ⇒Retention Time is to be
interpreted for the respective peak. Double-click the corresponding peaks
or press the F8 key in the Ret. Time column, to open the Retention Time
for... dialog box.
Actions in the QNT Editor
313
On the Interpretation tab page, determine the display of the retention time
(relative or absolute time) individually for each peak. Note that relative
times can be displayed either as difference or as percentage ratio to the
retention time of a reference peak.
The Reference peak dialog box allows you to define one (or several) of the
other peaks as reference peak. However, only peaks with absolute
reference times can be used as reference peaks. They are indicated with a
light blue background. It is not possible to delete reference peaks from a
peak table.
The retention time is re-calculated automatically when the retention time
interpretation is changed or when a different reference peak is selected.
Besides, you can also enter the retention time directly in the input field
(Peak Table page in the Ret. Time column) in the following manner:
[<Reference Peak>] <Ret. Time> [<Unit>]
You are free to choose the order in which the fields are entered.
If you do not enter a name for the reference peak, the time is interpreted as
absolute time.
Select min or % as the unit. min is the default if no entry is made. The unit
determines whether the time is indicated as the difference (min) or the ratio
(%) to the time of the reference peak. For absolute times, only min is
permitted.
314
Actions in the QNT Editor
Defining the Retention Index and the Kovats Index
(Ret. Index and Kovats Index columns)
Retention indexes can be used to generate generally comparable retention
times. Determine one or several marker peaks in the ⇒Ret. Index (= RI) or
⇒Kovats Index (= KI) column.
Enter a value between 0 and 99.999. Each peak for which a value is
entered is used as marker peak. Increment the values that you enter, i.e.,
either leave the field empty or enter any value above the value for the
predecessor peak. The column is 'empty' by default.
The retention index and the Kovats index are calculated based on these
variables. Both indexes are mainly used in GC to correct retention time
variations. Therefore, the peak table must contain certain marker peaks,
which usually have the following RI or KI values assigned:
Retention Index: 100, 200, 300, ...
Kovats Index: 100*number of carbon atoms of the alcane.
The RI or KI values of all other peaks remain empty; they are calculated by
means of interpolation.
Select Columns > Duplicate Column on the Edit or the context menu to
duplicate the column and thus, allow different values for the individual
channels.
Tip:
The Kovats index is calculated using the adjusted retention times.
Therefore, to enable calculation, enter a ⇒Dead Time on the General tab
page of the QNT Editor.
Entering Amount Values (Amount Column)
Standard substances are labeled by user input in the Amount column.
•
Search the peak table for the substance name of the standard
substance(s), or
•
Enter the name and the retention time as described in How to ...:
Identifying Peaks.
Actions in the QNT Editor
Actions in the QNT Editor
•
315
Type the amount value of the standard into the first Amount column.
This can be a concentration value (such as µg/ml) or the absolute value
(such as µg). If a standard is available in different concentrations, enter
the concentration of each vial in a separate ⇒Amount column. For
example, two concentrations result in two Amount column entries:
If injection is performed several times from the same sample vial
(multiple injection), one amount value is sufficient, even if a different
volume and thus, a different amount, are injected. Chromeleon
considers this automatically.
•
Repeat the procedure for each substance serving as a standard.
Inserting new Amount columns
•
Double-click the header of an existing Amount column (or select
Columns > Edit Amount Columns on the Edit or context menu) to
open the Edit Amount Columns dialog box.
•
From the Assign Standards on the basis of list, select the sample
variable (e.g., ⇒Name for the sample name, ⇒No. for the sample (vial)
number, or ⇒Ref. Amount Set, etc.) that shall be used to identify and
assign standards to the amount column.
•
Use one of the following methods to create new Amount columns:
Click New, type a unique column name in the edit field that appears in
the Amount Column window, and press Enter. Select an Amount
column (or Unassigned) in the Amount Column window to display the
associated standards. Drag the selected sample(s) ( = standard
sample;
= ¾Validation Sample or
= ¾Spiked Sample) from the
Standards window to the new column.
-orClick Auto-Generate. Select the preferred option from the drop-down
list box and click Apply. The following options are available: Select
Generate a separate column for EACH standard to generate a
separate column for all samples with the same value for the selected
sample variable. Or else, select Generate a single column to apply
to ALL standards to generate only one common column for all
standards.
316
Actions in the QNT Editor
Note:
The selected option will apply to all sequences that use the current
QNT File. For example: You select the Vial Number option, and then
assign vial number 5 to Amount column B. From now on, vial number
5 (regardless of its contents) will be assigned to Amount column B in
every sequence that uses this QNT File.
•
Click OK to close the dialog box and return to the peak table.
Selecting the Standard Method (Standard Column)
Use the Standard method to determine how calibration is performed.
Generally, a distinction is made between a calibration based on an internal
or an external standard.
External standard means that the calibration is performed based on one
or several standard samples. (This is the default.)
Using an internal standard substance means adding a standard to the
unknown sample. This can be either before (External/Internal) or after
(Internal) sample preparation. Either the standard (= ¾Internal Standard)
can be added to all samples or it can serve as a basis for a relative area
calculation. In this case, results are displayed only in relation to the amount
or area of the internal standard. (For more information, refer to Theory of
Calibration,
Standard Methods.)
•
In the Standard column, add the standard method for each peak to be
calibrated (external, internal/external Peak, internal Peak).
•
For the last two options, at least one peak of the peak table is used as
the internal standard. This is achieved by assigning it the Make current
Peak to ISTD Peak: intern or Make current Peak to ISTD Peak:
internal/external option.
•
Press the F8 key to enter the standard method assignment via an edit
dialog box.
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317
Selecting the Calibration Function
(Cal.Type and Int. Type Columns)
Via the Int. Type (⇒Integration Type) column, define how the individual
peaks are evaluated.
Press the F8 key or double-click a cell in the Int. Type column. The
following dialog box appears:
In addition to Area, Height, and CE-Area, you can select the relative area
or the relative height as the reference for the evaluation (Integration Type).
If you select Area, all amount calculations refer to the area of one peak.
This Peak Area Integration is the default setting. The peak height
integration is only used in exceptional cases.
The relative height is either calculated relating to all peaks or all identified
peaks. ISTD peaks can be considered for the calculation of the total area
(height).
There are several peak table columns, which define the conversion of the
determined area values into the calculated amount values. A separate
calibration and integration type must be assigned to each calibrated peak in
the peak table.
Enter a calibration function in the Cal. Type column. Press the F8 key to
receive a list of available functions and options. Apart from few exceptions,
the calibration types Linear or Linear with Offset are used.
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Actions in the QNT Editor
The ⇒Calibration Type determines which ¾Calibration Function is used for
deriving a valid amount/area assignment for a larger range from the
calibration points of the standard samples.
For more information about linear and non-linear calibration types, refer to
Theory of Calibration
Calibration Types (Linear), or
Calibration
Types (Non-linear).
Via the calibration type, you can also define the weighting and averaging of
calibration values (see How to ...: Actions in the QNT Editor
Weighting and Averaging Calibration Points.
Either the calibration curve leads through the origin, for example, with the
Lin and Quad calibration types, or the origin is not considered, for
example, with the LOff and QOff calibration types. For calibration types
with offset, the origin can be treated as a calibration point, using the
Include point (0,0) for curve fitting option. In this case, the calibration
curve will not be forced through the origin, but the origin will be considered
nevertheless.
Usually, all other columns of the peak table can be used with the default
settings. Press the F1 key to display more information. Press the F8 key to
open an edit box.
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319
Weighting and Averaging Calibration Points
Weighting
Chromeleon generally weights calibration points of higher concentrations
more strongly than lower concentrations, that is, the course of the
calibration curve is oriented towards the calibration points of higher
concentration. This makes sense as smaller concentrations also cause a
stronger dispersion of the determined area values, which would distort the
result beyond proportion.
To undo or even reverse this type of weighting, four additional weighting
functions have been introduced:
The weighting 1/Amount (or 1/Response) virtually undoes the "normal"
weighting described above; i.e., low and high concentrations are weighted
similarly. The weighting 1/Amount² (or 1/Response²) results in an overproportional weighting of smaller amounts.
By variation of the ¾Number of Replicates, this weighting can be avoided.
Smaller concentrations are injected more frequently than larger
concentrations, more calibration points in the low concentration range
support the calibration curve. Outliers are then less relevant.
Tips:
The stronger weighting of higher concentrations is valid in all ¾Calibration
Functions, with the exception of Point-to-Point.
Outliers can be explicitly "disabled" by excluding a specific standard sample
from the calculation. Exclude the sample on the Calibration tab page of the
quantification method.
When calculating the calibration values Variance, Var.Coeff, Std.Dev,
Rel.Std.Dev, and Corr.Coeff, averaging is not considered! Weighting only
influences the course of the calibration curve, the values are a measure for
the quality of the calibration.
Averaging
To determine the calibration curve, all available ¾Calibration Points are
normally used. As dispersion is stronger for the lower calibration levels,
many users verify the results by using a large number of calibration points.
The calibration curve is thus determined by a larger number of points on
the lower than on the higher level.
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Actions in the QNT Editor
If all points of a ¾Calibration Level are averaged before calculating the
calibration curve, and the subsequent calibration is performed based on
these average values only; the calibration curve is based on one point of
each calibration level only.
Defining the QNT Method for Several Detectors
1. If two detectors are connected in series, the retention times are delayed
against each other. As the flow normally remains unchanged, the shift of
the retention times is constant. Select the ⇒Delay Time parameter to
consider this fact.
First, enter the name of the second detector under Detector Name on the
General tab page of the QNT Editor. Click the arrow key and then select
the name from the list. Enter the measured delay time.
2. For some applications, it might be necessary to modify the QNT Method
for a second detector or another channel. For example, you cannot use the
Delay Time parameter when you are working with ¾Flow Gradients.
Instead, duplicate the following columns and assign the duplicate to one or
several channels:
⇒Calibration Type
⇒Integration Type
⇒Kovats Index
⇒Left/Right Limit
⇒Peak Type
⇒Response Factor
⇒Retention Index
⇒Retention Time
⇒Window
Select the column to duplicate in the Peak Table (Amount Table, Peak
Tracking table, respectively) and select Columns on the context or Edit
menu. Select Duplicate Column... to open the Duplicate Column dialog
box. Click Add Channel... to define the channel(s) for the new column.
If an individual peak is not detected in a channel, the corresponding cell in
the Retention Time column can be left empty. Thus, this channel will not
identify this peak. If you do not wish to include a detected peak into the
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321
report, just leave the corresponding cell in the Retention Time column
empty.
In addition, you can define more detection parameters for the respective
channel(s) in the Channel column on the Detection tab page. Also, refer to
Defining Detection
How to: ...: Actions in the QNT Editor
Parameters (Detection).
Entering Reference Spectra
(Reference Spectrum Column)
You can enter a reference spectrum for each peak in the peak table of the
QNT Editor:
Position the cursor in the Reference Spectrum column, press the F8 key
or left-click twice. In the dialog box, select the reference spectrum of the
peak. Click Browse to load any samples or spectra libraries for selecting
the reference spectrum. If samples are loaded, a list of all peaks in the
corresponding chromatogram is displayed. For spectra libraries, the list
displays the spectra included in the spectra library (see figure). The
selected reference spectrum is displayed in the right window. If the relevant
peak of the peak table is identified in the active chromatogram, the
spectrum is included as overlay. The corresponding match factor is
displayed in the upper corner of the plot frame.
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Actions in the QNT Editor
Click Select to copy the selected reference spectrum to the corresponding
line of the peak table.
Clipboard: Spectra can copied to the clipboard from the spectra plot
window or the spectra library by simultaneously pressing the CTRL+C keys
or by selecting Copy on the Edit menu. In the QNT Editor, either
simultaneously press the CTRL+V keys or select Paste on the Edit menu
to insert the copied spectrum as reference spectrum into the Reference
Spectra column.
Copy Peak Spectra as Reference: If one or several lines are selected in
the Reference Spectrum column, select Copy Peak Spectra as
Reference on the Edit or context menu to use the spectra of the active
chromatogram as the reference spectra.
Overlay of the Reference Spectrum on the Spectra Plot
To include the current reference spectrum from the peak table as Overlay
on the Spectra plot, place the cursor in the spectrum and right-click. Select
Decoration on the context menu. On the Peak Spectra tab page, click
Reference Spectrum in corresponding peak table. Click OK to receive
the spectrum from the Reference Spectrum column in addition to the
current one.
Autogenerating the Peak Table
To save the user from having to determine the retention time of each peak,
peak tables can be created automatically by selecting Autogenerate Peak
Table on the Edit or context menu.
Chromeleon automatically generates a peak table, entering the retention
times of all detected peaks of the currently open chromatogram as set
times. The user need only complete the table by entering component
names and altering the default window and other values as necessary.
Peaks, which are not of interest, can be deleted from the table. These will
consequently be excluded from the report, provided that the Including all
not detected peaks of the peak table parameter in the Integration
Report Properties (Table Properties command on the Edit or context
menus) is turned to OFF.
During automatic table generation, the variables described below are set as
follows:
Autogenerated Peak Table is entered as ⇒Comment.
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323
The peak names (⇒Name) are constructed from the name of the QNT
Method; for example, in the Test peak table, detected peaks will be
assigned the names Test-1, Test-2, Test-3, etc.
The ⇒Window values are entered as absolute values; they represent onethird of the distance from the nearest peak.
No ⇒Group.
The ⇒Amount values and the ⇒Response Factor are set to 1.0.
The peak type (⇒Sample Type) is Auto; that is, it is determined by
Chromeleon.
If you select Autogenerate Peak Table on the Edit menu to generate peak
tables, it is possible to use the results of the spectra library screening (type:
Use spectra library screening results) instead of enumerating all peaks
in the chromatogram (type: Enumerate peaks of current chromatogram).
In this case, library screening is performed based on the parameters
entered in the QNT File. The peak table will include all peaks for which at
least one reference spectrum was found and the name of the best hit.
The following options can also be used:
•
Apply only to peaks with .... With this option, only peaks exceeding a
selectable absolute or relative area or height will be included in the
peak table.
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Actions in the QNT Editor
•
Apply only to peaks in current time interval: Click this button to
include only peaks in the currently displayed section of the
chromatogram; for example, in the zoomed chromatogram section.
•
Copy reference spectrum from ...: This option simultaneously
includes the reference spectrum for each peak. Either the current peak
spectrum from the displayed chromatogram or the library spectrum of
the best hit from library screening can be used. If you want to use the
library spectrum of the best hit, make sure that the Use spectra library
screening results autogeneration option is enabled in the upper
window section.
•
Select Enable peak tracking using peak match type ... to enable
peak tracking for each included peak. Select Spectrum or Spectrum
and time as match type (see How to …: Actions in the QNT Editor
Peak Tracking).
•
Allow multiple best hits or Unique identification: The results of the
spectra library screening will produce a hit list of library spectra for
each peak in the chromatogram. If Unique Identification is selected,
these hit lists are handled as in Peak Tracking; that is, multiple identical
hits with smaller match factors are eliminated. In the case of Allow
multiple best hits, each peak in the chromatogram is copied to the
peak table with the best hit. If the best hits are identical, this can result
in identical entries in the peak table. In this case, adding a number to
the name generates a unique peak; for example 2-, -3.
If you have generated a new peak table via Autogenerate Peak Table, a
note is automatically included in the Comment column of the peak table:
•
If you have selected Enumerate peaks of current chromatogram, the
comment is: Autogenerated.
•
If you have selected Use spectra library screening results, the
comment is: Autogenerated. Spectrum: Name of reference
spectrum, Match: Match factor.
Tip:
If Use spectra library screening results is enabled, the settings made on
the Spectra Library Screening tab page are automatically entered in the
peak table (Peak Tracking tab page).
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325
Defining Detection Parameters
The detection parameters define how to integrate the chromatograms,
which are evaluated with the respective QNT Method, where to suppress
peaks, how to recognize peak start and peak end, etc. This enables the
user in many ways to define the integration and to minimize the reintegration effort for individual chromatograms.
You can either enter the detection parameters in the table on the Detection
tab page or define them graphically in the chromatogram.
In order to enter the parameters in the table on the Detection tab page of
the QNT Editor, express the desired conditions via the columns Parameter
Name and Parameter. They influence the integration of all chromatograms
to which the respective QNT Method applies. There are 26 different
⇒Detection Parameters, which can assume a new value at any time and
whenever desired. Only the value, which was defined, last will take effect,
however, only for the time of the chromatogram. The parameters are then
reset to their default values. If a parameter assumes a new value at a
specified time (Retention Time column), this is called an Event. You do
not have to enter these events in chronological order. They will be sorted
automatically when being saved. The Channel column allows you to define
whether this applies to an individual channel only or to all channels (default)
of a sample.
Each detection parameter has a default value assigned. Thus, in general
about 90% of all chromatograms are automatically integrated correctly by
the system. However, for critical applications, such as wavelength
switching, the user can influence the baseline or the peak type, for
example, rider peak or main peak, or disable the detection for defined
periods. Many parameters can only assume the values ON or OFF. They
are called switches. All other parameters are called variables. Parameter
tables can include up to 100 lines. For an example of how to enter
detection parameters in the table of the Detection tab page, refer to the
image below:
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Actions in the QNT Editor
In many cases, it may be easier to enter the detection parameters
graphically in the chromatogram (see How to ...: Actions in the
Defining Detection Parameters Graphically
Chromatogram
Usually, the chromatogram and the report table are immediately updated
when the detection parameters have been changed. However, you may
disable this function by deselecting the Autom. Re-Integerate option on
the View menu of the QNT Editor. If this option is disabled, save the QNT
Method or select Start Integration on the View menu to start re-integration
with the new integration parameters.
Tip:
Save the setting for this option in the corresponding ¾Report Definition File
(Select Save Report Definition on the Workspace menu.)
In addition, the QNT Editor provides more detection parameters. The
following sections describe how to edit the default values together with the
respective possibilities and advantages. Use the detection parameters for:
Reducing the Number of Evaluated Peaks
Excluding Certain Peaks
Inhibiting Peak Integration
Modifying the Baseline
Modifying the Peak Recognition Algorithm
Defining the Peak Start/End
Defining Rider Peaks
Defining the Area for PPA
Reducing the Number of Evaluated Peaks
To limit the integration report to a manageable size, only the most important
peaks (i.e., the largest peaks) should be included in the evaluation. Define
either the ⇒Minimum Height or the ⇒Minimum Area.
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327
How To
The following example refers to the minimum peak height:
•
Open a QNT File (select Open on the File menu) and click the
Detection tab page.
•
Look at the chromatogram and determine the minimum peak height to
be evaluated. You may also click the smallest peak to be integrated.
Then open the corresponding Peak Properties via the context menu.
The dialog box then indicates the width, height, and area of the peak.
•
Enter the name of the parameter to modify; for example, the
⇒Minimum Height in the Parameter Name column of the quantification
method.
•
Assign the smallest possible signal value, for example, in mAU, as the
new parameter value in the Parameter Value column.
•
In the Time column, determine from which time (relative to injection
time) the parameter becomes valid.
•
Alternatively, you can open an edit dialog box (press the F8 key) to
enter the parameter name, value, and time.
All peaks with an area smaller than the indicated %-value will not be
displayed.
As the parameters affect peak recognition and thus the baseline, you must
consider a tolerance of approximately 5% to ensure that all desired peaks
are recognized.
Another way to define, for example, the minimum area, is to graphically
define the corresponding parameter. For example, follow the steps below:
•
In the chromatogram, find the smallest peak just no longer to be
displayed.
•
Enlarge this peak to be clearly visible.
•
In the chromatogram, right-click to select an area whose area is a
slightly smaller than that of the selected peak.
•
Select Set Minimum Area on the context menu.
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Actions in the QNT Editor
This action enters the selected area as minimum area together with the
time of its left edge into the QNT Method. Enter the 0.000 as time into the
Detection tab page or move the parameter in the chromatogram with the
Detection Parameter Tool so that the minimum area is valid for the entire
chromatogram.
Note:
You can undo the graphical input of detection parameters. Click one of the
tables of the QNT Editor and select Undo on the Edit menu. (In the
chromatogram itself, you can only undo the modifications of the currently
open chromatogram.)
Tip:
To make the report clearer:
•
Select any cell in the report (integration tab page) and select Table
Properties on the Table menu.
•
Select the Reject peaks with smaller area than ...% check box and
specify below which size not to include a peak in the report.
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329
Excluding Certain Peaks
Spikes
Sometimes, very narrow peaks (so-called spikes) occur in chromatograms
due to, for example, air bubbles in the HPLC system. In such a case, the
problem (= the air bubbles in the HPLC system) should be solved first, of
course. However, to use the chromatogram, identification of these narrow
peaks can be suppressed with the QNT Method. Select the ⇒Minimum
Width parameter to define the minimum width for the peaks to be
integrated. Proceed as when entering the minimum height (see
Reducing the Number of Evaluated Peaks).
Peaks above the Detector Maximum
Sometimes peaks occur whose absorption maximum is above the detector
maximum so that integration does not make sense. You can then include
these peaks as unidentified peaks into the peak table by using the
⇒Maximum Peak Height parameter.
Broad Peaks
If an unusually broad peak occurs in the chromatogram, it may be from a
previous sample. Select the ⇒Maximum Width parameter to define this
peak as being unidentified.
Inhibiting Peak Integration
The ⇒Inhibit Integration parameter can be enabled at the time t1 and can
be disabled at the time t2. The peaks within the time window t1 - t2 are not
integrated. Whether this applies to all channels or to only one channel is
specified via the Channel column. To prevent the integration of all peaks in
the range from 0 to 1.0min (especially the injection peak), the following
input is necessary in the quantification method.
Peaks detected in this time span will not be integrated and will not be
included in an integration report.
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Actions in the QNT Editor
Modifying the Baseline
Modifying the baseline can be necessary especially for non-resolved peaks.
The baseline is normally defined via a mathematical procedure. For
calculating the individual peak areas, a perpendicular is dropped to the
baseline from each local minimum (standard).
If you think the integration is started too early or the peak end is delayed, a
better peak start or peak end can be forced by inserting a baseline point
(⇒Baseline Point detection parameter).
If a series of non-resolved peaks can be recognized on an "absorption
mound", for example, due to increased solvent absorption, the baseline can
be forced from minimum to minimum using the ⇒Valley to Valley detection
parameter.
Move baseline point
Default
Valley toValley
If single peaks are piled on a recognizable "absorption mound," these are
integrated individually. To be able to integrate the entire area, the baseline
can be fixed. The parameter ⇒Lock Baseline extrapolates the baseline
horizontally to the intersection with the signal curve. The Global Minimum
option searches for the absolute minimum in the direction of the peak end
or until the next Lock Baseline. Both parameters disable the Valley to
Valley option!
Lock Baseline current level
Default
Lock Baseline global minimum
Tip:
In all of these actions, the classification criterion Peak Type of the peak
table has priority! For a peak of the type Baseline-Main-Baseline, the peak
limits always have baseline contact!
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331
Modifying the Peak Recognition Algorithm
Whether signal variations are interpreted as peaks or not, is usually set
automatically. Manual modification of this "recognition sensitivity" is
possible via the combination of the ⇒Peak Slice and ⇒Sensitivity detection
parameters. Changing the parameters is required, for example, in
chromatograms with unusually wide (many minutes) or very narrow (<
0.1sec) peaks.
Another way to define the peak recognition algorithm is to define both
parameters graphically.
How To
•
Enlarge a baseline section so that the noise is clearly visible.
•
In the chromatogram, right-click to select an area from which the
baseline runs out neither at the top nor at the bottom.
•
Select Set Peak Slice & Sensitivity on the context menu.
The width of the selected area is entered into the QNT Method as peak
slice and the height is entered as sensitivity at the time of the left edge of
the area. Enter 0.000 as time onto the Detection tab page or move the
parameters in the chromatogram with the Detection Parameter Tool so
that this peak recognition algorithm applies to the entire chromatogram.
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Actions in the QNT Editor
Note:
You can undo the graphical input of detection parameters. Click one of the
tables of the QNT Editor and select Undo on the Edit menu. (In the
chromatogram itself, you can only undo the modifications of the currently
open chromatogram.)
The peak recognition algorithm considers signal variations only beyond the
adjusted sensitivity values. Peaks below this threshold are interpreted as
noise.
Both parameters affect peak recognition only, not integration! The area
calculation (integration) is not affected.
Defining Peak Start or Peak End
Depending on the chromatogram type, the peak start or the peak end can
be detected too early or too late. There are several ways to prevent this:
If you think that the integration is started too early or that the peak end is
delayed too much, select the Fronting Sensitivity Factor parameter for
the peak start and the ⇒Tailing Sensitivity Factor for the peak end. The
entered value multiplied with the left or right peak width determines the
peak start or the peak end.
Depending on the chromatogram type, different values may make sense.
'Try which value is best for your chromatograms. A value of 2 is often an
appropriate starting point for finding the best Fronting/Tailing Sensitivity
Factor.
You can also set a new ⇒Baseline Point to force the peak to start later or
to end earlier.
Caution:
However, when setting a baseline point keep in mind that this point will be
valid for all chromatograms, which are evaluated with the respective QNT
Method. If in one of these chromatograms a peak maximum occurs by
coincidence at the time of your hard entered baseline point, the peak
maximum will be defined as base point and the peak will not be detected.
Actions in the QNT Editor
333
Correct too late a peak start or too early a peak end (the latter can occur,
for example, with increased baseline noise as follows:
1. In recorded chromatograms: Select a higher ⇒Peak Slice (= about 20%
of the smallest peak width) and, in addition, a higher ⇒Sensitivity, if
necessary.
2. For samples that have not been processed yet: Change the data
acquisition ⇒Step in the program file. Select the step so that only about 20
data points are recorded for the smallest peak.
Defining Rider Peaks
The detection parameters ⇒Rider Threshold and ⇒Maximum Rider Ratio
allow you to define which peaks shall be detected as ¾Rider Peaks and
which shall be detected as main peaks. The following applies:
The smaller the rider threshold is, the smaller are the peaks that can be
detected as rider peaks. (Peaks below the rider threshold are always
regarded as main peaks. For peaks above the rider threshold, the
Maximum Rider Ratio defines whether a peak is a rider peak or a main
peak.) The larger the maximum rider ratio is, the larger the peaks that can
be defined as rider peaks.
Tip:
When defining rider peaks with these parameters please keep in mind that
the same peak may be detected in two different chromatograms as main
peak and as rider peak. This would result in considerable deviations; for
example, in the calibration. To prevent this, select the peak type Rider (or
Main) for the respective peak in the peak table. Thus, the peak is a rider
peak, if possible (or always a main peak).
In addition, you can define how to skim rider peaks by using the ⇒Rider
Skimming parameter. With the two options Tangential at lower peak end
and Tangential at both peak ends the peak is skimmed by a tangent.
Usually, there is hardly any difference between the results of the two
options.
With the Exponential option, the course of the baseline is approximated by
an exponential function; that is, the peak is skimmed by the exponential
function. This option clearly distinguishes from the two others. In most of
the cases, Exponential maps the actual baseline course very accurate.
With this option, the rider peak will usually receive a more realistic larger
area. To be able to use this option, make sure that a sufficient number of
data points is available.
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Actions in the QNT Editor
Defining the Area for PPA
When using ¾Photodiode Array Detectors and recording ¾3D Fields, you
may check the peak purity with the ¾Peak Purity Index (PPI).
Define the most expressive area of you spectrum by means of the ⇒Peak
Purity Start/End Wavelength parameters and limit the examination to this
area only.
The detection limit becomes especially apparent with very small peaks. The
influence (that is the noise, drift and limits of the measuring method) on the
peak spectrum is strongly developed at the peak start and peak end
because, in these areas, the concentration of the peak substance in the
flow cell is very low. Set the ⇒Peak Purity Threshold parameter to reduce
the influence on the PPI and the match factor and, thus, to prevent that
contaminated substances are indicated falsely. The PP threshold value
defines the percentage of the peak height starting as of which the spectra
will be considered for the respective purity examination.
If you work close to the detection limit, use higher PP threshold values to
reduce the influence of the detection limit on the purity examinations. If you
are mainly interested in the purity of large peaks, use lower PP threshold
values to examine the purity over the largest possible area of the peaks.
Grouping Peaks
If you are interested in the sum parameters for two or more peaks, you can
1. Define these peaks as a peak group (that is treated as one single peak).
2. Define them as a group of peaks.
1. If the peaks lie close together and are not baseline separated,
example, with overloaded columns, define the peak group start and
peak group end via the ⇒Peak Group Start/End parameters on
Detection tab page. The baseline will then be drawn from the start of
peak group to its end. Such a peak group is treated as one single peak.
for
the
the
the
2. To define a peak group in which the peaks do not necessarily succeed
one another, for example, to determine the amount/concentration of an
entire class of substances, take the following steps:
•
Identified peaks: Select Column > Display Column on the context
menu to insert the ⇒Group column into the peak table if the column is
not yet displayed. For those peaks that should belong to this group,
type the desired group name into this column.
Actions in the QNT Editor
335
•
Unidentified peaks: Click Unidentified peaks… on the General tab
page to define the period for which this group shall be valid. The
baseline will be drawn in the same way as for individual peaks.
•
Add the Group Amount column to the Report and the ¾Printer
Layout. This column shows the desired ⇒Amount value. In the report,
open the dialog box Insert/Add Report Column via the Insert Column
or Add Column commands on the context menu. Select the Peak
Results category and then choose Group Amount as variable.
Tip:
If the selected calibration type is, for example, LOff instead of Lin, the
value in the Group Amount column will not be identical to the sum of the
amount values of the individual peaks even if the group includes all peaks
of the chromatogram.
Subtracting a Blank Run Sample
On the General tab page of the QNT Editor, use the Blank Run & Matrix
Blank section to consider absorption values of a ¾Blank Run Sample, a
¾Matrix Blank Sample, or any other sample (¾Blank Run Subtraction).
•
Select No Blank Run Subtraction if no correction is to be performed.
If the absorption values of a specific sample are to be considered for
sample evaluation, determine which sample is to be used as Blank Run
Sample.
•
Select Subtract Recent Blank Run Sample in Corresponding
Sequence if the Blank Run Sample (sample type: Blank) that was
processed last in the current sequence before the current sample is to
be used.
•
Select Subtract a Fixed Sample to perform the correction with a
specific Blank Run Sample. Click Browse to search for the sample.
When subtracting a blank run sample, the chromatogram of the blank
sample is subtracted point by point from the active chromatogram. If
the current sample is a standard sample, the difference between the
two chromatograms is used for the calibration.
336
•
Actions in the QNT Editor
Enable Matrix Blank Subtraction enables the subtraction of matrix
blank samples. Contrary to the other options, the resulting peak areas
or peak heights are subtracted for peaks that have been identified for
the matrix blank sample and for the unknown sample.
Tip:
Although sample types other than "blank" can be subtracted as well,
this usually does not make sense because often-negative peaks would
occur in the resulting chromatogram.
Disabling Calibration Samples
The Calibration tab page provides information about the calibration and
allows you to determine which sample shall be used.
It depends on the ⇒Calibration Mode which sample ⇒Types available (for
the sample type, refer to the symbol in the Name column):
Usually standard samples
are used for calibration.
Only in the Standard Addition mode, i.e., if you use the ¾Standard
Addition method, you can use the sample types Unspiked and Spiked
(see ¾Spiked Sample) for calibration.
Click the box in the Enabled column to open the Disable (Enable)
Standard xyz dialog box and define whether the respective sample shall
be used for calibration purposes. You can exclude the respective
calibration sample from calibration either for all peaks and all channels
(default setting) or only for the selected peak and/or channel. For more
information about how to use this option, refer to the following examples:
The calibration sample was contaminated
Exclude the sample from the calibration. Accept the default setting: Disable
the sample for All Peaks and All Channels.
By mistake, too much of a substance was added to the calibration
sample
Do not exclude the sample completely from calibration, but disable the
calibration for this substance, instead. Click the peak and select Selected
Peaks and All Channels in the Disable (Enable) Standard xyz dialog
box.
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337
Contamination in the calibration sample that is detected in one
channel only
You do not need to exclude the sample completely from calibration. Select
the channel that detects the contamination and select All Peaks and
Selected Channel in the Disable (Enable) Standard xyz dialog box.
For more information about calibration, refer to How to ...: Actions in the
QNT Editor
Calibration
Calibration
The following topics provide a detailed description of the theory and
practical use of the available calibration possibilities. The first examples
describe the different applications:
Introduction and Example: 1 Standard and 1 Substance
Several Standards with Several Substances Each
Multiple-Point Calibration Using One Single Standard
Calibrating Using Standards of an Old Sequence
Standard Addition
Calibrating Unstable Substances
No Pure Substance Available - Known Relative Extinction
Coefficient
For an overview of the different calibration options for which an ¾Internal
Standard (ISTD) is used, refer to
Calibrating with an Internal
Standard Substance.
The ⇒Calibration Mode determines the standard samples that are used for
calibrating specific unknown samples. For an overview of the different
calibration modes and the topics that provide more information, refer to
Calibration Modes for External Calibration.
For more information about validation, refer to:
Entering the Concentration/Amount of the Validation Sample
Validating the Calibration Curve
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Actions in the QNT Editor
In addition, you may invert variables; see
Independent Variables.
Inverting Dependent and
Note:
To apply an existing calibration to a new sequence consisting of one or
several Unknown samples, set the ⇒Calibration Mode to Fixed. Always
perform calibration manually (Calibrate). The Auto-Recalibrate option is
not available.
Introduction and Example: 1 Standard and 1 Substance
Most calibrations in HPLC and IC are performed using external standard
samples. In the simplest case, the corresponding peak area for a known
amount of a substance is determined for one standard sample only.
Chromeleon then calculates the slope (c1) of the ¾Calibration Function
from the ratio of the amount and the peak area. (In this case, you can only
select the Linear without Offset calibration type). Chromeleon uses this
slope to calculate the ⇒Amount of this substance in unknown samples.
Example:
You want to determine the amount of substance A in two samples, Sample
1 and Sample 2. A standard sample (Standard) is available. For each
sample, an injection volume of 20 µl is injected by an autosampler. The
samples are located at the autosampler positions 1 (standard), 2, and 3
(unknown samples).
Sample List
In the Browser, create the following sequence using the Sequence Wizard:
(For information about the Browser, refer to Data Management
Browser.)
The
Note:
For more information about how to create a sample list, refer to How
to ...: Actions in the Browser
Creating a Sample List (Sequence).
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339
QNT Method/General Tab
The concentration of substance A in the standard sample is 12 mg/l. The
calibration curve for substance A shall be linear (calibration type: Lin) and
run through the origin. After processing this sequence, you have to create
the QNT Method for performing the calibration. On the General tab page of
the ⇒QNT Editor, enter the unit for all other entries in the ⇒Dimension of
Amounts field. (For this example, enter "mg/l"). In the Global Calibration
Settings section, keep the setting for the ⇒Calibration Mode, i.e., Total.
QNT Method/Peak Table Tab
Do not change the defaults in the Standard (External) and ⇒Calibration
Type (Lin) columns on the Peak Table tab page either. Chromeleon
automatically creates a "default" amount column. If only one standard
sample is available (as is in this case), you can keep this standard sample
as well. Enter the concentration in the Amount column:
Note:
If more than one substance is available in the samples, append a new line
to the table using the Lines > Append Line commands on the context
menu. Enter the concentration in the corresponding cell of the Amount
column.
Chromeleon now automatically calculates the amount for the two unknown
samples. You can display the results afterwards in the report on the
Integration tab page:
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
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Actions in the QNT Editor
Several Standards with Several Substances Each
Calibrations are usually performed using several standards. They often
contain more than one substance to be calibrated. The simplest case is two
standard samples that contain two substances each. In this case, you can
determine the slope and the offset and/or curvature of the calibration curve
(depending on the number of acquired data points).
Example:
You want to determine the amount of substances A and B in two samples,
Sample I and Sample II. Two standard samples, containing different levels
of Standard 1 and Standard 2, are available. Both standard samples
contain substance A and substance B. For each sample, an injection
volume of 20 µl is injected by an autosampler. The samples are located at
the autosampler positions 1 (Standard 1), 2 (Standard 2), 3 (Sample I), and
4 (Sample II).
Sample List
The sequence appears as follows (for more information, refer to How to ...:
Actions in the Browser
Creating a Sample List (Sequence)):
QNT Method/Peak Table Tab
In Standard 1, for example, the concentration of substance A is 10.2 mg/l
and the concentration of substance B is 20.1 mg/l. Standard 2 contains
30.5 mg/l of substance A and 49.7 mg/l of substance B. Thus, you have to
create the following peak table in the ⇒QNT Editor:
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341
Remove the default ⇒Amount column and add two new Amount columns
(one for each standard level):
•
Double-click the header of an existing Amount column (or select
Columns > Edit Amount Columns on the Edit or context menu) to
open the Edit Amount Columns dialog box.
•
From the Assign Standards on the basis of list, select the criterion
(Name, Vial Number, Sample ID, etc.) to use to identify and assign
standards to the amount columns.
Note:
The selected option will apply to all sequences that use the current QNT
File. For example: You select the Vial Number option, and then assign vial
number 5 to Amount column B. From now on, vial number 5 (regardless of
its contents) will be assigned to Amount column B in every sequence that
uses this QNT File.
•
Click Auto-Generate. Select Generate a separate column for EACH
standard in the drop-down combo box and click Apply. Two new
columns will appear in the Amount Column window. Double-click the
columns to rename them, if desired.
•
Click OK to close the dialog box and return to the peak table.
Note:
If one of the substances is not available in the standard sample, the
corresponding cell in the Amount column should be left empty.
If you are using several standards for calibration, you may prefer to select a
⇒Calibration Type other than Lin (Linear without Offset).
Using these settings, Chromeleon automatically calculates the
concentrations of substance A and substance B in the two unknown
samples.
For an overview of the calibration options provided by Chromeleon, refer to
How to ...: Actions in the QNT Editor
Calibration.
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Actions in the QNT Editor
Multiple-Point Calibration Using 1 Single Standard
If you want to perform a multiple-point calibration (see ¾Single-Point and
Multiple-Point Calibration) using only one standard sample, you can inject
different injection volumes (= quasi ¾Dilution Series).
Sample List
For example, if you inject 10, 20, and 40 µl of just one standard, the sample
list will appear as follows:
For Chromeleon, each injection is an individual sample. To distinguish
between different injections made from the same standard sample vial, you
may append the sample number and the injection number (see example
above).
QNT Method/General Tab
The standard sample contains, for example, 10 ml/l Uracil. As all injections
of a dilution series are made from the same sample vial with the same
concentration, you cannot represent the concentration in the calibration
curve. Instead, enter the actually injected amount in the ⇒Amount column
and enter a ⇒Dimension of Amounts. For example, enter ng on the
General tab page of the ⇒QNT Editor.
QNT Method/Peak Table Tab
Introduction and Example), you
As in the introduction example (see
do not need to change the default Amount column setting on the Peak
Table tab page.
If the calibration line does not run through the origin as in the example
below, select Linear with Offset (= LOff) as ⇒Calibration Type.
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343
These settings will then result in the following calibration line:
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Calibrating Using Standards of an Old Sequence
Calibration standards are often quite expensive. Thus, if the calibration
curve remains constant for weeks or months, the standards of a sequence
can be used for calibration for several weeks before the calibration
constancy needs to be checked again.
Sample List
In this case, the new sequence will neither contain standards nor
¾Validation Samples but unknown samples only (perhaps plus ¾Blank Run
Samples and/or ¾Matrix Blank Samples).
QNT Method/General Tab
Select Fixed as ⇒Calibration Mode in the Global Calibration Settings
section:
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Actions in the QNT Editor
In Fixed mode, manual calibration is possible only. That is, you have to add
standard samples on the Calibration tab page (see below). Then, click
Calibrate on the General tab page to include the newly entered standards
in the calibration.
QNT Method/Calibration Tab
If the Fixed calibration mode is selected, the Calibration table is empty at
first. Select Append Standard on the context menu to add the desired
standard(s). The Browse dialog box is opened. Select the desired standard
sample from any sequence:
The Calibration tab page then lists the desired standards:
After you have entered all standards, click Calibrate on the General page
to perform calibration with those standards. After each change, for
example, if you exclude a standard by disabling the Enabled check box or
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345
if you correct the injection volume in the sample list, you have to click
Calibrate again!
Note:
If you later edit the name of the sequence, from which the standards were
added or if you change the name of the directory that houses the
sequence, the reference becomes invalid. Then, you have to add the
standards again on the Calibration page.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Standard Addition
The sample matrix can considerably influence sample analysis. To consider
this, a known amount of one or more substances is added to the unknown
sample, particularly in ion and gas chromatography. In this way, the
concentration of these substances is increased by a value that is exactly
known. Afterward, the original and the ¾Spiked Sample are analyzed,
using ¾Standard Addition.
Sample List
In this case, the new sequence includes only unspiked, unknown samples
(⇒Type (Sample Type): Unspiked) and the associated Spiked samples.
For example, the new sequence could look as follows:
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Actions in the QNT Editor
Each unknown sample was spiked three times with the same known
amounts. In this example, several unknown samples are analyzed.
Therefore, the unknown samples must be assigned to the associated
spiked samples, via the ⇒Std. Add. Group column. In this column, the
samples are assigned to a common standard addition group. For example,
in this way, Sample 1 is calibrated with the spiked samples Spiked 1_1,
Spiked 1_2, and Spiked 1_3.
We recommend spiking unknown samples always with the same amount of
the same substances. In the ⇒Ref. Amount Set column, you can then
assign the spiked samples to the same amount values in the Amount
Table of the QNT Editor. Enter the same ID for the associated spiked
samples into this column (here: Spike1 for samples Spiked 1_1, Spiked
2_1, and Spiked 3_1).
QNT Method/General Tab
Select Standard Addition as ⇒Calibration Mode in the Global
Calibration Settings section:
Re-calibration is performed automatically; it cannot be disabled.
QNT Method/Amount Table Tab
On the Amount Table tab page, insert standard columns for every spiked
sample:
•
On the context menu, select Columns and then, select Edit Amount
Columns....
•
From the Assign Standards on the basis of list, select Ref. Amount
Set if you have used the associated column in the sample list (see
above) or, select another option from the list, e.g., Name.
•
Click Auto-Generate and select Generate a separate amount
column for EACH standard from the list:
Actions in the QNT Editor
•
347
Click Apply to add a column for all identically spiked samples in the
Amount Table.
Concentrations
If your dimension of amount is a concentration, enter the added
concentrations of the various substances in the spiked samples into the
associated Amount columns.
or
Amounts
If your dimension of amount is an amount, enter the added amounts of the
various substances into the associated Amount columns.
Chromeleon automatically analyzes the Unspiked sample(s) using the
Standard Addition method.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
348
Actions in the QNT Editor
Calibrating Unstable Substances
If you want to calibrate unstable substances, the concentration in the
samples that are analyzed later may be considerably lower than the
concentration in those samples that are analyzed first although originally
the concentration was the same. The instability of the substance makes
calibrating more difficult. Chromeleon provides two possible solutions:
Sample List
To consider the instability of substances one or several standard samples
are added to a series of unknown samples every now and then. The
sequence will then appear as follows, for example:
QNT Method/General Tab
The bracketed calibration illustrated in the above figure has been achieved
using the Bracketed ⇒Calibration Mode (set in the Global Calibration
Settings section). The four less decayed standards 1-4 (from positions 1,
2, 5, and 6) are used for calibrating the less decayed unknown samples
(samples 1 and 2 from positions 3 and 4). The more decayed standards 3-6
(from positions 5, 6, 9, and 10) are used to calibrate the higher decayed
samples 3 and 4 from positions 7 and 8. The calibration curve shows the
corresponding ¾Calibration Points, only.
For more information about the Bracketed mode see
Mode: Bracketed.
Calibration
Tip:
Knowing the half-life of an unstable substance (this is especially true for
radioactive substances) is a clever way to calculate the chromatogram, as it
would be without the decay. Use a virtual channel (¾Virtual Channel
Driver) to record all chromatograms in this special channel as if the
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349
substance would not decay. For a program example, refer to How
Program Examples for Virtual Channels.
to ...: Device Control
Having recorded these ¾Virtual Signals, perform the calibration as
described in the above examples for stable substances.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
No Pure Substance Available - Known Relative
Extinction Coefficient
If you wish to quantify substance A although the pure substance A is not
available, calibration can be performed nevertheless if the ratio of the
extinction coefficient to the extinction coefficient of a different substance B
is known. First, create the corresponding sample list following the
Several Standards with Several Substances Each.
description in
Then, create the ⇒Amount columns for your standards. As the pure
substance A is not available and thus is not contained in the standards, the
cells for substance A remain empty in the Amount table:
The entry made in the ⇒Response Factor column is the decisive entry.
This column allows you to use the calibration of substance B for substance
A. Double-click the selected cell (see above) to open the Response Factor
for Substance A dialog box and enter the factor of the extinction
coefficient at the measuring wavelength between the two substances:
Resp.Fact. =
Ext.Coeff A
Ext.Coeff B
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Actions in the QNT Editor
Set the interpretation to Relative to Peak and select substance B:
The resulting calibration curve is 75% of the ¾Calibration Function of
substance B. No calibration points are indicated in the calibration curve
because no points were acquired for substance A:
Extinction coefficients depend on the measuring wavelength. Thus, enter
the corresponding response factor needs for each channel. Create a
separate Response Factor column for each channel:
Select Columns... on the context menu of the existing Response Factor
column. Select Duplicate and then select the desired channel by clicking
the Add Channel button.
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351
Note:
The calibration curve of the reference peak is the decisive factor. If
possible, it should not have an offset. Otherwise, errors may occur when
calculating the amount using the response factor. This is especially true for
lower amounts.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Calibrating with an Internal Standard Substance
Especially in gas chromatography but also in HPLC or IC, calibration using
an internal standard substance, i.e., an ¾Internal Standard (= ISTD), is
used to eliminate possible measuring and sample preparation errors.
Chromeleon provides different calibration possibilities using one single
internal standard substance:
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Actions in the QNT Editor
If several internal standard substances are available, the flow chart
changes accordingly. For more information about the numbered options in
the flow chart, refer to:
1.
Compensating Measurement Errors (Internal Calibration)
2.
Correcting Sample Preparation Errors
(Internal/External Calibration)
3.
Using Different ISTD Amounts (Variable ISTD)
4.
Calibrating without Standard Samples
Compensating Measurement Errors
(Internal Calibration)
It is possible to compensate measuring errors, such as deviations that
occur during the injection, by calibrating with an internal standard
substance, i.e., the ¾Internal Standard. In the purely internal standard
method, calibration is performed relatively to the internal standard
substance, using area ratios instead of absolute areas.
Sample List
In the simplest case, only one standard sample is available. In this case,
the sample list looks, for example, as follows:
QNT Method/Amount Table Tab
Enter the amounts of the single substances contained in the standard
sample in the Amount column of the Amount table of the QNT Editor:
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353
QNT Method/Peak Table Tab
The internal standard substance is defined in the peak table. If all three
samples contain two relevant substances only as in the example, make the
following entries in the Standard column, using the F8 input box.
•
For substance 1, select Use this peak as Internal Standard and thus
define this substance as the internal standard. (The light yellow
background of the line and the ISTD: Internal entry indicate that the
assignment is correct.)
•
In the F8 input box of substance 2, select the option Internal. From the
Associated ISTD Peak field, select the standard substance serving as
internal standard (here: Subst. 1).
•
If necessary, repeat this operation for every additional substance that
should be calibrated based on the internal method.
After completing the input, the substance 2 is labeled Internal Subst. 1 in
the Standard column. In addition, substance 1 is labeled as the internal
standard for internal calibration:
Amount Calculation
Chromeleon automatically calculates the amount values for the "unknown"
substance. The calculation is performed according to the formula for
amount calculation (rel. to ISTD) (for more information, refer to ¾Formula
for Amount Calculation (Rel. to ISTD) in the Glossary section). Chromeleon
calculates the amount for substance 2 adapted by the deviation of
substance 1.
For detailed information about how to calculate the amount for unknown
substances using internal calibration, refer to
Internal Calibration:
Calculation.
For an overview of the different calibration possibilities with internal
standard substances provided by Chromeleon, refer to How to ...: Actions
Calibrating with an Internal Standard Substance.
in the QNT Editor
354
Actions in the QNT Editor
Internal Calibration: Calculation
In the purely internal standard method, calibration is performed only by
means of an internal standard substance, i.e., the ¾Internal Standard.
Calculation is performed using area ratios instead of absolute areas. That is
why in the ¾Formula for Amount Calculation (Rel. to ISTD)
⎛y
Amount p = f p ⎜⎜ Peak
⎝ y ISTD
⎞
Dil.Fact. n
⎟⎟ * Resp.Fact. p *
Weight n
⎠
Example
In a clinic lab, prepared urine samples of two patients are examined for the
catechol level. Adrenaline and dopamine are to be determined. Two
standard solutions of different concentrations are available (STD 1: 50 ng/µl
each; STD 2: 100 ng/µl adrenaline/dopamine each). To correct possible
inaccuracies regarding the precise dosing of the autosampler, catechol is
added. The internal standard method is selected. 20 µl of each unknown
sample and each standard sample is mixed with 20 µl of the catechol
solution. As the concentration of the added catechol solution is 10ng per µl,
each sample and standard vial (40 µl) contains 10 x 20 = 200 ng catechol.
This means that exactly 200/4 = 50 ng catechol is injected with each 10 µl
injection.
a) User Input
Sample List
In contrast to the examples in the external and internal/external calibration,
the second calibration point is not determined by means of the modified
injection volume but by means of a second standard sample with the
double concentration. As a result, two different autosampler positions are
used (1 and 2).
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355
QNT Method/Peak Table Tab
As the calibration of adrenaline and dopamine is performed with two
standard concentrations (STD 1, STD 2; different autosampler position in
the sample list), two amount values are entered for each peak. Then, the
internal standard substance is defined.
How to define the Internal Standard substance:
•
Select the Standard column in the Catechol line and open the F8 edit
box.
•
Select the Use this peak as internal Standard option and thus define
catechol as Internal Standard.
The yellow coloring of the line and the ISTD: Internal entry indicate the
correct assignment.
•
Change to the Standard column in the Adrenaline line and open the
F8 edit box again.
•
Select the option Internal and then, select the standard substance
serving as Internal Standard (here: catechol) in the Associated ISTD
Peak field.
•
Perform this operation for each peak that should be calibrated with the
Internal method (here dopamine).
After completing the input, the following occurs: In the Standard column,
alanine, and dopamine are labeled Internal Catechol. In addition, catechol
is marked as the internal standard substance by a darker shade of yellow.
Note:
In addition to the color changes from light yellow to dark yellow, there are
two other possible colors. If the retention time is expressed depending on a
selected reference peak (see ⇒Retention Time), a light blue background
highlights this reference peak in the peak table. If this reference peak is
also used as the internal standard peak, the corresponding line is displayed
in green (blue + yellow = green).
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Actions in the QNT Editor
QNT Method/General Tab
The Total mode is selected. This ensures that the calibration of all samples
(Sample I and II) is performed based on all standard samples (STD 1, STD
2).
QNT Method/Calibration Tab
This page shows all standard samples (of a sequence) that are inserted for
calibrating the current sample.
Press the F4 key or the SHIFT+F4 key combination to successively open
the sample of a sequence. The standard samples forming the basis for
calibration are shown for each sample.
Due to the selected mode, sample I and II appear as follows:
If you notice that an error occurred during the analysis of the standard
sample, you can "exclude" this standard sample. Remove the standard in
the Enable column on the Calibration tab page of the QNT Editor. Only
the standard samples labeled X are considered for the calibration.
b) Analysis Structure
Injection is four times. During the first run, the first calibration point of the
adrenaline and dopamine calibration curve is determined via STD 1, and
during the second run the second point is determined accordingly via STD
2. Run three serves to determine the concentration of adrenaline and
dopamine in sample I. In the fourth run, the concentrations of adrenaline
and dopamine in sample II are determined. In addition, the area of the
added catechol is determined in each run.
Chromeleon determines the following area values:
Name
Area Adrenaline
Area Dopamine
Area Catechol
STD 1
125
200
250
STD 2
250
400
250
Sample I
223
150
245
Sample II
178
380
255
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357
The area values determined for catechol reflect the ratio of the injected
volume or amounts (except for minor inaccuracies).
c) Calculation of
Amount p ,rel
and
x p , rel
The ratio of Area (Peak) to Area (ISTD) results in
x( prel ) =
x p , rel
:
Area p
Area ISTD
Substance
Area
Area (ISTD)
x (p, rel)
Adrenaline (STD 1)
125
250
0.5
Adrenaline (STD 2)
250
250
1.0
Dopamine (STD 1)
200
50
0.8
Dopamine (STD 2)
400
1.6
The ratio of Amount (Peak) to Amount (ISTD) results in
Amount ( p ,rel ) =
Substance
Amount p ,rel
:
Amount Peak
Amount ISTD
Amount
Amount (ISTD)
Adrenaline (STD 1)
50
50
Amount (p, rel)
1.0
Adrenaline (STD 2)
100
50
2.0
Dopamine (STD 1)
50
50
1.0
Dopamine (STD 2)
100
50
2.0
d) Calculation of the Calibration Coefficients
A linear calibration curve through the origin (calibration type: Linear) can
already be described by one calibration coefficient (c1). If the example is
selected so that the calibration points in each calibration curve are located
exactly on a straight line, that is, for example, in an exact measurement, c1
results as the y/x-quotient of each value pair Amount p rel to x p rel:
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Actions in the QNT Editor
Substance
Amount (p rel) / x (p rel)
c1
Adrenaline
1.0/0.5
2.000
Adrenaline
2.0/1.0
2.000
Dopamine
1.0/0.8
1.250
Dopamine
2.0/1.6
1.250
If the calibration points are not located exactly on one line, Chromeleon
calculates an optimized c1 approximate value for each substance. If a
different calibration type is selected, Chromeleon calculates the
corresponding calibration coefficients (c0, c1, and c2).
e) Amount Calculation
By means of the ¾Formula for Amount Calculation, the relative amount
(=relative to the amount of the ISTD) of the sample content adrenaline and
dopamine can be calculated from the known c1 and from the ratio peak
area (sample) to peak area (ISTD). If the ⇒Dil. Factor (Dilution Factor) and
⇒Weight (Sample Weight Factor) correction factors are assumed to be 1,
the following Amount/Amount ISTD values result:
Sample I
Calculation: Amount/Amount ISTD
Adrenaline
2.000 x (223/245) = 1.820
Dopamine
1.250 x (150/245) = 0.765
Sample II
Calculation: Amount/Amount ISTD
Adrenaline
2.000 x (178/255) = 1.396
Dopamine
1.250 x (380/255) = 1.863
By multiplication with the amount values of the internal standard substance,
the actual amount values for adrenaline and dopamine in the analysis
samples can be calculated.
Sample I
Calculation: Amount
Adrenaline
1.820 x 50 = 91.00 [ng]
Dopamine
0.765 x 50 = 38.25 [ng]
Sample II
Calculation: Amount
Adrenaline
1.396 x 50 = 69.80 [ng]
Dopamine
1.863 x 50 = 93.15 [ng]
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to...: Actions in the QNT Editor
Calibration.
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359
Correcting Sample Preparation Errors
(Internal/External Calibration)
It is possible to eliminate errors occurring during sample preparation by
calibrating using the internal/external method. If you use the
internal/external method, an internal standard substance, i.e., the ¾Internal
Standard (= ISTD), is used to adapt the external calibration to the
corresponding sample. Exactly that amount of the internal standard
substance is added to each standard sample and each unknown sample
that makes sure that the concentration is identical in all sample vials. The
following entries are required:
Sample List
In the simplest case, only one standard sample is available. In this case,
the sample list looks, for example, as follows:
QNT Method/Amount Table Tab
Enter the amounts of the single substances contained in the standard
sample in the Amount column of the Amount table of the QNT Editor:
QNT Method/Peak Table Tab
The internal standard substance is defined in the peak table. If all three
samples contain two relevant substances only as in the example, make the
following entries in the Standard column, using the F8 input box.
•
For substance 1, select Use this peak as Internal Standard and thus
define this substance as the internal standard substance. (The light
yellow background of the line and the ISTD: Internal entry indicate that
the assignment is correct.)
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Actions in the QNT Editor
•
In the F8 input box of substance 2, select the option Internal/External.
In the Associated ISTD Peak field, select the standard substance
serving as internal/external standard (here: Subst. 1).
•
If necessary, repeat this operation for every additional substance that
should be calibrated based on the internal/external method.
After completing the input, the substance 2 is labeled Int/Ext Subst. 1 in
the Standard column. In addition, substance 1 is labeled as the internal
standard for internal/external calibration. The description ISTD: Internal is
changed to ISTD: Int/Ext and the yellow coloring of the Subst. 1 line is
intensified:
Amount Calculation
Chromeleon automatically calculates the amount values for the substance
2. The calculation is performed according to the formula for amount
calculation (for more information, refer to ¾Formula for Amount Calculation
in the Glossary section). In case of internal/external calibration, the ISTD
factor is used to correct the external calibration, using the ISTD amount
determined for the respective sample.
The ISTD factor considers the ratio of the (nominal) amount entered in the
peak table to the amount that was determined for the respective sample
due to the peak area:
ISTD Factor =
Amount ISTD ( PeakTable )
Amount ISTD ( Sample )
Using this formula, Chromeleon calculates the amount for substance 2 that
has been adapted by the deviation of substance 1.
For detailed information about how to calculate the amount of unknown
substances using internal/external calibration, refer to
Internal/External
Calibration: Calculation.
For an overview of the different calibration possibilities with internal
standard substances provided by Chromeleon, refer to How to ... Actions
Calibrating with an Internal Standard Substance.
in the QNT Editor
Actions in the QNT Editor
361
Internal/External Calibration: Calculation
In a calibration with the internal/external method, external calibration is
adapted to the corresponding sample, by using an internal standard
substance, i.e., an ¾Internal Standard (= ISTD):
Each standard sample and each unknown sample is added exactly that
amount of internal standard substance to make sure the concentration is
identical in each vial. The internal standard substance and the substance to
be determined are calibrated using known standard solutions; that is, the
¾Calibration Coefficients are determined from the ⇒Amount values of the
standard sample and the corresponding peak area values by means of the
¾Calibration Function.
Thus, the amounts of all substances (including the ISTD) can be
determined. As the concentration of the internal standard substance is
identical in all samples, the same ISTD amount should result.
If this is not the case, an error occurred in the chromatography system
(sample preparation, injection, carry-over, etc). The deviation of the actual
ISTD amount from the nominal ISTD quantifies of the error. If the
substances to be determined and the internal standard substance are
similar, it can be assumed that the values of the remaining contents of the
sample deviate in the same way; that is, they are incorrect. A correction by
the deviation of the nominal and the actual internal standard substance
supplies the actual values.
Example:
You want to determine the concentration of alanine and glycine in two
samples. One standard sample is available. The internal standard
substance norvaline is added to all three sample vials, so that the final
concentration is 10 mmol/l. During the subsequent pre-column
derivatization, 10 µl sample + 20 µl OPA reagent + 20 µl stop reagent are
pipetted together. A constant concentration of 2 mmol/l is added from the
norvaline.
The chromatographic separation follows the derivatization of the amino
acids in OPA derivatives. 10 and 20 µl of the standard solution (¾Dilution
Series) and 10 µl of each sample (autosampler position 2 and 3) are
injected.
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Actions in the QNT Editor
a) User Input:
Sample List
QNT Method/Peak Table Tab
As the calibration of alanine and glycine is performed with only one (STD 1)
standard concentration (same autosampler position in the sample list), only
one amount value is be entered for each peak. Then, the internal standard
substance is defined.
How to define the Internal Standard substance
•
In the Norvaline line, select the Standard column and open the F8 edit
box.
•
Select Use this peak as internal Standard and thus define norvaline
as the internal standard substance.
A light yellow background and the ISTD: Internal entry indicate that the
assignment is correct.
•
In the Alanine line, change to the Standard column and open the F8
edit box again.
•
Select the option Internal/External. In the Associated ISTD Peak
field, select the standard substance serving as Internal/External
Standard (here: norvaline).
•
Repeat this operation for each peak that should be calibrated using the
Internal/External method (here: glycine).
Actions in the QNT Editor
363
After completing the input, the following occurs: In the Standard column,
alanine and glycine are labeled Int/Ext Norvaline. In addition, norvaline is
labeled as the internal standard for the Internal/External calibration. The
ISTD: Internal description is changed to ISTD: Int/Ext and the yellow
coloring of the norvaline line intensifies.
Note:
In addition to the color changes from light yellow to dark yellow, there are
two other possible colors. If the retention time is expressed depending on a
selected reference peak (see ⇒Retention Time), a light blue background
highlights this reference peak in the peak table. If this reference peak is
also used as the internal standard peak, the corresponding line is displayed
in green (blue + yellow = green).
QNT Method/General Tab
The Total mode is selected. This ensures that the calibration of all samples
(Samples I and II) is performed based on all standard samples (STD 1).
QNT Method/Calibration Tab
The page shows all standard samples (of a sequence) that are used for
calibrating the current sample.
Press the F4 key or the SHIFT+F4 key combination to successively open
the samples of a sequence. The standard samples forming the basis for
calibration are shown for each sample.
Due to the selected mode, samples I and II appear as follows:
If you notice that an error occurred during the analysis of the standard
sample, you can "exclude" this standard sample. Remove the standard in
the Enable column on the Calibration tab page of the QNT Editor. Only
the standard samples labeled X are considered for the calibration.
364
Actions in the QNT Editor
b) Analysis Structure:
Injection is four times. During the first run, the first calibration point of the
calibration curve is determined, and during the second run, the second
point is determined. Run three serves to determine the concentration of
alanine and glycine in sample I. In the fourth run, the concentrations of
alanine and glycine in sample II are determined. Chromeleon determines
the following area values:
Name
Area Alanine
Area Glycine
55
80
40
110
160
80
Sample I
45
75
39
Sample II
80
150
41
STD 1 (first run)
STD 1 (second run)
Area Norvaline
The determined area values of the internal standard substance norvaline
reflect the ratio of the injected volumes (amounts), except for minor
inaccuracies.
c) Calibration Points
From the known amount values and from the determined area values of the
standard samples, the value pairs of the individual calibration points can be
established:
Substance
Area value
Alanine
45
Alanine
90
Glycine
80
Glycine
160
Norvaline
40
Norvaline
80
Amount value [1]
Amount value [2]
50
100
50
100
20
40
Chromeleon determines all calibration coefficients, depending on the
selected calibration function.
d) Calculation of the Calibration Coefficients
A linear calibration curve through the origin (calibration type Linear) can
already be described by one calibration coefficient (c1). If the example is
selected so that the calibration points in each calibration curve are located
exactly on a straight line, that is, for example, in an exact measurement, c1
results as the y/x-quotient of each value pair (= slope of the calibration
curve).
Actions in the QNT Editor
Substance
y/x-Value pair
365
c1
Alanine
50/45
1.111
Alanine
100/90
1.111
Glycine
50/80
0.625
Glycine
100/160
0.625
Norvaline
20/40
0.500
Norvaline
40/80
0.500
If the calibration points are not located exactly on one line, Chromeleon
calculates an optimized c1 approximate value for each substance. If a
different calibration type were selected, Chromeleon would also calculate
the remaining calibration coefficients (c0 and c2) according to the
calibration function.
e) Amount Calculation: Internal Standard Substance in Unknown
Samples
If the area values of the internal standard substances from samples I and II
are known, the amount of the internal standard substance norvaline can be
determined in the two samples by means of the calibration coefficient c1
(here = 0.5) established for the calibration curve of the norvaline.
Sample
Calculation
Amount (ISTD)
I
39 x 0.5
19.50
II
41 x 0.5
20.50
The ratio between the (nominal) amount value entered in the peak table
and the ⇒ISTD Amount (Amount of the Internal Standard) of the internal
standard in the corresponding sample is referred to as ISTD factor.
ISTD − Factor =
Amount ISTD ( Peak −Tab.)
Amount ISTD ( Sample )
The following values are resulting:
Sample
Calculation
ISTD Factor
I
20 / 19.5 =
1.026
II
20 / 20.5 =
0.976
The result states that an error was made by 1.026 (sample 1) or 0.976
(sample 2). The actual amounts of alanine and glycine deviate in all
probability by 2.6 or 2.4% from the "real" values. They are corrected by this
amount.
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Actions in the QNT Editor
f) Amount Calculation: Alanine and Glycine
The amount values of glycine and alanine are calculated by means of the
¾Formula for Amount Calculation. In contrast to an external calibration,
ISTD Factor is not equal to 0; i.e., the results are corrected by the
calculated ISTD factor.
Sample
Calculation (Area x c1 x ISTD Fact. =)
Amount
I
45 x 1.111 x 1.026 =
51.30 (Alanine)
I
90 x 0.625 x 1.026 =
54.90 (Glycine)
II
80 x 1.111 x 0.976 =
86.75 (Alanine)
II
160 x 0.625 x 0.976 =
97.60 (Glycine)
The alanine or glycine amount values corrected by the norvaline deviation
are resulting.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Using Different ISTD Amounts (Variable ISTD)
Due to the method itself or the properties of the internal standard
substance, i.e., the ¾Internal Standard, it is sometimes impossible to add
exactly the same amount of ISTD. To solve this problem, Chromeleon
provides the Use sample amount as reference (Variable Internal
Standard) option. Use this option to define the ISTD as variable internal
standard substance. (Access to the option is via the F8 box of the
Standard column in the peak table).
In this case, you do not need to enter the amount of the internal standard
substance in the Amount column of the peak table (this is not possible),
but in the ⇒ISTD Amount column of the sample list. It is not important
whether Internal/External or Internal is selected as calibration method. In
this way, it is possible to enter the amount of an internal standard
substance separately for each sample. The following entries are required:
Actions in the QNT Editor
367
Sample List
Enter the amount of the internal standard substance directly in the ISTD
Amount column of the sample list:
QNT Method/Peak Table Tab
Except the substance serving as internal standard, enter all amount values
as before. Then, select the internal standard substance as follows:
If all three samples contain two relevant substances only as in the example,
make the following entries in the Standard column, using the F8 input box.
•
For substance 1, select Use this peak as Internal Standard and thus
define this substance as the internal standard.
•
In addition, enable the Use sample amount as reference (Variable
Internal Standard) option to define the ISTD as variable internal
standard substance.
The yellow coloring of the line and the ISTD: Var. Internal or ISTD: Var.
Int/Ext entry indicate that the assignment is correct. The corresponding
Amount column cells are automatically set to 1.
Make all other QNT File entries as before. The peak table appears as
follows:
QNT Method/Amount Table Tab
Enter the amount values for the unknown substances contained in the
standard sample in the Amount column of the Amount table of the QNT
Editor:
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Actions in the QNT Editor
Evaluation
Evaluation is similar to the corresponding evaluation with a constant
amount of the internal standard substance. However, slightly different
calibration and evaluation formulas are used.
For a detailed description of the differences between standard methods,
refer to Calibration
Evaluation with Various Standard Methods.
For an overview of the different calibration possibilities with internal
standard substances provided by Chromeleon, refer to How to ...: Actions
Calibrating with an Internal Standard Substance.
in the QNT Editor
Calibrating without Standard Sample
If you calibrate with an internal standard substance, i.e., an ¾Internal
Standard, you may as well perform the calibration without standard
samples. The prerequisite is that you know the relative ⇒Response Factor
of the respective substance for the ISTD. The following entries are
required:
Sample List
Assign all samples the Unknown sample type. Enter the amount for the
ISTD in the ISTD Amount column:
QNT Method/General Tab
On the General tab page, select Fixed as ⇒Calibration Mode.
QNT Method/Peak Table Tab
The internal standard substance is defined in the peak table. If all three
samples contain two relevant substances only as in the example, make the
following entries in the Standard column, using the F8 input box.
Actions in the QNT Editor
369
•
For substance 1, select Use this peak as Internal Standard and
enable the Use sample amount as reference (Variable Internal
Standard).
•
In the F8 input box of substance 2, select the option Internal. In the
Associated ISTD Peak field, select the standard substance serving as
internal standard (here: Subst. 1).
•
If necessary, repeat this operation for every additional substance that
should be calibrated based on the internal method.
After completing the input, the substance 2 is labeled Var. Internal Subst.
1 in the Standard column. In addition, substance 1 is labeled ISTD Var.
Internal to indicate that substance 1 is used as internal standard for the
internal calibration:
QNT Method/Amount Table Tab
Define the corresponding calibration curve in the Amount table of the QNT
Editor:
•
Enter =1.000 as C1 value for both the ISTD and the unknown
substance.
Tip:
Do not recalibrate afterwards by clicking the Calibrate button on the
General tab page because this action overwrites the values for the
¾Calibration Coefficient.
•
Enter the known response factor. This factor automatically refers to the
ISTD because the ISTD is used for calibration. It is not necessary to
define the ISTD as reference.
370
Actions in the QNT Editor
Amount Calculation
Chromeleon automatically calculates the amount values for the substance
2. The calculation is performed according to the formula for amount
calculation (rel. to ISTD) (for more information, refer to ¾Formula for
Amount Calculation (Rel. to ISTD) in the Glossary section). In case of
internal calibration, the external calibration of the internal standard
substance is adapted to the corresponding sample, using the area ratio of
the peak to the ISTD in the corresponding sample. In addition, in this
special case, the response factor is used to take the absorption of
substance 2 in relation to the ISTD (here: Subst. 1) into account.
Chromeleon calculates the amount for substance 2 adapted by the
deviation of substance 1.
For detailed information about how to calculate the amount for unknown
substance using internal calibration, refer to
Internal Calibration:
Calculation.
For an overview of the different calibration possibilities with internal
standard substances provided by Chromeleon, refer to How to ...: Actions
Calibrating with an Internal Standard Substance.
in the QNT Editor
Calibration Modes for External Calibration
The ⇒Calibration Mode determines the standard samples that are used for
calibrating specific unknown samples. The following calibration modes are
available:
Calibration
Mode
Description
Total
All standard samples of the current sequence
Group
Grouping calibration
Additional
Additional standard samples for the samples that appear later in the
sequence.
Bracketed
Unknown samples are "bracketed" by standard samples.
Fixed
Specific standard samples (also from other sequences)
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371
For a schematic representation of the Group, Additional, and Bracketed
calibration modes, refer to the example below:
For more information about the different calibration modes including
calibration curve calculation, refer to the topics below:
Calibration Mode: Total
Calibration Mode: Additional
Calibration Mode: Group
Calibration Mode: Bracketed
Calibration Mode: Fixed
Note:
Please note that the procedure is described for the Total mode. All other
examples describe the differences between the calibration modes only.
Calibration Mode: Total
If standard samples exist, the ratio between the amount and the peak area
can be used to calculate the calibration coefficients c0 (offset), c1 (slope),
and c2 (curvature) by means of the selected ¾Calibration Function. The
resulting values are entered in the ¾Formula for Amount Calculation,
together with the area values of the substance of an unknown sample. As
the result, Chromeleon provides the ⇒Amount of substance A in the
unknown sample. You can also use the concentration instead of the
amount. However, in this case, the concentration must be seen in relation
to the injection volume.
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Actions in the QNT Editor
Example:
You want to determine the concentration of the substances A and B in two
samples (Sample 1 and Sample 2). One standard solution (Std 1) is
available containing substance A in a concentration of 12 mg/l and
substance B in a concentration of 17 mg/l. The calibration curve for
substances A and B shall be linear and run through the origin (calibration
type: linear). The curve shall show two calibration points for each
substance. As only one standard solution is available, two different volumes
(10 and 20 µl) must be injected (¾Dilution Series). Two ¾Calibration Points
are resulting. The autosampler injects a volume of 10 µl for the two
unknown samples. The standard and analysis samples occupy the
autosampler positions R99, RA1, and RA2.
a) User Input
Sample List
For more information, refer to How to ...: Actions in the Browser
Creating a Sample List (Sequence).
QNT Method/Peak Table Tab
Regarding this example, note the following:
•
Select the External standard method in the Standard column using the
F8 edit box.
•
Only one standard concentration (Std 1) is available for calibrating the
substances A and B. That is why one concentration value for each
substance is entered in the Amount column of the peak table. The
same applies if several sample list entries are generated by analyzing
the same standard sample several times (twice, in this case). If the
injection volume is identical, the calibration is referred to as 1-point
Actions in the QNT Editor
373
calibration with several replicates. If the injection volume varies (10 and
20 µl) as in this case, this is referred to as multiple-point calibration,
that is, 2-point calibration. This is a special case of the ¾Dilution Series
("concentration series").
However, if two separate standards with different concentrations are
available (two vials, different autosampler positions), two concentration
values need to be entered in the Amount column of the peak table.
All other entries in the peak table are based on the criteria described in
How to ...: Actions in the QNT Editor
Creating a Peak Table.
QNT Method/General Tab
To calibrate all samples (here: samples 1 and 2) with the two standard
samples, select the Total ⇒Calibration Mode.
QNT Method/Calibration Tab
This page shows all standard samples (of a sequence) that are used for
calibrating the current sample.
Press the F4 key or the SHIFT+F4 key combination to successively open
all samples of a sequence. The standard samples used for calibrating the
sample are shown for each sample.
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Actions in the QNT Editor
If you notice that an error occurred during the analysis of the standard
sample, you can "exclude" this standard. Remove the standard in the
Enable column on the Calibration tab page of the QNT Editor. Only the
standard samples labeled X are considered for the calibration.
b) Analysis Structure
Injection is four times. During the first run, the first calibration point of the
calibration curves of the substances A and B is determined on the basis of
the determined area values. In the second run, the second point is
determined accordingly. Run three serves to determine the area values of
the substances A and B of sample 1. In the fourth run, the area values of A
and B of sample 2 are determined. The following area values are resulting:
Name
Area Subst. A
Area Subst. B
STD 1 (first run)
150
200
STD 2 (second run)
300
400
Sample I
175
150
Sample II
95
180
c) Calculation of the Calibration Coefficients
From the area values of the standard samples and the corresponding
concentration values, you can determine the four value pairs. The
intersection of each value pair represents a calibration point. Based on the
selected calibration type (in the example:
Calibration Type (Linear)),
Chromeleon calculates the optimum course of the calibration curve; that is,
the system tempts to find a course with four calibration points on or near
the curve. If the course of the curve is established, the corresponding
calibration coefficients (c0, c1, c2) can be calculated.
Substance
y/x-value pair
c1
A
12/150
0.08
A
24/300
0.08
B
17/200
0.085
B
34/400
0.085
With a linear course through the origin, the calibration curve can be
described by one single coefficient (c1). C1 expresses the slope of the
curve. If all calibration points are located exactly on the calibration line, the
resulting c1 calibration coefficient is the direct y/x-quotient of each value
pair.
Actions in the QNT Editor
375
calculated
c1 = 0.085
c1 = 0.080
[ng]
30
B
calculated
A
25
20
15
10
5
detected area
0
0
100
200
300
400
[mAU x min]
If the calibration points are not located exactly on one line, Chromeleon
calculates an optimized approximate c1 value for each substance from the
¾Calibration Function.
If a different calibration type (Theory of Calibration
Calibration Type
(Non-Linear)) is used, Chromeleon also calculates the remaining
calibration coefficients (c0 and c2).
d) Amount Calculation
If the calibration coefficients of a substance A are known, each area value
from an unknown sample for substance A can be converted into an amount
value by inserting the values in the calibration function. When inserting this
value into the ¾Formula for Amount Calculation, the actual amount value
will result.
When you perform this action for the peak areas of the substances A and B
in samples I and II (the correction factors of the formula for amount
calculation are assumed with 1.0), the following amount values are
calculated:
Sample
Calculation
Amount. A
I
175 x 0.08
14.00
I
150 x 0.085
II
95 x 0.08
II
180 x 0.085
Amount B
12.75
7.60
15.30
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
376
Actions in the QNT Editor
Calibration Mode: Additional
The following example illustrates the difference between the Total and
Additional ⇒Calibration Modes.
Independently from the standard method, this mode determines which
standard samples are used for evaluating a specific unknown sample. The
position of the unknown sample in the sample list is decisive here.
Extending the Example: External Calibration/Mode: Total
Two standard samples are injected after two unknown autosampler have
been injected. This results in an alternating list of two standard samples:
two unknown samples, two standard samples, etc. All other settings are
maintained; that is, two-point calibration is performed. The calibration is
verified by additional replicates.
a) User Input:
Sample List
QNT Method/Peak Table Tab
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377
QNT Method/General Tab
If the user selects the Additional mode, each unknown sample is
evaluated based on the standard samples analyzed so far.
This means: As before, samples I and II are evaluated by means of a twopoint calibration. However, four analyzed standard samples are available
for the samples III and IV. The result is a two-point calibration with two
replicates each. Finally, the samples V and VI are evaluated by means of a
two-point calibration with three replicates each.
QNT Method/Calibration Tab
The page shows all standard samples (of one sequence) that can be used
for calibrating the current sample.
Press the F4 key or the SHIFT+F4 key combination to successively open
the samples. The standard samples forming the basis for calibration are
shown for each sample.
Thus, two standard samples are displayed for the samples I and II, four
standard samples are displayed for the samples III and IV, and six standard
samples are displayed for the samples V and VI (see below).
378
Actions in the QNT Editor
If you notice that an error occurred during the analysis of the standard
sample, you can "exclude" this standard. Remove the standard in the
Enable column on the Calibration tab page of the QNT Editor. Only the
standard samples labeled X are considered for the calibration.
b) Evaluation:
Evaluation is similar to the example with external calibration (mode: Total).
The only difference is:
The calibration curve is created for the different samples based on a
different number of replicates.
This can affect the calculated calibration coefficients and thus the result,
but does not necessarily do so. Normally this type of calibration is used to
adapt to changed column conditions. Thus, it may happen that after a
several samples, a specific substance cannot elute 100 percent from the
column. As this will also be the case with the standard substance, the result
is automatically corrected.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Calibration Mode: Group
The Group ⇒Calibration Mode completes the Total and Additional
modes. The Group mode is used when there are time-dependent
modifications during the analysis, for example, decomposition of the
analyzed substance.
The mode is described using the example of the Additional mode (sample
list of alternating sample pairs (2 standards, 2 samples, 2 standards, etc.).
Actions in the QNT Editor
379
a) User Input:
Sample List
QNT Method/Peak Table Tab
QNT Method/General Tab
If the user selects the Group mode, each sample is evaluated based on the
standard sample in the sample list that has been analyzed immediately
before.
QNT Method/Calibration Tab
This page indicates all standard samples (of a sequence) that are used for
calibrating the current sample.
Press the F4 key or the SHIFT+F4 key combination to successively open
the sample of a sequence. The standard samples forming the basis for
calibration are shown for each sample.
This means in the example that in the Group mode the standard samples
from the lines 1 and 2 are displayed for the samples I and II. The standard
samples from lines 5 and 6 are displayed for the samples III and IV, and the
standard samples from lines 9 and 10 for the samples V and VI.
380
Actions in the QNT Editor
b) Evaluation:
Evaluation is similar to the examples with the external calibration (modes:
Total and Additional). The only difference is that the calibration curve is
constantly updated without considering the previously analyzed standard
samples.
For the example, this means:
Samples I and II are evaluated by means of a two-point calibration of the
standard samples in the lines no. 1 and 2.
In contrast to the Additional mode, samples III and IV are also evaluated
by means of a two-point calibration. However, only the standard samples
STD 1 from lines no. 5 and 6 are used.
Correspondingly, the samples V and VI are evaluated by means of the
standard samples STD 1 in the lines no. 9 and no. 10.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Calibration Mode: Bracketed
In a bracketed calibration, a standard sample is included in a series of
unknown samples so that modifications, such as column or detector drift,
can also be considered in the calibration function. Amount calculation of an
unknown sample is always performed using the calibration coefficients of
the surrounding standard samples.
New Example
You want to determine the concentration of substance A in four samples
(Samples I-IV). Two standard solutions (STD 1, STD 2) of substance A with
different concentrations (50 and 100ng/µl) are available for external
calibration. At the beginning, after the second and after the fourth sample, a
two-point calibration is to be performed. To receive exacter results, a
bracketed calibration is performed. The basis for calibrating samples I and
II is provided by standards STD 1 and STD 2 from lines no. 1, 2, 5, and 6,
while the samples III and IV are calibrated on the basis of the standards no.
5, 6, 9, and 10.
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381
a) User Input:
Sample List
QNT Method/Peak Table Tab
The external calibration (Standard = External) is performed via two
standard samples (autosampler positions 10 and 11) of different
concentrations. In contrast to the Total, Additional, and Group examples,
two different ⇒Amount values must be entered in the corresponding
amount columns [1] and [2] of the peak table.
QNT Method/General Tab
To evaluate each sample based on the neighboring standard samples in
the sample list, select the Bracketed ⇒Calibration Mode.
QNT Method/Calibration Tab
This page shows all standard samples (of a sequence) that are used for
calibrating the current sample.
Press the F4 key or the SHIFT+F4 key combination to successively open
the samples of a sequence. The standard samples forming the basis for
calibration are shown for each sample.
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Actions in the QNT Editor
For the samples I and II, the page will look is as follows:
For the samples III and IV, the page will look as follows:
Note that the standards in line 5 and 6 are determined only once.
b) Analysis Structure:
Injection is eight times. The following area values are determined:
Name
Area substance A
STD 1 (conc.1)
218
STD 2 (conc.2)
439
Sample I
167
Sample II
152
STD 1 (conc.1)
224
STD 2 (conc.2)
442
Sample III
283
Sample IV
305
STD 1 (conc.1)
219
STD 2 (conc.2)
441
c) Calculation of the Calibration Coefficients
From the determined area values of the standard samples and the amount
values from the peak table, the value pairs of the six calibration points can
be listed. Note that depending on the standard (STD 1 or STD 2) the
amount value is once taken from the Amount [1] peak table column and
once from the Amount[2] column.
Actions in the QNT Editor
383
No.
Substance
y/x-value pair
y/x
c1 (K1)
1
STD 1 (conc.1)
50/218
0.2294
0.2267
c1 (K2)
2
STD 2 (conc.2)
100/439
0.2278
0.2267
5
STD 1 (conc.1)
50/224
0.2232
0.2267
0.2261
6
STD 2 (conc.2)
100/442
0.2262
0.2267
0.2261
9
STD 1 (conc.1)
50/219
0.2283
0.2261
10
STD 2 (conc.2)
100/441
0.2268
0.2261
If all calibration points are used simultaneously, a two-point calibration with
three replicates of the same calibration level results.
As amount determination of samples I and II or III and IV is performed only
according to the neighboring standard samples, there are two calibrations
for substance A instead of one. Each calibration is a two-point calibration
with two replicates each. That is why Chromeleon calculates two different
sets of calibration coefficients.
If a linear calibration function without offset is assumed as in the previous
examples, Chromeleon determines two different c1-values. One value (K1)
is calculated for the numbers 1, 2, 5, and 6, and another value (K2) is
calculated for the numbers 5, 6, 9, and 10.
d) Amount Calculation
If the calibration coefficients of a substance A are known, the amount of
substance A contained in each sample can be calculated by inserting the
peak areas determined from the unknown samples in the calibration
function. Inserting this value in the ¾Formula for Amount Calculation
returns the actual amount value.
If this action is performed for the determined peak areas of substance A in
the samples I and II (taking K1 into account), the following amount values
are calculated (the correction factors of the formula for amount calculation
are assumed to be 1.0):
Sample
Calculation
Amount A
I
167 x 0.2267
37.86
II
152 x 0.2267
34.46
For the samples III and IV, the result is as follows (taking K2 into account):
Sample
Calculation
Amount A
III
283 x 0.2261
63.99
IV
305 x 0.2261
68.96
384
Actions in the QNT Editor
Tip:
Calculation of the amount values is continuously; that is, after analyzing an
unknown sample, the values are calculated based on the standard samples
calculated so far!
Calculation of the area values of samples I and II is performed depending
on the current processing status, first with one, then with two and three,
and finally with four standard samples. The same applies to the samples III
and IV that are evaluated based on the standard samples in the lines 5,6,9,
and 10 of the sample list.
For example, if a ¾Report is generated after each sample, it will only
include the results from the samples analyzed so far. The result may differ
from the final result. Therefore, the results of a calibration with the
Bracketed mode should be printed after completing all standard samples.
In the Bracketed ⇒Calibration Mode, processing of all standard samples
should be completed before printing the results.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Calibration Mode: Fixed
To use any desired standard samples of an existing calibration for
determining current unknown samples, perform calibration based on the
Fixed ⇒Calibration Mode.
Caution:
Always perform the calibration manually (Calibrate). The Auto-Recalibrate
option is not available.
a) User Input:
Sample List
Only unknown samples are entered.
If the sample list is generated using the Sequence Wizard, decide whether
to refer to an existing QNT File (which is recommended) or whether to
leave out this information. When closing the Wizard, Chromeleon
Actions in the QNT Editor
385
recognizes that the sequence does not contain any standard samples and
copies the QNT File with the entire information to the sequence directory.
The Fixed mode is selected at the same time.
If no QNT File is defined in the Sequence Wizard, enter the file name in the
Method column of the sample list after the QNT File has been created.
QNT Method/Peak Table Tab
If an existing QNT File is copied, a peak table already exists. Change the
table according to your requirements.
However, if a new (empty) QNT File is used, a new peak table must also be
created. It is not necessary to enter the ⇒Amount because calibration is
based on an existing calibration.
QNT Method/General Tab
If an existing QNT File is copied in which the Fixed calibration mode is
selected, this mode is automatically selected.
If a new QNT File was generated, select the Fixed mode. Determine the
standard samples to be used for calibration on the Calibration tab page.
QNT Method/Calibration Tab
If a QNT File was copied, this page lists all standard samples to which the
QNT-File originally referred.
With each newly created QNT File, no standard samples are listed. Select
Insert Standard or Append Standard on the context menu to insert the
standard samples to be used for calibration and to evaluate the current
samples according to their results.
The standard samples listed here always apply to all samples of a
sequence. Press the F4 key or the SHIFT+F4 key combination to display
the same maximum number of standard samples.
b) Analysis and Evaluation
Performing the analysis and the evaluation of the individual samples is
similar to the previous examples.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
386
Actions in the QNT Editor
Entering the Concentration/Amount of the Validation
Sample
¾Validation Samples are used to check the calibration. They correspond to
standard samples in as much as the amount of analyte is known. They are
used to validate the calibration, but not to determine the calibration curve.
Enter the known concentration (or amounts) of the different analytes in the
validation samples in the QNT Editor. The two sections below explain the
ways in which the validation and standard samples can interact and the
most common means to implement them. It is up to each user to decide
which method is appropriate for a particular application.
1. The validation sample is equal to the standard sample
All
individual
concentrations/amounts
correspond
to
the
concentrations/amounts of a standard sample that has already been used
for the calibration curve, especially if the validation sample is injected from
a vial containing standard sample.
In this case, an ⇒Amount column for the corresponding standard sample is
already available in the QNT Editor. The validation sample should have
already been assigned to the correct vial. To verify this, proceed as follows:
•
On the Amount Table tab page, place the cursor in the header of the
Amount column of the corresponding standard sample. Double-click
the column header to open the Edit Amount Columns dialog box.
•
Select an Amount column (or Unassigned) in the Amount Column
window to display the associated standards.
•
Select one or more validation samples ( ) in the Standards window
and drag them to the appropriate column in the Amount Column
window.
•
Click OK to close the dialog box and return to the amount table.
All values in the Amount column now apply to the validation sample(s),
also.
2. The validation sample is not equal to the standard sample
When using a validation sample whose composition is different from all
standard samples (if only in its concentration of amount of one analyte), a
separate Amount column must be created for the validation sample. Follow
the steps below:
Actions in the QNT Editor
387
•
On the Amount Table tab page of the QNT Editor, double-click the
header of an existing Amount column (or select Columns > Edit
Amount Columns on the Edit or context menu) to open the Edit
Amount Columns dialog box.
•
Click New, type a unique column name in the edit field that appears in
the Amount Column window, and press Enter.
•
Select an Amount column (or Unassigned) in the Amount Column
window to display the associated standards.
•
Select one or more validation samples ( ) in the Standards window
and drag them to the new column in the Amount Column window.
•
If the column created for the validation sample is valid for other
validation samples or for standard samples, assign the additional
standards as described in The validation sample is equal to the
standard sample.
•
Click OK to close the dialog box.
For information about how to use the validation sample to check the
Validating the Calibration Curve.
calibration curve, refer to
For an overview of the calibration options provided by Chromeleon, refer to
Calibration.
How to ...: Actions in the QNT Editor
Validating the Calibration Curve
To check the calibration using the respective values of the ¾Validation
Samples (perhaps of the standard sample as well) you have to compare
the expected concentration or amount values to the measured/calculated
values either numerically by means of the Amount Deviation report variable
or visually by means of the calibration function.
1. Numerically, by means of the Amount Deviation report variable:
The determined (actual) area values are converted to concentration or
amount values by means of the ¾Calibration Function and compared to the
expected (nominal) values of the ⇒Amount table.
•
Add a new column to a report or a ¾Printer Layout page or change the
assignment of an existing column as described in How to ...: Actions
Defining the Contents of a Report.
in the Report Table
388
•
Actions in the QNT Editor
Select the Amount Deviation variable in the ⇒Peak Results report
category. Click the Parameter button to define whether the result of the
variance comparison shall be expressed as absolute amount value or
as deviation in percent (Relative in % of the expected Amount).
2. Visually, by means of the Calibration Curve
For checking the calibration curve visually, the validation samples can be
displayed (specially marked) in the curve:
•
Select Decoration on the context menu of the calibration curve
(respectively, the Calibration Plot Properties command in the Printer
Layout).
•
Select the Draw validation sample points check box on the Options
tab page.
Note:
Even if this action is performed in the QNT Editor, these validation
samples will not be considered for the calibration. Nevertheless, if you
wish to use a validation sample as standard sample later and consider
it for calculating the calibration curve, set the ⇒Type (Sample Type) to
Standard in the sample list of the Browser.
3. By Means of the Confidence Interval
a) In addition, the confidence region interval can be indicated in the
calibration curve. Select the Draw upper and lower confidence limit
check box on the Options tab page and define the desired confidence
region to check whether your validation and standard samples are within
the corresponding confidence interval (also, refer to How to ...: Actions in
the Calibration Curve
Indicating the Confidence Interval).
b) For single values, you can also indicate the limits for the respective
¾Confidence Interval in the Report using the Upper/Lower Confidence
Limit variables of the ⇒Peak Calibration report category.
For information about how to enter the concentration/amount, refer to How
to ...: Actions
in
the
QNT
Editor
Entering
the
Concentration/Amount of the Validation Sample.
For an overview of the different calibration possibilities provided by
Chromeleon, refer to How to ...: Actions in the QNT Editor
Calibration.
Actions in the QNT Editor
389
Inverting Dependent and Independent Variables
Normally, calibration curves are fitted using normal regression analysis, in
which the known amount (or concentration) of the analyte is treated as the
independent variable (x) and the corresponding response (peak area or
peak height) is treated as the dependent variable (y):
Many legacy data systems use inverted curve fitting, which treats the
response as if it were the independent variable (x), and amount as if it were
the dependent variable (y):
390
Actions in the QNT Editor
You can select the Inverted curve fitting option on the General tab page:
Although inverted curve fitting is not supported by standard statistical
theory, it was often taken because it makes amounts of analytes in
unknowns easier to calculate when quadratic and cubic calibration
functions are used. With linear regression, the differences between normal
and inverted curve fitting are usually small, but the differences can be
significant with higher-order polynomials, In a few applications, most
notably the determination of ammonium by suppressed conductivity
detection, a better fit to the empirical data may actually be achieved using
inverted curve fitting.
To provide consistency of results for customers migrating from legacy
systems, and to satisfy users of special applications, Chromeleon supports
inverted curve fitting via an option in the General tab page of the QNT
Editor. Methods imported from PeakNet 5 default to using inverted curve
fitting, the approach used by PeakNet 5. Normal curve fitting remains the
default setting for new methods created in Chromeleon.
Caution:
It is the user's responsibility to decide whether inverted curve fitting is
sensible for their application.
If you select Inverted, the dependent and independent variables are
inverted. Thus, different values are usually obtained for the following
variables:
⇒Peak Results Category
•
⇒Amount (concentration or amount)
•
Amount Deviation (deviation from the expected concentration or
amount)
Actions in the QNT Editor
•
Concentration (concentration, if Amount is really an amount)
•
Relative Amount (relative concentration or amount)
•
Group Amount (concentration or amount of a group)
⇒Peak Calibration Category
•
¾Offset c0 (intersection with the y-axis)
•
¾Slope c1
•
¾Curve c2 (curvature)
•
¾RF Value (1/slope)
•
¾Variance
•
¾Variance Coefficient
•
¾Standard Deviation
•
¾Relative Standard Deviation
•
¾Coefficient of Determination
•
¾DOF-Adjusted Coefficient of Determination
•
Calibration Point X
•
Calibration Point Y
•
Evaluation of Cal.Function for X ()
•
Residual for Calibration Point X
•
Upper Confidence Limit (upper limit for the ¾Confidence Interval)
•
Lower Confidence Limit (lower limit for the confidence interval)
391
392
Actions in the QNT Editor
Spectra Library Screening
Use Spectra Library Screening page of the QNT Editor to search spectra
libraries for reference spectra. The spectra search is also possible from the
spectra plot, as described in How to ...: Actions in UV Spectra and/or
Starting a UV Spectra Search.
Mass Spectra
For more information, refer to:
Entering Criteria for the Spectra Library Screening
Starting Library Screening and Viewing Results
Integrating Screening Results in Reports and Peak Labels
Entering Criteria for the Spectra Library Screening
Enter the criteria for a spectra library search on the Spectra Library
Screening (= SLS) page of the QNT Editor:
•
Select a spectra library (LIB file) from the Spectra Library to be
searched in ... field. If the required LIB file is not included, click the
Browse button to search for the file.
Actions in the QNT Editor
393
•
In the Match Criterion field, select the method (see ⇒Match Criterion)
used for comparing the original spectrum and the library. The best
search results are usually received via the setting Least Square.
•
In the Hit Threshold field, enter a ¾Match Factor between 0 and 1000;
for example, 950. Only spectra with a match value above 950 will be
shown. Spectra with less similarity will not be included. Unless
derivatives of spectra are compared with each other, reference spectra
with a match value below 900 are usually spectra of other substances.
Exceptions to this rule are only acceptable, if the signal-to-noise ratio is
low.
Note:
To be able to compare UV spectra, the spectrum and the reference
spectrum should be recorded under identical conditions. For best
results, create your own library of spectra you recorded yourself.
Additional conditions are possible to perform a more exact search:
Usually, the Use Spectrum Derivative option is disabled; that is, the
comparison of spectra is based on their actual curve. If the 1st derivative
option is selected, the comparison of two spectra is based on the first
derivative. As a result, the curve characteristics increase (shoulders
become extrema), allowing a more exact comparison of extrema. However,
the signal-to-noise ratio will considerably decrease; and sections with lower
signals will be less significant.
Select Restrict Wavelength Range to limit the spectra comparison to the
relevant spectral range.
Select Check Greatest Rel. Max. to use only spectra with the greatest
relative maximum at the same position. Select Allowed Deviation to
specify a tolerance range. The range should not exceed 10 nm to avoid
that the criterion loses significance.
Click Maximum Retention Time Deviation to determine the maximum
retention time deviation of the reference spectrum in percent. This prevents
including substances with very similar spectra that are eluted at different
retention times.
If you select Check Number of Relative Extrema, Chromeleon checks the
number of relative extrema of the reference spectrum. Select this option to
exclude spectra that are very similar but have an additional maximum.
394
Actions in the QNT Editor
Select Maximum Retention Index Deviation: or Maximum Kovats Index
Deviation: to include only those spectra in the comparison for which the
retention index or, respectively, the ⇒Kovats Index is identical with the
index of the sample substance. The respective tolerances are defined in
the right field.
Tip
With an increased noise level, noise peaks can be considered extrema. In
this case, we recommended disabling Check Number of Relative
Extrema.
On the right side of the window, you can further restrict the resulting library
spectra. Open the dialog box Restrict Library Search with Add. Via Field
Name, you can then select type of parameter to be determined from the
following search criteria:
Solvent Composition
Detector Serial Number
Control Program
Timebase
Substance Name
Sequence Name
Unique ID
Sample Name
Comment
Extract Operator
Detector Name
These items can be linked (partly) via the following operators (Conditions)
with a freely selectable value:
Operator
Restricts the search to spectra with parameters that
is equal to:
fulfill the specified condition.
starts with:
start with the entered string.
does not start with:
do not start with the entered string.
ends with:
end with the entered string.
does not end with:
do not end with the entered string.
contains:
contain the entered string.
does not contain:
do not contain the entered string.
Note:
If you want to search all libraries in a directory, simply enter the character *
as the file name. The wildcard characters (known from MSDOS) * and ?
are valid. Example: LIB::\CMDATA\LIB\D* searches all libraries starting
with the letter D in the directory LIB of the datasource CMDATA.
Actions in the QNT Editor
395
Starting Library Screening and Viewing the Results
Click Apply to use the entered search parameters. This command starts
the library search for each peak in the active chromatogram.
The spectra plot is displayed in addition to the chromatogram. The
reference spectrum (hit) with the corresponding match criterion will be
shown. The displayed spectrum is the reference spectrum of the selected
peak. You can display the search results for the other substances by
clicking the corresponding peaks in the chromatogram.
If more than one library spectrum fulfills the entered criteria, you can
display the other hits. Place the cursor on the spectrum, open the context
menu, and open the Spectra Decoration tab page. Use the Peak Spectra
tab page to enter the number of reference spectra to overlay:
In this example, you will receive a list of three reference spectra. The first
spectrum has the highest match value and thus the greatest similarity to the
original spectrum.
396
Actions in the QNT Editor
Inserting Screening Results in Reports and Peak Labels
If you save the search parameters in the QNT File, the results of the
Spectra Library Screening can be used for the peak label or in report
tables or templates (¾Printer Layout).
Inserting Screening Results in ¾Report Definition Files
To display the screening results in the report, the Peak Purity report
category supports the following variables:
Number of SLS Hits calculates the number of library screening hits for a
peak. SLS Hit opens the ⇒Hit Spectrum report category that includes all
variables of the corresponding library spectrum:
Designation
Description
Substance Name
Match Factor
¾Match Factor
Library Name
Name of the spectra library
Library Record
Opens the ⇒Spectra Library category
Number of rel. Extrema
Solvents
Comment
¾Comment
Sequence Name
Sequence Header Record
Sample Name
Sample Record
Branches to the ⇒Sample category
Acquisition Time
Acquisition date and time
Timebase
¾Timebase
¾PGM File
Program
Sample Rate
Retention Time
⇒Retention Time
Lambda Min.
Minimum wavelength
Lambda Max.
Maximum wavelength
Lambda Range
Wavelength range
Lambda Resolution
Spectral wavelength resolution
Detector Name
Detector Serial Nr.
Extract Time
Time when the sample was added to the library
Extract Operator
User who added the sample to the library
Retention Index
Linear ⇒Retention Index
Kovats Index
⇒Kovats Index
Actions in the QNT Editor
397
The Formula field shows a short version of your selection; for example,
peak.hitSpec(1.)name for the spectra name of the best hit. The digit
indicates the hit: 1 is the best, 2 is the second best hit, etc.
Including Screening Results in the Peak Label
To include the screening results in the chromatogram, follow the steps
below:
•
Position the cursor in the chromatogram.
•
Select Chromatogram Decoration on the context menu.
•
On the Peak Label tab page of the Chromatogram Decoration dialog
box, click {...} to reach the Insert Variable into Peak Label Formula
dialog box. Select the Number of SLS Hits and SLS Hit variables (see
Report above) from the Peak Purity category.
Example
To label each peak in the chromatogram with the name of the best hit and
the corresponding match factor, follow the steps below:
•
From the category Peak Purity, select the Substance Name under
SLS Hit.
•
In the Formula field, enter " - " after the formula, then click Match
Factor under SLS Hit.
•
Click OK. You will receive the following Formula input on the Peak
Label tab page of the Chromatogram Decoration dialog box:
398
Actions in the QNT Editor
Tip:
If you have saved the search parameters in the QNT File, it is sufficient to
enable Spectra library screening results; for example, on the spectra
plot. Just open the context menu and select the option via Spectra
Decoration and Peak Spectra. In this case, you do not need to open the
Spectra Library Screening dialog box and click Apply.
Defining the System Suitability Test
Use the SST page (see ¾System Suitability Test (SST)) of the ⇒QNT
Editor to check your system's performance for individual samples. The
number of tests is only limited by the capacity of your computer. Each test
is defined in a separate line. Use the arrow key ↓ to append additional lines
to the table. This action automatically opens the SST Wizard, which guides
you through all further steps.
Tip:
In order to perform a System Suitability Test, enter the QNT File into the
sample list before you start the analysis. If you start the analysis and the
QNT File has not yet been entered, the test is not performed during the
batch run. Thus, the ¾Batch cannot be aborted in case of Fail Action Abort Batch.
For more information, refer to:
SST Wizard: Overview and Start Conditions
SST Wizard: Sample and Test Conditions
SST Wizard: Other Wizard Pages
Modifying the System Suitability Test
SST Example: Is the Amount in the Calibrated Range?
SST Example: Amount Deviation on Reinjection
Inserting SST Results in the Printer Layout
Actions in the QNT Editor
399
SST Wizard: Overview and Start Conditions
The SST Wizard supports you in inserting a new ¾System Suitability Test
(SST). To open the SST Wizard, double-click a cell or press the F8 key. Or
else, select Lines… Append Line or Insert Line on the context menu or
press the ↓ key. (Note: To open the SST Wizard via the ↓ key, place the
cursor on the bottom line.)
The SST Wizard provides the following pages:
•
Start
•
Sample Condition
•
Test Condition
•
Aggregated (optional)
•
Peak & Channel Condition (optional)
•
N.A. & Fail Action
On the first Wizard page, select a test from the Predefined Tests list:
400
Actions in the QNT Editor
All predefined values on the following pages depend on this selection.
To copy the previous test, select <Copy Previous Test> from the list. If
there is no previous test, i.e., if the test is the first on the list, this entry is
not available.
If no other conditions are required, you can already complete the Wizard on
this page by clicking Finish. Clicking Next takes you to the following
Wizard pages. Use these pages to check and edit the predefined
conditions as necessary.
For more information, refer to:
SST Wizard: Sample and Test Conditions
SST Wizard: Other Wizard Pages
SST Wizard: Sample and Test Conditions
Use the Sample Condition page to specify for which sample(s) the test is
performed. Select the desired option:
Actions in the QNT Editor
401
Select Apply on all Samples to perform the test for each sample. Select
Sample Type to perform the test for a certain sample type only. In the
Sample Number(s) and/or Vial Number(s) input fields, enter the sample
or vial number(s) for which to perform the test; for example 1,3,7-10. (Note:
To separate the entries, you can use either a comma or a semicolon.)
Select Sample Property to perform the test only for samples with the
specified property. Enter the property in the corresponding input fields. The
User defined condition option is reserved for advanced users. Select this
option to enter any kind of formula in the report format. The test is
performed only for those samples for which this condition is true.
Test Condition
Use Test Condition tab page to enter the basic conditions for the single
system suitability tests:
First, enter a unique name in the Test Name input field. Specify the Test
Condition by clicking the "..." (Browse) button. Select the desired variable
from the Edit Result Formula dialog box. Select the Operator from the
drop-down list box, and then enter the compare value in the Value input
field. The following operators are available:
402
Actions in the QNT Editor
Operator:
Description:
Sample Condition: The SST if performed for all samples for which
the property .....
Test Condition: The SST checks whether the sample property ....
=
equals the entered string of characters.
<>
does not equal the entered string of characters.
>
is larger than the entered string of characters.
<
is smaller than the entered string of characters.
>=
is larger than or equals the entered string of characters.
<=
is smaller than or equals the entered string of characters.
contains:
contains the entered string of characters.
does not contain:
does not contain the entered string of characters.
starts with:
starts with the entered string of characters
does not start with:
does not start with the entered string of characters.
ends with:
ends with the entered string of characters
does not end with:
does not end with the entered string of characters.
Note:
If the operators >, <, >=, or <= apply to text variables, the lexicographical
order is observed; for example, A<B.
The Value can be either a value or a report variable or formula. Select the
Use aggregation check box to aggregate the test conditions over several
samples. Selecting this check box takes you to the Aggregate page.
For more information, refer to:
SST Wizard: Overview and Start Conditions
SST Wizard: Other Wizard Pages
SST Wizard: Other Wizard Pages
Aggregate
Use this page to define the function, sample(s), and condition for sample
aggregation. (Refer to the online Help for information about the Aggregate
tab page.)
Actions in the QNT Editor
403
Peak & Channel
This page is optional and appears only if the test condition requires that
you enter a peak or channel. (Refer to the online Help for information about
the Peak & Channel tab page.)
N.A. & Fail Action
Use this page to determine what the test result is if the test cannot be
performed (N.A.). Also, determine which action is taken if the test fails (Fail
Action).
For more information, refer to:
SST Wizard: Overview and Start Conditions
SST Wizard: Sample and Test Conditions
Modifying the System Suitability Test
To modify an existing ¾System Suitability Test (SST), double-click a cell or
press the F8 key. The SST Properties dialog box provides the following
tab pages:
•
Sample Condition
•
Test Condition
•
Aggregate
•
Peak & Channel Condition
•
N.A. & Fail Action
These pages correspond to the individual SST Wizard pages. For more
SST
information, refer to How to ...: Actions in the QNT Editor
Wizard: Overview and Start Conditions.
SST Example: Is the Amount in the Calibrated Range?
It is only possible to determine the amount of an unknown sample if it is in
the calibrated range, i.e., between the smallest and the largest amount of
the standard samples. You can check this using a System Suitability Test.
Follow the description below:
404
Actions in the QNT Editor
Create a test that checks whether the current Amount is either equal or
larger than the smallest Amount of all standard samples:
•
If no System Suitability Test has been created yet, double-click the
empty line on the SST tab page to open the SST Wizard. For an
existing System Suitability Test, select the bottom line of the existing
test and press the arrow down key on the keyboard. From the
Predefined Tests list, select the Minimum Peak Amount test. The
Wizard guides you through test creation. Clicking Next> takes you to
the next Wizard page.
•
On the Sample Condition page, specify for which samples the test is
performed. The default setting is Apply on all Samples.
•
You can accept the settings on the Test Condition page:
•
Use the next Wizard page to determine the peak(s) and the channel for
which the test is performed.
•
Finally, determine what the test result is if the test cannot be performed
and which action is taken if the test fails.
In the same way, create a second test that checks whether the current
Amount is either equal or smaller than the largest Amount of all standard
samples. In this case, select the Maximum Peak Amount test from the
Predefined Tests list.
Actions in the QNT Editor
405
SST Example: Amount Deviation on Reinjection
For reinjections from the same vial, the relative standard deviation of the
determined Amount values should be as small as possible. You can use a
System Suitability Test to check whether the standard deviation is below a
specified minimum value. Follow the description below:
•
If no System Suitability Test has been created yet, double-click the
empty line on the SST tab page to open the SST Wizard. For an
existing System Suitability Test, select the bottom line of the existing
test and press the arrow down key on the keyboard. The Wizard guides
you through test creation. Clicking Next> takes you to the next Wizard
page.
•
On the Sample Condition page, specify for which samples the test is
performed. The default setting is Apply on all Samples.
•
On the Test Condition page, enter an appropriate name for the new
System Suitability Test, e.g., Amount Deviation.
•
Determine the Test Condition, e.g., peak.amount >= 1. (In this case,
1 is the maximum allowed deviation in percent.)
•
Click the "…" button behind the Test Condition input field. In the
dialog box, select ⇒Peak Results from the Categories list, and
then select Amount from the Variables list.
•
Click OK to confirm your selection. This returns you to the SST
Wizard.
•
In the Operator input field, select >=. In the Value input field, enter
1.
406
Actions in the QNT Editor
•
Select the Use aggregation check box. Clicking Next> takes you to
the Aggregate page.
•
On the Aggregate page, select RSD% from the Aggregate Function
drop-down list.
•
Enter the Maximum number of samples to aggregate, e.g., 5.
•
For Only aggregate samples with, determine Replicate ID =
smp.replicate. This searches for a maximum of four previous samples
for which the replicate ID matches the replicate ID of the current
sample. The relative standard deviation of the Amount value is
determined for a maximum of five samples--for the current sample and
up to four others.
•
Use the next Wizard page to determine the peak(s) and the channel for
which the test is performed.
•
Finally, determine what the test result is if the test cannot be performed
and which action is taken if the test fails.
Actions in the QNT Editor
407
Inserting SST Results in the Printer Layout
If you save the ¾System Suitability Test (SST) parameters in the QNT File,
you can use the test results for peak labeling or as individual variables in
the ¾Printer Layout (not in tables).
Use the Report category System Suitability Test to display the SST
results in the Printer Layout. The following variables are available:
Variable
Description
Number
Name
Sample Condition
Test Condition
Aggregate
Operator
Value
Channel
Peak
N.A.
User-defined test result if the test is not
performed
Fail Action
Aggregated Samples
Sample Condition Results
Test Results
Aggregated Sample List
Aggregated Sample Result List
Result of Test Condition or Aggregate
Result of Compare Value
The Formula field indicates your selection in short; for example,
sst.test_condition for the test condition.
Processing Mass Traces
¾Mass Traces have a relatively high noise level. Therefore, they must be
processed before being used further. Chromeleon uses the following
algorithm:
•
First, the spectrum of the peak maximum is determined by averaging
several spectra in the peak maximum on both ends.
408
Actions in the QNT Editor
•
Then, the background spectra of both peak ends are determined, also
by averaging several spectra.
•
The background peak spectrum (usually at the peak maximum) is then
determined via linear interpolation of these two background spectra
and is finally subtracted from the single spectrum (usually of the peak
maximum).
Tip:
Only mass spectra recorded with the same filter settings are averaged and
subtracted. The filter settings of the Apex mass spectrum of the current
peak are used for this. For time spectra, the filter settings of the mass
spectrum nearest to the selected retention time are used.
In the ⇒QNT Editor, use the MS tab page to specify how mass spectra are
formed from individual spectra. Select the Enable Background
Subtraction check box to enable spectrum subtraction/bunching:
In the combo box under Peak Spectrum Bunch, enter the number of
single spectra that shall be bunched to form the spectrum at the peak
maximum. A maximum of 99 single spectra can be averaged. For
symmetry reasons, it is possible to enter odd numbers only.
Select Peak Dependent Background Subtraction to allow automatic
background subtraction for each peak.
Actions in the QNT Editor
409
In the edit fields under Left Region Bunch and Right Region Bunch,
enter the number of spectra to be used to form the two background spectra.
You can select up to 99 single spectra. Zero and even numbers are
permitted as well. These settings apply to peak spectra and time spectra
below peaks.
Select Fixed Background Subtraction Ranges to define two fixed ranges
for background subtraction for the entire chromatogram. It usually makes
sense to set one range at the beginning and the other one at the end of the
chromatogram. These settings apply to peak spectra and to all time
spectra.
Click Apply to accept the settings and calculate the resulting mass
spectrum. When the Show Spectra view is enabled, the newly calculated
mass spectrum is displayed at once.
Note:
Defining the background subtraction manually in the chromatogram affects
the settings on the MS tab page. For example, if you select MS
Background Subtraction on the context menu of the chromatogram, and
then select Fixed Background Ranges, the corresponding option is
automatically selected on the MS tab page of the QNT Editor.
Processing UV Channels
For a better comparability of UV spectra, it is useful to subtract the
background spectrum (of the solvent). Chromeleon performs this
automatically, using the following algorithm:
•
The background spectra are determined for both peak ends. At both
peak ends, several UV spectra can be averaged over a specified
range.
•
The background peak spectrum (usually at the peak maximum) is then
determined via linear interpolation of these two background spectra
and is finally subtracted from the single spectrum (usually of the peak
maximum).
410
Actions in the QNT Editor
Tip:
In addition, it is possible to combine several spectra to the Apex spectrum,
and then subtract the background spectrum from this averaged spectrum.
However, this is usually not necessary for UV spectra as they have a
relatively low noise level compared to ¾Mass Spectra.
Use the UV tab of the ⇒QNT Editor to determine background subtraction
for UV spectra. The Enable Background Subtraction check box is
enabled indicating that spectrum subtraction is active:
Only for UV spectra with a high noise level, it is necessary to average
spectra to reduce the noise level. For example, spectra averaging may be
required for very small peaks that are only slightly above the ¾Limit of
Detection. In the combo box under Peak Spectrum Bunch, enter the
number of single spectra that shall be bunched to form the spectrum at the
peak maximum. A maximum of 99 single spectra can be averaged. For
symmetry reasons, it is possible to enter odd numbers only. Therefore, by
default, only one spectrum is selected as Width of the Peak Spectrum
Bunch.
Peak Dependent Background Subtraction allows automatic background
subtraction for each peak. In the edit fields under Left Region Bunch and
Right Region Bunch, enter the number of spectra to be used for forming
the two background spectra. You can select up to 99 single spectra. Zero
and even numbers are permitted as well. These settings apply to peak
Actions in the QNT Editor
411
spectra and time spectra below peaks. The default setting is that one
spectrum each is subtracted on both sides of the peak.
Select Fixed Background Subtraction Ranges to define two fixed ranges
for background subtraction for the entire chromatogram. It usually makes
sense to set one range at the beginning and the other one at the end of the
chromatogram. These settings apply to peak spectra and to all time
spectra.
Click Apply to accept the settings and calculate the resulting UV spectrum.
When the Show Spectra view is enabled, the newly calculated UV
spectrum is displayed at once.
Note:
Defining the background subtraction manually in the chromatogram affects
the settings on the UV tab page. For example, if you select UV
Background Subtraction on the context menu of the chromatogram, and
then select Fixed Background Ranges, the corresponding option is
automatically selected on the UV tab page of the QNT Editor.
412
Actions in the QNT Editor
Actions in the Chromatogram
413
Actions in the Chromatogram
The window shows the integrated chromatogram of a sample. The peak
areas are determined and integrated automatically, based on the
⇒Detection Parameters.
If the representation and/or evaluation of single samples do not correspond
to the expected result, you can select another chromatogram section to
specifically change the peak delimiters and the baseline manually. It is also
possible to display more chromatograms for comparison. In addition, you
can assign the peaks manually and define a baseline for the entire
chromatogram (averaged baseline). You can also apply a data filter to
smooth the chromatogram and to improve the reproducibility of the peak
baselines.
For more information, refer to:
Using Hotkeys in the Chromatogram
Manual Re-Integration
Manual Peak Assignment
Comparing Chromatograms
Performing Data Smoothing
Due to the increased noise, we recommend performing background
subtraction for mass spectra. This is also possible for UV channels. In both
cases, determine background subtraction in the chromatogram (see
Subtracting Background Spectra).
You can also generate additional channels after data acquisition, see:
Copying a Channel
Combining Channels via Arithmetic Operations
Also, refer to
Displaying Peak Information in a Separate Area.
To graphically move the retention window, refer to
Window Graphically.
Moving a Retention
If you want to change the ⇒Detection Parameters, for example, because
too small or unimportant peaks are integrated or because the baseline does
not meet the requirements, you can perform these changes graphically in
the chromatogram; see
Defining Detection Parameters Graphically.
414
Actions in the Chromatogram
You can define the detection parameters according to your requirements.
All detection parameters are available in the ⇒QNT-Editor on the
Detection tab page (see How to ...: Actions in the QNT Editor
Defining Detection Parameters).
For more information about how to display a ¾Report or a spectra plot,
refer to How to ...:
Actions in UV Spectra and/or Mass Spectra
Actions in the Report Table
Using Hotkeys in the Chromatogram
Often, it may be necessary to display specified chromatogram areas only.
In this case, use the mouse to zoom out this area. In addition, so-called
hotkeys are provided for user-friendly operation. These keys refer to the
number and navigation area on the keyboard.
Scrolling
<←> = <LEFT> scrolls all chromatograms to the right; <→> = <RIGHT>
scrolls them to the left. If you press the respective key, the chromatogram is
scrolled by 5% of the window width.
<↑>= <UP> (<↓> = <DOWN>) scrolls the active chromatogram plot up
(down). If you press the respective key, the plot is scrolled by 10% of the
plot height.
Tip:
Scrolling beyond the chromatogram limits is supported for both cases.
<HOME> (<END>) brings you to the first (last) data point of the active
chromatogram. The entry is ignored if the chromatogram is already
displayed in full-size mode.
Zoom/UnZoom
Press the <x> (Multiply) key to zoom in horizontally by 20%. The anchor
point is the center of the time axis; for example, 0.0 to 10.0 min > 1.0 to 9.0
min.
Actions in the Chromatogram
415
Press the <÷> (Divide) key to unzoom horizontally by 25%. The anchor
point is the center of the time axis.
Press the <CRTL><x> or <CRTL><÷> key to display all chromatograms in
full size. This action corresponds to an Autoscale of the time axis.
Press the <+> (Add) key to zoom in vertically by 20%. The anchor point is
10% of the signal height because the baseline region at the beginning or
end of a peak is usually more interesting than the course of the peak at its
maximum; for example, 0.0 - 100.0 mAU > 2.0 - 82.0 mAU.
Press the <-> (Subtract) key to vertically unzoom by 25%. The anchor point
is near the baseline as described above.
Tip:
If the unzoom operation is performed after the zoom operation, the original
chromatogram view will be restored.
Press the <CTRL><+> or <CTRL><-> keys to autoscale the active
chromatogram.
Press the <NUM5> key to autoscale the time and signal axes in all
currently open plots. This operation corresponds to the Full Size command
on the View menu.
Peak Selection
Press the <CRTL><←> keys to select the previous peak of the active
chromatogram. If no peak is selected, the first peak of the chromatogram
will be selected.
Press the <CRTL><→> keys to select the next peak of the active
chromatogram. If no peak is selected, the first peak of the chromatogram
will be selected.
Press the <CTRL><POS1> keys to select the first peak of the active
chromatogram.
Press the <CTRL><Home> keys to select the last peak of the active
chromatogram.
Tip:
If the newly selected peak is outside the current view, the peak is
automatically scrolled into the view.
416
Actions in the Chromatogram
Chromatogram Selection
The following hotkeys are available for chromatogram ¾Overlay:
Press the <Page Up ↑> or <CTRL><↑> keys to activate the next upper
chromatogram; that is, the chromatogram whose caption is found above the
active chromatogram.
Press the <Page Down ↓> or <CTRL><↓> keys to activate the next lower
chromatogram; that is, the chromatogram whose caption is found below the
active chromatogram.
Chromeleon provides many additional hotkeys and shortcuts. For a
summary of the most important keys, refer to the table in Basic Operation
Keyboard Shortcuts.
Manual Re-Integration
All actions that can be performed directly on the chromatogram via the
different icons on the Integration ¾Toolbar are referred to as manual reintegration. They only affect the active chromatogram:
Moving Peak Delimiters
Modifying the Baseline Manually
Inserting or Deleting Peaks
Changing the Peak Type
Defining an Averaged Baseline
Prerequisite
Manual re-integration is possible only for integrated chromatograms. They
have a red baseline below the peaks and blue peak delimiters on the left
and right ends of the baseline (= integration limit).
Tip:
To display these elements on the chromatogram, select Decoration >
Peak Decoration on the context menu.
Actions in the Chromatogram
417
For manual re-integration, we recommend zooming the area in which you
want to make the changes. Select Automatic on the context menu. All
places in the chromatogram that can be changed manually will then be
marked by the corresponding mouse cursor. The shape of the mouse
cursor indicates which action can be performed.
If you have not yet saved the modifications, you can undo them by
selecting Delete Manipulations on the Edit menu.
Moving Peak Delimiters
Near the peak delimiters (blue color by default), the mouse cursor changes
its appearance (if the Automatic Tool or the Delimiter Tool are selected).
The
cursor indicates the peak start and the
cursor indicates the peak
end.
Left-click and move the mouse cursor to the new peak start or peak end.
It is not possible to cross another peak delimiter. This peak delimiter, too,
will then be moved in the same direction. After positioning, Chromeleon
draws a new baseline. The modified peak properties (Area, Width, Amount,
etc.) are immediately updated and displayed in the integration report.
Note:
You can also move the peak delimiters of negative peaks.
Modifying the Baseline Manually
To individually integrate two peaks that are not completely separated in the
chromatogram, drop a perpendicular line from the minimum between the
two peaks to the baseline. The intersection with the baseline is referred to
as "baseline node" (a).
Baseline node
a)
b)
c)
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Actions in the Chromatogram
To draw the baseline from peak end to peak end, enable the ⇒Valley to
Valley detection parameter. The baseline node automatically moves
towards the signal curve until it rests on the curve in the minimum between
two peaks (b).
You can move each baseline node along the perpendicular line and
position it at the desired position ("freely floating baseline node" (c)). If you
approach state (a) or (b), the mouse cursor automatically clicks into place.
The shape of the mouse cursor (( , ) indicates which action is currently
performed. Select the Automatic Tool or the Baseline Tool.
At the end of a baseline, you can generate freely floating baseline nodes
(d, e).
d)
e)
f)
Depending on the direction in which the mouse cursor is drawn, the system
automatically distinguishes between moving a peak delimiter horizontally
and moving a baseline node vertically. Between two baseline nodes, you
can move the entire baseline in vertical direction (f). The shape of the
mouse cursor ( ) indicates whether you can perform this action.
Inserting or Deleting Peaks
Inserting a Peak
You can later insert a new peak, i.e., a baseline and two peak delimiters, at
any free position on the chromatogram and on the leading and trailing
edges of a peak.
If a small peak ( ) appears on the right side of the mouse cursor, you can
insert a new peak at this position. If a warning sign ( ) appears instead,
you cannot insert a new peak.
•
Select Insert Peak Tool on the context menu to have the mouse
cursor indicate only those positions where you can or cannot insert a
peak. If the Insert Peak Tool is active, you cannot perform any other
actions (edit modes).
Actions in the Chromatogram
419
Deleting a Peak
To delete a peak, place the mouse cursor on the peak.
Select Delete on the context menu to remove the peak's delimiter and its
baseline.
Splitting Peaks
Sometimes only one peak is recorded when two substances elute
approximately at the same time. It may happen that one peak is detected
instead of two. This depends on the concentration ratio of the substances
and the arrangement of the single peaks. You can then add a second peak
Inserting or Deleting Peaks). The
below the existing one (also, refer to
following commands are available:
Split Peak
Move the cursor to the position where you want to split the existing peak.
Select Split Peak on the context menu to split the peak into two main
peaks. The perpendicular line splits the peak exactly at the cursor position.
Note:
Splitting the peak does not change the baseline.
Shape Shoulder
Select Shape Shoulder on the context menu to exponentially skim a
¾Peak Shoulder from the existing peak. First, move the cursor to the
position for the peak start of the peak shoulder and select Shape
Shoulder. The exponential-skimming algorithm automatically calculates the
peak end for the peak shoulder.
Note:
The shoulder maximum is calculated relative to the baseline. Thus, it must
not correspond to the highest signal value.
We recommend using the ⇒Peak Shoulder Threshold to define shoulder
shaping for the entire quantification method. Use the ⇒Rider Threshold
and ⇒Maximum Rider Ratio detection parameters to define the peaks that
will be detected as rider peaks. For more information, refer to How to ....
Defining Rider Peaks.
Actions in the QNT Editor
420
Actions in the Chromatogram
Changing the Peak Type
To change the peak type, position the mouse cursor on the respective
peak.
•
Select Change to Main Peak or Change to Rider on the context
menu.
Use this function to change a main peak into a ¾Rider Peak and vice
versa. Single peaks are always interpreted as main peaks. That is why you
cannot convert them into rider peaks. The context menu indicates: Can't
Change Peak Type.
With automatic classification, the ⇒Rider Threshold and ⇒Maximum Rider
Ratio parameters determine whether a peak is classified as a main peak or
a rider.
Defining an Averaged Baseline
By means of two points entered in the chromatogram, you can define a
baseline for the entire chromatogram (= averaged baseline):
How To
•
In the chromatogram, right-click to select an area at the beginning of
the chromatogram. The first ⇒Baseline Point shall be in the middle of
this area.
•
Select Set Averaged Baseline Start on the context menu.
Actions in the Chromatogram
421
This sets the first baseline point. The x-value is the middle of the selected
area while the y-value is the averaged value of the signal values weighted
by the data rate. This first value will not be marked, as it is not sufficient for
setting the baseline. A second point is required as well.
Note:
If you exit Chromeleon without having entered the second point, the first
point will be deleted.
Enter the second point as follows:
•
In the chromatogram, right-click and select an area somewhere at the
end of the chromatogram. The second baseline point shall be in the
middle of this area.
•
Select Set Averaged Baseline End on the context menu.
This action sets the second baseline point. The new baseline is drawn
through both points, which are marked by a red cross.
You can modify the averaged baseline later in two ways:
1. Select a new baseline point, which then replaces one of the former ones.
2. Use the baseline tool on the Integration ¾Toolbar, see How to ...:
Actions in the Chromatogram
Modifying the Baseline Manually.
Manual Peak Assignment
(Dialog box "Properties of Peak No. x")
If you have inserted a new peak, or wish to name existing or unidentified
peaks, or rename incorrectly identified peaks, you can do this in the
The
chromatogram either via the QNT Editor (see Data Reprocessing
QNT Editor) or in a report.
•
Click the respective peak and select Peak Properties on the context
menu to open the dialog box Properties of Peak No. x or double-click
the peak.
•
Enter the corresponding name under Component or select one of the
names listed in the combo box. Click <Return> or the lowest symbol
bottom to insert the peak into the peak table of the QNT Method using
the retention time of the active chromatogram and the settings of the
previous peak (as far as sensible).
422
•
Actions in the Chromatogram
It is also possible to rename an identified peak by manually assigning it
a different name. Enter the new name or select one of the names listed
in the combo box and confirm your entry by clicking the first symbol
button. Via the additional symbol buttons, you can cancel this action or
delete all manual peak assignments (see quick info for the buttons).
Manual peak assignments of identified peaks are not transferred to the
QNT Method but apply to the currently open chromatogram only.
You do not have to close the dialog box to continue working in the
chromatogram. <Return> accepts a new entry in the peak table of the QNT
Editor (provided that the peak has not yet been assigned there). The dialog
box then indicates the values of the next peak to be assigned. The dialog
box remains open until explicitly closed or until the chromatogram is closed.
Note:
The manual peak assignments are saved in the ¾Quantification Method.
However, manual peak assignments for identified peaks are not included in
the QNT Method and are valid for the currently open chromatogram only.
Actions in the Chromatogram
423
Comparing Chromatograms
Chromeleon allows you to compare several chromatograms by
simultaneously displaying different samples or several ¾Channels of the
same sample. Besides, you can also compare different channels of
different samples. To compare chromatograms, select the samples or
channels and relate them to each other.
For more information, refer to:
Selecting the Samples and Channels
Displaying Several Chromatograms
Mirroring Chromatograms
Normalizing Chromatograms
Selecting the Samples and Channels
Different alternatives are available to select samples and channels that are
to be displayed as ¾Overlays:
•
Display the chromatogram of a sample in an integration window by
opening the sample, for example, by double-clicking its name in the
¾Browser.
Select one or several samples of one ⇒Sequence in the Browser and
drag the sample(s) into an open integration window while holding down
the left mouse button. The action is indicated by a + sign on the mouse
cursor. When you release the mouse button, the chromatogram of the
sample(s) is displayed. The same channel is used as for the reference
sample.
Instead of selecting a sample in the Browser and dragging it into an
open integration window, you may also select the sample in the
integration view using the Add Overlay command on the File menu.
The chromatogram of the first sample will then be overlaid by the
chromatogram of the selected one.
Or:
•
Select several samples in the Browser and then select Compare on
the context menu. The Compare submenu lists all recorded channel
types. After selecting a channel, the chromatograms of all samples with
raw data for this channel are compared.
424
Actions in the Chromatogram
Or:
• Select a sample in the Browser, and then select All Channels on the
context menu to compare all channels of a sample in a separate
window. Press the F4 key or the Shift+F4 key combination to display
the channels of all samples in a sequence successively.
If several samples are selected, all channels of the first selected
sample are displayed. Press the F4 key or the Shift+F4 key
combination to browse through the selected samples.
Or:
•
Click the Next/Previous Chromatogram icons ( / ) while pressing
the Ctrl key to insert the chromatogram of the next or previous sample.
Or:
• Perform a ¾Query over several sequences or ¾Datasources. Select
the Compare command on the context menu. The Compare submenu
lists all recorded channel types. After selecting a channel, the
chromatograms of all samples with raw data for this channel are
compared.
Or:
•
Click the Next Channel icon ( ) while holding down the Ctrl key to
insert the next channel of the same sample.
•
Click the Previous Channel icon ( ) while holding down the Ctrl key
to insert the previous channel of the same sample.
Displaying Several Chromatograms
After adding an ¾Overlay (= an additional chromatogram), you can define
the display arrangement for the two chromatograms.
How To
•
Select Decoration... on the context menu of the chromatogram plot.
•
In the Chromatogram Decoration dialog box, select the Comparison
tab page and define the chromatogram Arrangement:
Select Overlay to overlay the single chromatograms on one single plot
(see the left section of the screenshot below).
Select Stack to display the chromatograms one below the other in
different plots (see the right section of the screenshot below).
Actions in the Chromatogram
425
Select Mixed to combine the Overlay and the Stack option.
Chromatograms of different detectors are displayed as single plots.
Use the Overlay view to add an additional signal axis (on the right). The
axis refers to the overlaid chromatogram that was added last using the
Overlay with right signal axis command. This command, too, is available
on the Comparison tab page of the Chromatogram Decoration dialog
box. Thus, you can select a different signal range for the chromatogram
that was added last. Use the Stack view for individual scaling of the signal
axis for each single chromatogram.
To offset the chromatograms in x- and/or y-direction in the Overlay view,
set the signal or time offset on the Comparison tab page.
•
Select the Time check box to move the active chromatogram in the xdirection.
•
Select the Signal check box to move the active chromatogram in the ydirection.
The offset is specified in percent of the signal or time axis. A signal offset of
5% shifts each of the following chromatograms upwards by 5%.
Accordingly, a time offset causes a percentage alteration in the x-direction.
This results in a "pseudo-3D" presentation.
426
Actions in the Chromatogram
Mirroring Chromatograms
If you have inserted an ¾Overlay (= an additional chromatogram), you can
mirror the overlay.
• Select Decoration on the context menu on the chromatogram.
• In the Chromatogram Decoration dialog box, select the Comparison
tab page. Verify that Overlay is selected in the Arrangement section.
• You can mirror the chromatogram that was added first by selecting the
Mirror chromatogram check box in the Overlay section.
If you have also selected the Overlay with right signal axis check box,
this setting will be ignored. In this case, the overlay is displayed with the
right signal axis; it is not mirrored. However, if you add another
chromatogram, it is displayed with the right signal axis and the previous
chromatogram is mirrored.
Normalizing Chromatograms
For a sensible comparison of different chromatograms, often a common
reference point is chosen for the overlaid chromatograms (see ¾Overlay).
The chromatograms are normalized. As the default, the ⇒Retention Time
is used for the normalization, that is, all chromatograms are arranged so
that their start points match (t = 0).
Actions in the Chromatogram
427
Alternatively, chromatograms can use a common peak as their reference
point.
•
Select Decoration on the context menu and open the Comparison tab
page.
•
Select the reference peak from the At Peak list box.
•
Select Shift if this is to be performed independently from the length of
the chromatogram.
•
Select Stretch if the chromatograms are to be stretched or
compressed in x-direction, so that there is a time scaling in all
chromatograms. The stretch 0 to 1min (or n min) has the same length
for all chromatograms.
•
Enable the Normalize Signal check box if the height of the desired
chromatogram peak should match in addition. Depending on how much
the reference peak must be stretched or compressed, all other peaks in
the chromatogram are also stretched or compressed. This enables
amount estimation for the same peaks from different chromatograms.
This option is not available in the Stack view.
Performing Data Smoothing
Data ¾Smoothing can help improve the appearance of chromatograms and
the reproducibility of peak baselines by reducing noise through digital
filtering. Smoothing affects both, the display and the integration of the
chromatogram. When smoothing is completed, the new chromatogram is
displayed overlaid on the original chromatogram. The original data file is
not altered and the smoothed data is saved separately.
Tip:
You can define data smoothing also in the ¾Post-Acquisition Steps view of
The PGM Editor). Please observe the
the PGM Editor (see Control
corresponding hints below.
428
Actions in the Chromatogram
How To
1. Display the sample's chromatogram by double-clicking its name in the
¾Browser.
Tip:
Open the PGM File in which you want to define data smoothing and
select the Post-acquisition steps view, instead.
2. Select Smoothing on the context menu. The Smoothing dialog box
appears:
Tip:
In the PGM Editor, follow the steps below: Select Insert line on the
context menu to add a new post-acquisition step. The New postacquisition step dialog box is opened. Select Smooth data to open the
Smoothing dialog box.
3. Select the Filter Type, Filter Size, and Iterations. For more
information, refer to
Data Smoothing.
4. In the Smoothed Channel box, enter the name for the smoothed
channel. The suggested name is the current channel name, followed by
the type of filter (MA for Moving Average, OL for Olympic, and SG for
Savitzky-Golay), the filter size, and the number of iterations.
Actions in the Chromatogram
429
5. To smooth all the samples in the sequence or ¾Query, select the Apply
to all samples in the current sequence or query check box.
Tip:
This option is not provided in the Smoothing dialog box of the PGM
Editor.
6. Click OK to start the smoothing.
For information about how to smooth MS chromatograms during mass
trace extraction, refer to How to ...: Actions Related to Mass
Spectrometers
Extracting Mass Traces Afterward.
Copying a Channel
Chromeleon allows you to copy a channel to evaluate the same raw data in
different ways, for example, to determine both, the ingredients and the
contaminations of a pharmaceutical product in one chromatogram. To do
so, evaluate the original channel with one QNT Method and then, evaluate
the copied channel with a different QNT Method.
Tip:
You may also define copying of channels in the ¾Post-Acquisition Steps
The PGM Editor). Please observe
view of the PGM Editor (see Control
the corresponding hints below.
How To
1. Display the sample's chromatogram by double-clicking its name in the
¾Browser.
Tip:
Open the PGM File in which you want to define that a channel is copied
and select the Post-acquisition steps view, instead.
2. Select Copy Channel... on the context menu. The Copy Channel...
dialog box appears.
430
Actions in the Chromatogram
Tip:
In the PGM Editor, follow the steps below: Select Insert line on the
context menu to add a new post-acquisition step. The New postacquisition step dialog box is opened. Select Copy Channel to open
the Copy Channel dialog box.
3. In the New Channel box, enter the name for the new channel. The
suggested name is the name of the current channel plus _COPY.
However, you are free to enter any name of your choice as well.
4. To copy the channel for all samples in the sequence or ¾Query, select
the Apply to all samples in the current sequence or query check
box.
Tip:
This option is not provided in the Copy Channel dialog box of the PGM
Editor.
5. Click OK to start the procedure.
Combining Channels via Arithmetic
Operations
Chromeleon also allows you to combine two channels of different samples,
using arithmetic operations. Each data point is created by combining the
associated two data points from the existing channels (i.e., the data points
at the corresponding time), using the desired operation.
Arithmetic combinations are not restricted to chromatograms in the strict
sense. They can be used for all 2D channels, except for temporary
channels.
Tip:
You may also define the combination of two channels in the ¾PostThe PGM
Acquisition Steps view of the PGM Editor (see Control
Editor). Please observe the corresponding hints below.
Actions in the Chromatogram
431
How To
1. Display the sample's chromatogram by double-clicking its name in the
¾Browser.
Tip:
Open the PGM File in which you want to define that two channels are
combined and select the Post-acquisition steps view, instead.
2. Select Arithmetic combination of channels... on the context menu.
The Arithmetic combination of channels... dialog box appears.
Tip:
In the PGM Editor, follow the steps below: Select Insert line on the
context menu to add a new post-acquisition step. The New postacquisition step dialog box is opened. Select Arithmetic combination
of channels to open the Arithmetic combination of channels dialog
box.
3. The current channel is Channel A. Select the factor with which the
channel shall be multiplied.
432
Actions in the Chromatogram
Tip:
In the PGM Editor, select the channel of the corresponding sample from
the channels defined in the PGM File. Channels defined in previous
post-acquisition steps are also available for selection.
4. Select a channel of the same sample or of any other sample of your
choice as Channel B. Also, determine the factor with which Channel B
shall be multiplied.
5. Select the desired Operation.
6. In the Result Channel box, enter the name for the new channel. The
suggested name is the abbreviation for the operation plus the names of
the two channels. However, you are free to enter any name of your
choice as well.
7. To combine the selected channels for all samples in the sequence or
¾Query, select the Apply to all samples in the current sequence or
query check box.
Tip:
This option is not provided in the Arithmetic combination of channels
dialog box of the PGM Editor.
8. Click OK to start the procedure.
Subtracting Background Spectra
¾Mass Spectra usually have a higher noise level than UV spectra.
Therefore, you may want to subtract the background spectra. (However, for
UV channels, it is usually not necessary to change the default background
subtraction.) There are two ways to subtract background spectra.
Peak Dependent Background Subtraction
Enable Background Subtraction on the context menu of the
chromatogram. Return to the context menu and select Peak Dependent
Ranges. Chromeleon automatically determines two ranges for calculating
the background and then subtracts the entire mass or UV spectrum of
these ranges. On the MS or UV tab page of the ⇒QNT Editor, define the
number of mass spectra or UV spectra to be used for the two ranges. (For
more information, refer to How to …: Actions in the QNT Editor
Processing Mass Traces and/or
Processing UV Channels.)
Actions in the Chromatogram
433
Fixed Background Subtraction for the Entire Chromatogram
At the beginning of the chromatogram, right-click and select the baseline
range for which the mass spectra (UV spectra) will be subtracted. A context
menu will appear. Select Set Background Subtraction Range 1 on the
context menu to use the defined range as the first range for which to
subtract the mass spectra (UV spectra) from the mass spectra (UV spectra)
of the single peaks and/or from the retention time spectrum.
Note:
Selecting Set Background Subtraction to Range 1 automatically enables
the Fixed Background Ranges option of the Background Subtraction
command on the context menu.
In the same way, select a baseline range at the end of the chromatogram.
Select Set Background Subtraction Range 2 on the context menu to
define this range as the second range for which mass spectra (UV spectra)
will be subtracted from the mass spectra (UV spectra) of the single peaks.
Note:
It is useful, but not imperative, to define a second range; background
subtraction can also be performed using only the mass spectra (UV
spectra) of the first range.
The two defined ranges are marked by a horizontal line (in the same color
as the spectrum, or in blue if no spectrum is available) and labeled "SB1" or
"SB2" (SB = subtracted background). If the background subtraction range
was used, the number of single mass spectra or UV spectra that were
averaged is indicated in parentheses:
434
Actions in the Chromatogram
Note:
It is possible to set the two ranges for background subtraction of mass
spectra in a UV channel. However, we recommend setting the two ranges
in the corresponding MS channel because the respective peaks are visible
only there.
Clear Background Subtraction Ranges removes the previously defined
ranges.
Effects
The settings selected in the chromatogram are saved in the QNT File of the
current sample. These settings overwrite the settings on the MS or UV tab
page of the QNT Editor. It makes no difference whether background
subtraction is defined on the Integration plot or in the QNT Editor. Thus,
your input affects all samples that are evaluated using this QNT File.
Tip:
Make sure that no peak of another sample is within the retention time used
as the Background Subtraction Range in Fixed mode.
For a mass spectrum (UV spectrum) that was recorded between two
ranges, the ranges are averaged and the result is subtracted. The mass
spectra (UV spectra) of the two ranges are weighted based on the time
distance from where the respective mass spectrum (UV spectrum) was
recorded; that is, the range that is nearer to the respective mass spectrum
(UV spectrum) is considered more.
If a mass spectrum (UV spectrum) is not located between the two ranges,
only the averaged mass spectrum (UV spectrum) of the range next to the
spectrum is subtracted.
Actions in the Chromatogram
435
Displaying Peak Information in a Separate
Area
Chromeleon supports displaying the peak information in a separate area
above the actual chromatogram:
•
Double-click the chromatogram
Decoration dialog box.
•
On the Peak Calipers tab page, select Show peak calipers to display
the peak information.
to
open
the
Chromatogram
In addition, you can also display the following information:
•
The name of the current QNT method: Show QNT info.
•
The lines indicating the width of the retention time ⇒Window of the
associated substance: Show all caliper drop lines.
The chromatogram looks as follows:
In addition, you move the retention time window graphically. (For more
Moving a Retention Window Graphically.
information, refer to
436
Actions in the Chromatogram
Moving a Retention Window Graphically
Chromeleon allows you to change the retention window, i.e., the expected
⇒Retention Time and the time ⇒Window, for single peaks in the
chromatogram:
•
Go to the Peak Table tab page of the QNT Editor to check the
graphical move of the retention window described below:
•
Option: Add the lines that indicate the time limit of the window to the
chromatogram: In the Chromatogram Decoration dialog box, select
the Show all caliper drop lines check box on the Peak Calipers tab
Showing the Peak
page. (For more information, refer to
Information in a Separate Area.)
To graphically move the retention window, perform one of the steps below:
1. Move the entire window: To move the retention window of a peak,
place the cursor on the center of the associated peak caliper. The
cursor changes its appearance and looks as follows:
.
Hold down the left mouse button and drag the caliper to the desired
position, i.e., to the desired time. The entry in the Retention Time
column of the peak table is updated automatically.
2. Move the start or end time: To change the time of a specific line,
place the cursor on the edge of the caliper or on the associated dotted
line. The cursor changes its appearance and looks as follows .
Hold down the left mouse button and drag the line to the desired
position, i.e., to the desired time. The entries in the Window and
Retention Time columns of the peak table are updated automatically.
3. Move the start or end time symmetrically: Hold down the Shift key
when the cursor is placed on the edge of a caliper or on a dotted line.
The cursor changes its appearance and looks as follows . Hold down
the left mouse button and drag the lines to the desired position, i.e., to
the desired time. The entry in the Window column of the peak table is
updated automatically.
Tip:
In the same way, you can change the start and end time of an area for
groups of unidentified peaks.
Actions in the Chromatogram
437
Defining Detection Parameters Graphically
Chromeleon supports entering detection parameters graphically: This
considerably facilitates defining the time for the ⇒Detection Parameters.
How To
•
If no parameter is displayed in the chromatogram, use the Detection
Parameter Tool. Either select this command on the context menu or
click the following icon on the integration ¾Toolbar:
.
•
In the chromatogram, move to the exact location where you want to
enter a parameter.
•
Select Detection Parameters on the context menu.
•
You can then select
Insert Inhibit Integration On or Off to inhibit or allow integration (see
Inhibiting Peak
How to ...: Actions in the QNT Editor
Integration).
Insert Valley to Valley On or Off to enable or disable integration from
valley to valley.
Insert Detect Negative Peaks On or Off to enable or disable
integration of negative peaks.
Insert Baseline Point to insert a new baseline point (for more
information about the last three commands, refer to How to ...: Actions
in the QNT Editor
Modifying the Baseline).
Insert to insert a new parameter.
•
The Detection Parameter Tools moves the entered parameters to the
desired location in the chromatogram.
The example on the next page corresponds to the table input in How to ...:
Defining Detection Parameters.
Actions in the QNT Editor
438
Actions in the Chromatogram
A dotted line indicates the parameters in the chromatogram. The short form
for the parameters and their values are displayed along the line. The
picture shows, for example, the Rider Threshold (in short: RidThd), Rider
Skimming (RidSki), and Maximum Rider Ratio (RidRat) parameter values at
1.000 min.
To enter the detection parameters graphically, you also select a
chromatogram area while holding the right mouse key. The context menu is
opened automatically; the menu contains the following commands:
•
Set Averaged Baseline Start and Set Averaged Baseline Start End
to set an averaged baseline (see How to ...: Actions in the
Chromatogram
Defining an Averaged Baseline).
•
Set Minimum Area to define the minimum peak area.
•
Set Minimum Height to define the minimum peak height.
•
Set Minimum Width to define the minimum peak width (for more
information about the Set Minimum ... commands, refer to How to ...:
Reducing the Number of Evaluated
Actions in the QNT Editor
Peaks).
Actions in the Chromatogram
439
•
Set Peak Slice & Sensitivity to define the peak recognition algorithm
Modifying the Peak
(see How to ...: Actions in the QNT Editor
Recognition Algorithm).
•
Set Inhibit Integration Range to select a range for the peak inhibition.
•
Set Void Volume Treatment Range to determine the range for
recognition of the negative water peak. This overwrites all previous
ranges for recognition of the negative water peak.
Note:
You can undo the graphical input of detection parameters. Click one of the
QNT Editor tables, and then select Undo on the Edit menu. (In the
chromatogram itself, you can only undo the modifications made for the
active chromatogram.)
When new detection parameters are entered graphically, they are
automatically copied to the Detection sheet of the ⇒QNT Editor. They will
be used for all samples that are evaluated with the QNT File of the current
sample. As an alternative, you can also enter the detection parameters
directly on the Detection sheet. (For more information, refer to How to ...:
Actions in the QNT Editor
Defining Detection Parameters).
440
Actions in the Chromatogram
Actions in the UV Spectra and/or Mass Spectra
441
Actions in UV Spectra and/or
Mass Spectra
Actions in the UV Spectra and/or Mass Spectra
To enable the Spectra plot, select Show Spectra on the View menu or
click
The Spectra plot represents UV spectra and their identification by means of
a spectra library.
The Mass Spectra plot is very similar to the Spectra plot. To enable the
Mass Spectra plot, select Show Mass Spectra or click
For more information, refer to:
Displaying a Peak Spectrum (UV or MS)
Displaying and Overlaying Single (UV or MS) Spectra
Match Factor, Difference Spectra, 1st /2nd Derivatives of UV Spectra
Starting a UV Spectra Search
Also, refer to How to ...: Actions Related to Mass Spectrometers
Extracting Mass Traces Afterward.
Tip:
In order to represent spectra, make sure that the corresponding raw data is
available. Obtain the raw data by recording a 3D field using a ¾Photodiode
Array Detector.
Displaying a Peak Spectrum (UV or MS)
•
In the neighboring chromatogram window, select the peak of which you
want to display the UV or ¾Mass Spectrum.
The spectrum of the peak is visible. (Note: If the corresponding raw data is
not available, an error message is displayed.)
•
Select Decorations.. on the context menu of the (Mass) Spectra
window and determine the peak height at which to extract and display
spectra via Peak Spectra.
442
Actions in the UV Spectra and/or Mass Spectra
Depending on the settings, up to five spectra of the same peak are
displayed in different colors. Normally, the spectra are extracted at 10 and
50% peak height from the leading and the trailing peak edges and at the
run time of the peak. The representation of the spectrum is normalized. If
the spectra largely match, this can be a criterion for peak purity.
Displaying and Overlaying Single (UV or MS) Spectra
In addition to peak spectra (= UV or ¾Mass Spectrum in the peak
maximum or spectrum at a defined peak height), Chromeleon is capable of
displaying any other spectrum of a chromatogram at the time t. To extract
any spectra of a chromatogram via a mouse click, follow the steps below:
•
Enable the Spectra Tool on the context menu or click the
corresponding icon on the Integration toolbar.
•
The changed mode is indicated by a spectra symbol that is added to
the mouse cursor.
•
Click anywhere in the chromatogram to display the corresponding
spectrum.
•
Repeat the action while pressing the Shift key to overlay several
spectra.
Displaying Spectra of Different Samples
To objectively compare spectra of different samples, it is necessary to
perform a chromatogram comparison.
•
Compare two chromatograms by displaying an additional
chromatogram in the Integration window (see Integration
Chromatogram Comparison).
•
Enable the Spectra Tool and select single spectra by clicking various
places in the chromatogram while pressing the Shift key.
Note:
UV spectra are overlaid in one window. MS spectra are displayed one
below the other to enhance the clearness of representation. For a large
enough representation, enlarge the upper section of the entire window, if
necessary.
Actions in the UV Spectra and/or Mass Spectra
443
Match Factor, Difference Spectra, 1st/2nd Derivatives
of UV Spectra
When two or more spectra are represented on the spectra plot, the
similarity of the UV spectra becomes an issue.
The match factor can express the similarity, by forming difference spectra
or by representing the first or second derivative of a UV spectrum.
•
Select Decorations on the View or context menu and select the Show
match check box on the Label tab page. Chromeleon issues a value
for each represented spectrum, expressing the match degree relative
to the main spectrum (0 = no match; 1000 = perfect match).
•
On the Analysis tab, define whether the difference spectrum or the first
or second derivative of a spectrum shall be displayed in a second
window in addition to the actual spectra.
In the case of the match factor and the difference spectrum, the question
which UV spectrum is considered a main spectrum is especially important,
as this is the basis of comparison or the basis for all calculations.
Usually, this is the peak spectrum extracted at the retention time. If there is
no peak spectrum, distinguish two situations: If you select the spectra tool
to extract the single UV spectra from the chromatogram, the spectrum that
is first extracted is the main spectrum. If spectra are extracted automatically
at different peak heights, the spectrum with the "oldest" retention time is
considered the main spectrum. When representing difference spectra, the
Difference to ... entry indicates the basis of calculation.
Starting a UV Spectra Search
To facilitate substance identification, the peak spectrum, which is displayed
on the Spectra Plots of the Integration or PPA method, can be compared
to the UV spectra of a spectra library.
Based on the available library, Chromeleon creates a spectra list sorted by
the degree of similarity, the Hit List. The number of possible hits can be
limited via comprehensive search criteria.
•
If the cursor is positioned in a spectra window, select Library Search
... on the context menu to start the spectra search.
444
Actions in the UV Spectra and/or Mass Spectra
In the edit box, specify the library that should be searched. The following
are minimum entries required to receive a valid search result:
•
Select a spectra library (LIB file) from the Spectra Library to be
searched in ... list. If no LIB file is displayed, click Browse to search
for the file.
•
In the ⇒Match Criterion field, determine the mathematical method
based on which the original spectrum and the library spectrum are
compared with each other. The best search results are generally
received with Least Squares.
•
Click OK to start a spectra search.
Note:
To be able to compare UV spectra with each other, the spectrum and the
reference spectrum should be recorded under identical conditions. For best
results, compare spectra to your own spectra library.
Result
A list of possible candidates will be displayed. The top spectrum has the
highest match value and the best similarity to the original spectrum.
Detailed Search
To accelerate the search and to receive very specific results, there are
numerous options:
•
Normally, the Spectrum Derivative option is disabled, that is, spectra
comparison is based on the actual curve shape. If the 1´st Derivative
option is selected, the comparison of the two spectra is performed
based on the first derivative. Consequently, the curve characteristics
are more significant (shoulders become real extremes), which allows a
more precise comparison. The drawback of this option is the reduction
of the signal-to-noise ratio, which causes sections with weaker signals
to loose significance.
•
Select Hit Threshold and enter a value between 0 and 1000, for
example, 950. Only the spectra with a match above 950 will be
displayed. Spectra with a lower match value will not be displayed.
Spectra with a match value below 900 are usually spectra of other
substances (if derivatives are not used for the comparison). Exceptions
to this are acceptable only if, for example, the signal-to-noise ratio is
low.
Actions in the UV Spectra and/or Mass Spectra
445
Select the following options to ensure precise search results:
•
Enter the number of relative extremes the spectrum should have
(Number of Relative Extrema). This option is useful to exclude
spectra that are similar but have an additional side maximum.
•
Select Check Greatest Relative Maximum to use only spectra with
the greatest relative maximum at the same position. Select Allowed
Deviation to define a tolerance range. The range should not be more
than 10 nm to avoid that the criterion weakens.
•
Via Retention Time Window, define a time window that includes the
retention time of the peak. Use this option to exclude substances with
very similar spectra, but which are eluted at very different retention
times.
Additional criteria are only required in special cases. However, they are
useful for searching large libraries containing numerous spectra of the
same substance, but extracted under different conditions; for example,
different solvents and detectors, different users, different date, etc.
Note:
When creating your own spectra library, please enter information in all
fields, even in the fields that may seem unnecessary. In the course of time
and with a growing library, this may become a major advantage.
446
Actions in the UV Spectra and/or Mass Spectra
Actions in the Calibration Curve
447
Actions in the Calibration Curve
The Calibration Curve displays the ¾Calibration Curve calculated from the
¾Calibration Points. The Calibration tab page of the Report allows you to
display the calibration data of the peaks of the active chromatogram.
If a single standard was excluded from the calculations (Enabled column)
in the Calibration settings made in the QNT Editor, the corresponding
calibration point is red. Calibration points of this type will not be used for
calculating the calibration curve for which they were disabled. The display
color of the calibration points of the current sample is purple.
In the Calibration Curve, select Disable Standard on the context menu to
exclude standards, completely or in part, from the calibration. The Disable
(Enable) Standard xyz dialog box appears for the selected calibration
point. (This point refers to the selected peak in the active chromatogram.
Double-click a different calibration point to select a different standard.)
Define whether the corresponding standard sample shall be used for
calibration purposes. You can exclude the respective standard from the
calibration for all peaks and all channels (default) or for the selected peak
and/or channel only. (Also, refer to How to ...: Actions in the QNT Editor
Disabling Calibration Samples.)
Select Decoration on the context menu to perform the following actions on
the different tab pages:
•
Select the peak for which to show the calibration curve (Peaks tab
page).
•
Determine the axis scale (Scale tab page).
•
Define the display and decoration of the axes, frames, and grids
(Frame & Axes tab).
•
Show validation samples (select Draw validation sample points on
the Options tab). Validation samples are indicated by the following
symbol: .
•
Draw lines for the ¾Confidence Interval (Options tab, see
Confidence Interval)
•
Change the colors of the single elements (Colors tab).
Show
In addition, you can select Next Peak or Prev. Peak to display the
calibration curve of the next/previous peak.
448
Actions in the Calibration Curve
Displaying Values
A purple cross marks the standard sample or the ¾Spiked Sample that is
currently used:
. (The sample has been selected on the Calibration tab
page of the ⇒QNT Editor.) If you disable a specific sample in the Enabled
column on the Calibration tab page of the QNT Editor, e.g., because an
error occurred during determination of the area value, this sample is
excluded from calibration. The corresponding calibration point is then
. This kind of calibration points is not
marked by a red asterisk:
considered when the calibration curve is determined. To exclude standards,
completely or in part, from calibration, select Disable Standard on the
context menu of the Calibration Curve.
To display validation samples, Select Decoration on the context menu.
The Calibration Plot Decoration dialog box is opened. On the Options
tab page, select the Draw validation sample points check box. Validation
samples are indicated by the following symbol: . They are not considered
for calibration.
Actions in the Calibration Curve
449
Indicating the Confidence Interval
The confidence interval describes the range in which the "true" calibration
curve will be found with a given probability. The range of the ¾Confidence
Interval grows linearly with growing standard deviation. The easiest way is
to indicate the confidence interval graphically.
How To
•
Open the Calibration Plot Decoration dialog box by selecting
Decoration ... on the context menu.
•
Select Draw upper & lower confidence limits on the Options tab
page.
•
Select the probability with which the values should be within the
confidence interval. The following options are available: 90%, 95%,
98%, 99%, 99.8%, 99.9%, and 99.99%.
The following example shows the confidence interval of a calibration at a
probability of 99%:
You can indicate the limits of the confidence interval in the Report using the
Upper and/or Lower Confidence Limit variables of the ⇒Peak Calibration
peak category.
For more information, refer to How to …: Actions in the Report Table
Setting Parameters for Variables (e.g., for the confidence interval).
450
Actions in the Calibration Curve
Actions in the Report Table
451
Actions in the Report Table
Chromeleon provides numerous options to work with reports. It is possible
to use existing default reports or create special ¾Reports to meet individual
requirements. For more information, refer to:
Displaying a Report
Defining the Contents of a Report
Defining the Appearance of a Report
Adjusting the Line Height Automatically
Saving a Report
Linking Report Variables
Calculating the Peak Variable "Amount"
Adding and/or Renaming a Worksheet
Displaying the Peak Summary
Displaying an Audit Trail
Creating a History Report
Displaying MS Reports
Selecting Other Special Reports
Setting Parameters for Variables (e.g., for the confidence interval)
Displaying a Report
A numerical ¾Report can be included in each of the six method windows
(Integration, PPA, QNT Editor, Spectra, Calibration Curve, and Spectra
Calibration).
•
Select Report on the View menu.
If you have not generated a report before, the default report (called
Default) will be opened. The default report contains the following
worksheets: Integration, Calibration, Peak Analysis, Summary, and
Audit. If you are working with a ¾Photodiode Array Detector, select the
default report, DEFLTDAD. This report contains two additional worksheets:
Peak Purity and Lib Search.
452
•
Actions in the Report Table
To select a worksheet, click the corresponding tab.
Each worksheet includes several default variables that are specific to the
selected report type. For example, in an integration report, there are
columns for Ret.Time, Area, and ⇒Amount. A calibration report contains
columns for Offset (c0), Slope (c1), and Curve (c2), etc.
These predefined report sheets can be used for various purposes; no
additional input is necessary.
Defining the Contents of a Report
To change the contents of a ⇒Report Definition File, select Table on the
context menu or double-click a table column.
•
Select Insert Column to insert a column in the report table on the left
of the current cursor position.
•
Select Add Column to add a column to the report table on the utmost
right.
Note:
A new column that has been created using the Add Column command
has no format yet.
•
Select Fix Column to move the selected columns to the far left.
Columns of this type are permanently visible, even when scrolling.
•
Select Delete Column to delete one or several columns.
•
Select Column Properties to modify the column properties. Thus, the
current column contents, for example, number of theoretical plates, can
be replaced by a different variable.
•
To change the formula of a cell, enable the Layout Mode on the Table
menu. An edit line appears above the table. In the table, click the cell
for which you want to edit the formula. To the left of the edit line, the
number of the cell is displayed. Edit the formula as desired. You can
also use the ¾Additional Functions of the ¾Report Publisher module, if
your Chromeleon license supports this.
Tip:
When Layout Mode is enabled, do not change the format.
Actions in the Report Table
453
Tip:
Each column usually shows one report variable. However, it is possible to
link several report variables (see How to ...: Actions in the Report Table
Linking Report Variables). Mathematical and statistical functions such
as SUM, AVERAGE, etc. are only available in the Report and the ¾Printer
Layout together with the ¾Report Publisher!
•
Select Table Properties to modify the properties of the entire report.
Use this command to sort the table in groups or remove peaks below a
certain area value.
Defining the Appearance of a Report
To modify the appearance of a report, select the Table menu. Then, select
Format >...
•
Alignment to position the text horizontally and vertically in a cell or a
column.
•
Font to determine the font, font style, and size.
•
Border to determine the location, the color, and the shape of the
frame.
•
Pattern to determine the color and the pattern of the cell background.
•
Number Format to determine the format of the represented values.
•
Autoformat to design the entire report by selecting a ready-made
default template. The Preview window shows the appearance of the
report in the currently selected format.
Tip:
When Layout Mode is enabled, do not change the format.
454
Actions in the Report Table
Adjusting the Line Height Automatically
Chromeleon allows you to adjust the line height automatically. Do this
especially for sample variables, for which the single values are displayed in
several lines in one cell:
• Enable Layout Mode on the Table menu.
• In the left column, double-click the separation line under the line for
which you want to adjust the height automatically. (In the picture below,
for example, double-click the line marked in blue separating lines 6 and
7. This automatically adjusts the height of line 6 so that the entry in the
List of Aggr. Smp. column is completely displayed.)
In the example in the picture, the heights of lines 3 to 5 are adjusted
automatically. Thus, the list of aggregated samples is completely displayed
in each line. The height of lines 6 to 9 is defined by a fixed value. In this
case, the lists are not completely displayed.
Saving a New Report Definition File
You can save all modifications made in a report to a ⇒Report Definition
File (RDF):
•
Select Save Report Definition ... and then enter the file name under
which to save the RDF.
•
If you change a default Report Definition File from the Dionex
Templates > Report directory, you usually cannot save the new file to
this Report folder because the Dionex Templates directory is locked.
Save the modified file to a different directory; for example, create a new
'Report' directory under your local datasource.
Actions in the Report Table
455
Tip:
If you do not save the RDF before you exit the screen report, any changes
made to the report's layout are lost. There will be no hints as to the loss of
these changes because a warning would then have to appear for every
other change, as well, e.g., when resizing single windows.
Linking Report Variables
To generate user-defined report variables, use the four basic arithmetic
operations or powers to link two or more report variables. Link the single
variables, using the formulas of the ⇒Report Categories.
How to:
•
Select a report column, and then select Add Column on the Table
menu.
•
In the Formula field of the edit box (Add Report Column), enter the
formulas you wish to link by mathematical operators.
•
Note that the variable names are not identical with their "formulas"; for
example, peak height and peak.height). For the correct syntax, refer
to ⇒Report Categories. You can copy the syntax by selecting the
required formula and pressing the CTRL+C key combination. Change
to the edit box again and insert the formula by pressing CTRL+V.
Example: To put the peak height in relation to the corresponding
⇒Amount, use the following expression:
peak.height / peak.amount
•
You can also use the following operators to link expressions: +, -, x,
and ^ (for powers)
•
Enter the desired column header in the Header field.
•
Click Customize to include newly defined variables in the report
variables list.
•
Click OK to complete your input.
Tip:
Mathematical and statistical functions such as SUM, AVERAGE, etc. are
only available in the Report and in the ¾Printer Layout together with the
¾Report Publisher!
456
Actions in the Report Table
Calculating the Peak Variable "Amount"
If you wish to check the indicated ⇒Amount values, have a look at the
respective coefficients of the calibration function first. Then, add the
columns offset (c0), slope (c1), and curve (c2), which are all part of the
Peak Calibration category.
For the amount calculation of an unknown sample, the inverse function f(y)
of the calibration function F(x) must be calculated. Calibration functions are
calculated as follows:
f ( y) =
Linear:
1
y
c1
f ( y) = −
Linear with offset:
f ( y) =
Quadratic:
1
* ⎛⎜ − c1 ± c12 + 4 * c2 * y ⎞⎟
⎠
2 * c2 ⎝
f ( y) =
Quadratic with offset:
c0 1
+ y
c1 c1
1
* ⎛⎜ − c1 ± c12 − 4 * c2 * ( c0 − y ) ⎞⎟
⎠
2 * c2 ⎝
⎛ 1⎞
⎜ ⎟
Exponential:
⎛ 1⎞
⎜ ⎟
⎛ 1 ⎞ ⎝ c1 ⎠
c
f ( y) = ⎜ ⎟
* y⎝ 1 ⎠
c
⎝ 0⎠
Note:
For ambiguous inverse functions (two possible values for quadratic with or
without offset) always use the value which comes "closest" to the X values
of the respective calibration.
If it is not possible to calculate the expression (c1=0 for linear with or
without offset, the radiant < 0 for quadratic with or without offset, or c0=0, or
c2=0 for exponential), "n.a." is returned as amount.
If the response factor, dilution factor, weight, and a factor for the ¾Internal
Standard are available, that is, if they do not equal 1, they have to be
considered for the calculation as well (see ¾Formula for Amount
Calculation).
Actions in the Report Table
457
Adding and/or Renaming a Worksheet
You can add new worksheets to the ⇒Report Definition File and define
their contents and appearance.
•
Select Insert Report on the Table menu and determine the type of
report to be created.
The following reports are available (the order is the same as in dialog
box):
Result Tables
Peak Results
Calibration
Report
Displays all variables required for creating a calibration report on the
right.
Integration
Report
Displays all variables required for creating an integration report.
Sample Results
Calibration
History
Displays all variables documenting the course of the calibration on
the right.
Peak Summary
Displays all variables required for creating a peak summary.
SST Summary
Report
Displays all variables required for documenting the results of the
¾System Suitability Test for the entire sequence.
458
Actions in the Report Table
Audit Trails
Audit Trail
(Commands,
SST, ...)
Displays the Audit Trail of the current sample (see Data
MS Instrument
Info Report
Displays information about the ¾Mass Spectrometer.
MS Status Log
Report
Displays the mass spectrometer settings.
MS Tune Data
Report
Displays the tune data of the ¾Xcalibur raw data file.
MS Instr. Method
Report (XRaw)
Displays the MS method.
Management
Audit Trails).
Spectra Results
MS Raw Report
Displays the raw data of the current ¾Mass Spectrum.
Fraction Collection Results
Fraction Report
Displays all variables required to create a fraction report.
Tube Report
Displays the different variables describing the single fraction
collection tubes. This also includes the fraction report variables.
However, please note that these variables are not selected by
default.
QNT Tables
Detection
Parameter
Displays all ⇒Detection Parameters of the current sample.
Peak Table
Displays all variables that are required for displaying the peak table
of the QNT Editor.
SST Table
Displays all variables that are required for the representation of the
¾System Suitability Test.
PGM Tables
Commands
Displays the ¾Program for the current sample.
Post Acq.
Processing
Displays the ¾Post-Acquisition Steps for the current sample.
MS Method
Displays information about the current method of the ¾Mass
Spectrometer.
Miscellaneous Tables
Database Query
Inserts any kind of database queries into the report.
History Report
Displays all variables required for creating a ¾History report.
•
From the variables that are available on the right-hand side, click those
you wish to include in the new report. Click OK to create the desired
worksheet.
•
In the report, double-click the newly added tab and edit the name.
Actions in the Report Table
459
Saving
To save the content and appearance of a report, select Save Report
Definition.
Tip:
Please note that the appearance and the contents of the on-screen report
needs not to be identical with the actually printed report, the Report
Templates. The printout is defined in the ¾Printer Layout. Thus, it allows
you to initiate printing independently of what is displayed on screen.
Displaying the Peak Summary
Use the Summary worksheet in the Report (see Reports
Summary Report) to show certain results from all samples.
The Peak
The required steps are described in How to ...: Actions in the Report
Table:
Displaying a Report and
Adding and/or Renaming a Worksheet.
In contrast to the Integration and Calibration worksheets of the ⇒Report
Definition File that list the data of the peaks from one sample, the
Summary worksheet represents, e.g., data of a specific peak from all
samples (generally from a ⇒Sequence).
To display, for example, the amount of different peaks of all samples of a
sequence in one table, select Column Properties on the context menu.
Select Peak Results from the Categories list, and then select Amount
from the Variables list and have Chromeleon display the value for certain
peaks (Fixed Peak(s)). The default setting shows the values for the
currently Selected Peak. Determine the peak for which the data are
displayed by clicking the peak in the above chromatogram.
The default Summary report contains several columns. In addition to the
sample name and the retention time, it also includes the Area, Height,
⇒Amount, Types, and Plates columns. When you select a peak in the
chromatogram, the values of this peak are included in the Summary report.
If the selected peak is not contained in a sample, this line remains empty
(n.a. = not available).
460
Actions in the Report Table
Below the sample list, there are the lines Average and Relative Standard
Deviation. The average value of a column is calculated and displayed. The
relative standard deviation from this value is indicated in percent.
The user can modify the default Summary report at any time. However, it is
recommended to keep this report page. If another report is required, create
an additional Peak Summary report page with new column assignments.
Note:
Instead of a single sequence, the Summary report can also be started
based on a ¾Query. Then, the Summary may include different samples
from different sequences.
Displaying an Audit Trail
You can include the sample audit trail (see Data Management
The
Sample Audit Trail) as a worksheet in a ⇒Report Definition File. The
worksheet always shows the audit trail for the currently selected sample.
•
The Day Time column indicates the time of a command or message.
•
The Ret.Time column indicates the corresponding retention time.
•
The Command/Message column command shows the command itself,
the text of a message, or an event.
To make further settings, select the Table Properties command on the
context menu. The Audit Trail Properties dialog box is opened; define
which the data shall be displayed.
In addition, you can select one of the following Display options:
•
Run only to display only the entries for the sample run
•
Preconditions only to display only the conditions before a sample run
•
Preconditions and Run to display all entries
Actions in the Report Table
461
The display filter (Filter Level) defines the type and extent of the Audit Trail
entries for a sample run.
Normal:
Only the most important commands and properties
are displayed.
Advanced:
Normal and Advanced level commands and
properties are displayed.
Expert:
Normal, Advanced, and Expert level commands
and properties are displayed (for experts only).
Errors and Warnings: Only error messages and warnings but no
commands are displayed.
You can add the contents of the worksheet to other documents, using the
Cut and Paste commands. It is also possible to print the entire audit trail in
the ¾Printer Layout.
Using Audit Trail Variables in Other Worksheets
Certain events (such as performing a ⇒Trigger or changing the wavelength
at a specific time) can be included in any Report worksheet.
•
Open the corresponding worksheet and select Column Properties on
the context menu.
•
Select Audit Trail from the Categories list.
•
In the Variables field, select one of the audit trail variables. The
variables that are available for selection depend on the events listed in
the sample audit trail.
•
Use the Formula field to display events that are not listed (for example,
the system pressure that is recorded using a Log command). In this
case, the event variable is appended to the name AUDIT; separated by
a period (AUDIT.pressure).
•
Click OK to confirm your input.
Your report now includes an additional column for an audit trail variable.
Normally, the value for the corresponding peak at the retention time is
entered in each line. If there is no value at this time, the value that was
recorded last is entered. When forming a gradient, Chromeleon calculates
the corresponding values (%A, %B, %C . . .).
462
Actions in the Report Table
If you want to display a specific audit trail event for all peaks in a report at a
specific time:
•
Select Column Properties and then select an Audit Trail variable.
•
Click Parameter....
A dialog box is opened allowing you to enter a retention time. Click OK to
confirm the entry. A report column with a fixed retention time is generated.
For example, the Wavelength audit trail variable displays the wavelength
at time t.
However, you can also search for the next associated entry in the Audit
Trail. Starting point of the search is the entered retention time. Select
backward as Search Direction to find the previous entry before the
retention time. Select forward to find the next entry after the entered
retention time.
Creating a History Report
The ¾History report is not part of a default report. Therefore, select Insert
Report to add a history worksheet to a ⇒Report Definition File (see How
Adding and/or Renaming a
to ...: Actions in the Report Table
Worksheet). By default, the worksheet shows the history of the current
sample. Select Table Properties on the context menu to open the History
Report Properties dialog box where you can change the settings:
•
On the History Objects tab page, select the object for which to display
history entries.
•
On the Time Restrictions tab page, specify the time when the history
entries to be displayed must have been made.
•
On the Operations tab page, determine the changes to be displayed.
•
On the Users tab page, determine the user(s) whose changes shall be
displayed.
•
On the Sorting tab page, define the sorting order for the history
entries.
•
A special layout mode is provided for the history report. Select the
Layout Mode on Layout tab page. However, this is only possible if
detail columns are available. If detail columns exist, select the Design
template mode to display a shortened history report, thus, simplifying
layout definition.
Actions in the Report Table
463
Displaying MS Reports
Similar to the ¾History report, the different MS reports are not part of the
default ⇒Report Definition Files. Select Insert Report to add them as
separate worksheets to a report definition file (see How to ...: Actions in
the Report Table
Adding and/or Renaming a Worksheet):
•
Select the MS Instrument Info Report to display information about the
¾Mass Spectrometer. If there is no MS data, the following message
appears in the report: "No MS Instrument Info found".
•
The MS Instrument Method Report indicates the MS method. If there
is no MS data, the following message appears in the report: "No MS
Instrument Method found".
Note:
The MS Method (under PGM tables) indicates the current method. It is
possible that both MS methods are identical.
•
The MS Raw Report shows the raw data (mass, intensity, and relative
intensity) of the ¾Mass Spectrum of the current sample. If no MS data
is available, the following message appears in the report: "No MS Raw
Data found".
•
The MS Status Log Report shows the mass spectrometer settings. If
there is no MS data, the following message appears in the report: "No
MS Status Log found".
•
Select the MS Tune Data Report to display the tune data of the
¾Xcalibur raw data file. If there is no MS data, the following message
appears in the report: "No Tune Data found."
Notes:
If the MS Control option is disabled on your PC, the MS reports will not be
displayed in the Insert Report dialog.
All MS reports comprise only the default columns. It is not possible to add
more columns.
464
Actions in the Report Table
Selecting Other Special Reports
As with the ¾History report, the following reports are not part of the default
⇒Report Definition Files either. Select Insert Report to add them as
separate worksheets to a report definition file (see How to ...: Actions in
the Report Table
Adding and/or Renaming a Worksheet):
Result Tables
•
The Calibration History documents the calibration run.
•
The SST Summary Report shows the results of the ¾System
Suitability Test for the entire sequence. The default columns are part of
the ⇒Sample and ⇒System Suitability Test categories. It is also
possible to edit the columns or to use different columns for the report.
QNT Tables
•
The Detection Parameter Report shows the ⇒Detection Parameters
used for the current sample.
•
The Peak Table report shows certain entries of the peak table of the
QNT Editor (such as the expected ¾Retention Time or the retention
⇒Window).
•
The SST Report shows the parameters used and the results of the
System Suitability Test. The default columns are part of the System
Suitability Test category.
PGM Tables
•
The Commands report shows the ¾Program of the current sample.
•
The Post-Acquisition Processing report shows the ¾Post-Acquisition
Steps of the current sample.
•
The MS Method shows the current method of the ¾Mass
Spectrometer.
Note:
The MS Instrument Method Report (under Result Tables) indicates
the method that is used for data acquisition. It is possible that both MS
method reports are identical.
Actions in the Report Table
465
Miscellaneous Tables
•
Select the Database Query report to integrate any kind of database
query in the report. Select Table Properties on the context menu to
open the table properties and specify the datasource in which the query
shall be performed. Use ¾SQL statements to define the properties for
which the query shall be performed.
Note:
The Detection Parameter Report and the Commands, Post-Acquisition
Processing, MS Method, and Database Query reports comprise only the
default columns. It is not possible to add more columns.
Setting Parameters for Variables
(e.g., for the confidence interval)
You can set special parameters for many of the report variables. For an
example, refer to the description below for the limits of the ¾Confidence
Interval. The Parameter button is enabled for those variables for which
special parameters can be set.
466
Actions in the Report Table
Click the Parameter button to open the Parameter Input … dialog box.
Use this dialog box to make the settings for the displayed report variable:
The Parameter Input for Upper Confidence Limit dialog box allows you,
for example, to define the probability and the calibration level at which the
upper limit of the confidence interval will be computed:
Confidence
probability
Select the probability with which the indicated values shall apply via
the arrow key. The following probability values are available:
90%, 95%, 98%, 99%, 99.7%, 99.8%, 99.9%, and 99.99%.
Compute confidence
limit at
Define the level for which to compute the respective limit of the
confidence interval. Select one of the following options:
•
Lowest calibration level
•
Highest calibration level
•
Average of all calibration levels
•
Reference amount (standard and validation samples only) for
reference values. The corresponding values are given for
standard and validation samples only if an entry for the respective
peak is available in the Amount column. If a peak has no entry or
if the Amount column is not available for a standard or validation
sample, n.a. is returned.
•
Computed amount (for the computed amount of the respective
peak in the single samples).
•
Any fixed value. Define the Amount value in the right-hand field.
Actions in the Report Table
467
The graphical representation of the confidence interval in the calibration
curve is possible as well. For more information, refer to How to ...: Actions
Indicating the Confidence Interval.
in the Calibration Curve
The limits of the confidence interval, which are defined using the
corresponding Report variables, are determined as follows: For a given
amount, the system determines the intersections with the limiting curves of
the confidence interval In the height of the value that is defined via the
calibration curve.
The image shows the calibration curve together with the corresponding
confidence interval at a probability of 99%. In the above example, the upper
and lower limits belonging to a concentration of 6.3 µg/ml are derived. Their
values are 6.0 and 6.6 µg/ml, respectively. (The distance between the
limiting values and the given Amount value must not necessarily be
symmetrical.)
468
Actions in the Report Table
Actions in the Printer Layout
469
Actions in the Printer Layout
Use the Printer Layout to prepare your data for the printout. It is saved in
the ⇒Report Definition File (= RDF). To assist you in preparing the first
printout and in creating your own report definition files (also called report
templates below), Chromeleon provides some default RDF templates.
During the Chromeleon installation, the default RDFs are written to the
Dionex Templates > Reports directory of your local datasource.
To printout more data that is not included in the default report definition
files, Chromeleon provides the possibility to create individual worksheets
via the ¾Printer Layout window. These worksheets can be combined and
saved as a report template. The results of sample processing can thus be
printed in a number of layouts.
For more information, refer to:
Opening and Editing the Printer Layout
Saving/Loading a Report Definition File
Specifying the Pages to be Printed
Specifying the Printout
Printing the Results of a Single Sample
Printing the Results of a Sequence or a Sample Batch
Setting Print Area and/or Print Title(s)
Automatically Repeating the Output for the Selected Objects
Saving the Contents of a Printer Layout Page
Users who have the ¾Report Publisher add-on product can use the
additional features described in
Entering User-defined Formulas.
Thus, they have numerous possibilities, e.g., for
Calculating the Amount Percentage (for Identified Peaks)
Calculating the Concentration Percentage
(in Relation to the Total Concentration)
Creating Dynamic Columns That Contain Flexible Formulas
Creating Dynamic Links to Lines Other than the Current Line
470
Actions in the Printer Layout
In addition, you can use the Printer Layout to create charts (see
Creating Charts).
For assistance to change the page format and to create headers and
footers refer to:
Changing the Page Format
Notes on the Page Setup
Opening and Editing the Printer Layout
To access the ¾Printer Layout, first open the desired sample and then
change to the Printer Layout:
•
Select and double-click the desired sample in the Browser. The
(screen) report for the sample appears.
•
on the Method toolbar to change to
Click the Printer Layout icon
the Printer Layout. The Printer Layout of the corresponding report
definition file (RDF) is opened, i.e., the Preferred RDF File of the
sequence to which the sample belongs.
Note:
If you open the report definition file in the Browser, e.g. by double-clicking,
the Printer Layout is opened with the data that were saved last. In this
case, it is not possible to load the desired sample data.
The Printer Layout consists of several (work)sheets. To change to a
different sheet, click the desired tab on the bottom edge. The 'default' report
definition contains, for example, an Integration tab, a Calibration (Curr.
Peak) tab, and a Calibration (Batch) tab. The area of the single sheets is
virtually unlimited in horizontal and vertical direction. However, depending
on the amount of information, it may comprise different print pages. The
different sheets comprise chromatograms, tables, diagrams, calibration
curves, or other elements indicating the corresponding values of the current
sample.
If you need more variables, chromatograms, tables, etc. than those
available in the open Report Definition File, you can insert them into the
existing worksheets.
Actions in the Printer Layout
471
Or else, create a new worksheet:
•
Enable Layout Mode on the Edit menu.
•
Select Insert Sheet... on the Edit menu. An additional worksheet is
inserted before the open sheet.
For information about how to edit existing worksheets, refer to:
Inserting a Chromatogram
Inserting a Trend Plot
Inserting and Editing a Table
Inserting a Column into an Existing Table
Inserting and Deleting Individual Variables
Inserting Text
Inserting a Chromatogram
•
Enable ¾Layout Mode on the Edit menu.
•
Select Insert > Chromatogram on the Edit menu or context menu.
The mouse cursor changes to a + sign.
•
Draw a rectangular frame of any size. The frame reserves this area for
the chromatogram of the current sample.
•
To indicate that the chromatogram is a Chromeleon object all four
corners of the chromatogram inserted in this way are marked by a red
triangle.
•
Click the chromatogram to select it completely. You can then move it,
or reduce or enlarge it in size.
•
Select Chromatogram Properties on the Edit menu or context menu.
Format the chromatogram as desired on the corresponding tab pages
of the Chromatogram Decoration dialog box (caption, axes, font size,
etc.).
•
Press the F4 key or the Shift+F4 key combination to display the
chromatogram of the next or previous sample and to check whether the
selected settings are appropriate for all chromatograms.
472
Actions in the Printer Layout
Inserting a Trend Plot
•
Open the Printer Layout (see
Layout).
•
Enable ¾Layout Mode on the Edit menu.
•
Select the worksheet to which you want to insert the trend plot, or add
a new worksheet (select Insert Sheet... on the Edit menu).
•
Select Insert > Trend Plot on the Edit menu or context menu. The
mouse cursor changes to a + sign.
•
Draw a rectangular frame of any size. The frame reserves this area for
the trend plot.
•
To indicate that the ¾Trend Plot is a Chromeleon object all four corners
of the plot are marked by a red triangle.
•
To select the complete area, click inside the frame.
•
You can now move the frame and reduce or enlarge it in size, as in
Windows.
•
Select Trend Plot Properties on the Edit menu or context menu.
Define the data and format of the trend plot as desired on the
corresponding tab pages of the Trend Decoration dialog box
(Statistics, Data Labels, Data, Caption, Frame & Axes, etc.).
Opening and Editing the Printer
Inserting and Editing a Table
•
Enable ¾Layout Mode on the Edit menu.
•
Select Insert > Chromeleon Report Table on the Edit or context
menu. The Insert Report Table dialog box appears.
•
In the Report Tables field, mark the report table you want to insert. If
necessary, click the + character beside the report table groups to
display the tables underneath. The columns available for the selected
table are displayed in the Columns field.
•
Some columns are already marked by default. Select the columns that
should be included in the table and deselect all others. Press and hold
the Ctrl key to select or deselect several columns simultaneously.
•
Clicking OK inserts the table into the worksheet.
Actions in the Printer Layout
•
473
To indicate that the table is a Chromeleon object all four corners of the
table are marked by red triangles. The last row(s) are not marked by
red triangles because they are no Chromeleon objects.
Caution:
If the worksheet already contains a report table, insert the new table
above or below the existing one. It is not possible to insert several
report tables next to each other.
Tip:
You cannot move or copy an entire table. When moving or copying a
table, only the current content (= the values) is copied to the clipboard
but not the underlying variables! (The red triangles are missing
indicating that this table is no longer a Chromeleon object.) Besides, it
is not possible to move single columns. Instead, insert a new column at
the corresponding position and delete the 'old' column.
•
Select a column header and then select Report Column Properties
on the context menu. Determine the header, the dimension, and the
format of the column or of the column values.
•
To modify the appearance of a single cell, a table area, or the entire
table (font size and style, frame, color, etc.), select the cell, the area or
the entire table and then select the corresponding command on the
Format menu.
•
Select the column or the row you want to delete and then, select
Delete Column(s) or Delete Row(s) on the Edit menu. To insert
additional rows or columns in front of the selected area, select Insert
Row(s) or Insert Column(s) on the Edit menu.
Inserting a "Total" Row
The new table does not comprise a Total row. Insert the Total row as
follows:
•
Copy any cell from another Total row; for example, from another
¾Printer Layout table or from an integration report.
•
Select the cells of the new table, which should indicate the sum of all
cell values of the corresponding column.
•
Select Paste.
474
Actions in the Printer Layout
Tip:
Proceed in the same way to update any existing entries in the rows
Average and/or Rel.Std.Dev rows. These rows are not marked by red
triangles, because they are no Chromeleon objects.
Creating the correct cell reference
The values in the Total row must receive the correct cell reference. This is
especially important when the new table is longer than the table from which
the cell was copied.
•
Enable ¾Layout Mode on the Edit menu. The edit line is displayed.
•
Select the first value in the Total row. In the edit line, the corresponding
formula appears; for example, =SUM(C10:C22).
•
Select the cell range indicated in parentheses in the edit line with the
mouse; for example, C10:C22.
•
Then, select the actual cell range in the table with the mouse or type
the cell range in the edit line via the keyboard.
•
Press <Enter> to confirm your input.
The sum cell value is recalculated based on the new cell range. Then
perform the individual steps for the remaining sum cell values.
For more information, also see
Table.
Inserting a Column into an Existing
Inserting a Column into an Existing Table
To insert a new column into an existing table:
• Enable ¾Layout Mode on the Edit menu.
• Select the entire column, including its header and it’s the cell Total, to
the left of which you want insert the new column:
Actions in the Printer Layout
475
• Select Insert Column(s) on the context menu. A new table column is
inserted that also contains a Total cell.
• Double-click the header of the new column to open the Report Column
Properties dialog box. Select the desired variable from the Variables
list (Please note: The picture only shows part of the dialog box):
• Click OK to assign the selected variable to the new column.
• Fill the cell Total. Click a different cell in this line, which already contains
a value. Copy the cell reference by pressing the Ctrl + C keys and then
paste it into the new cell by the pressing Ctrl + V keys. (The format of
the copied cell is automatically transferred, too. Adapt the format to the
new column if necessary.)
For more information, refer to
Inserting and Editing Tables.
476
Actions in the Printer Layout
Inserting and Deleting Individual Variables
Usually, general sample data, such as the sequence name, the
corresponding ¾Datasource, the user name, etc. appear at the beginning
of a printout or in addition to a table or a chromatogram.
To insert more variables:
•
Enable ¾Layout Mode on the Edit menu.
•
Select a single cell and then select Insert > Chromeleon Report
Variable on the Edit menu.
•
In the Select Report Variable dialog box, select the variable category
from the Categories list and then, select the desired variable from the
Variables list.
•
Clicking OK includes the variable into the selected cell.
•
To indicate that the variable is a Chromeleon object, a red triangle
appears in the upper right corner of the cell.
•
To modify the appearance of the variable, select the respective cell and
then select the corresponding command on the Format menu.
Deleting a Variable
To delete a variable, it is not sufficient to delete the value indicated in the
edit line. Follow the steps below:
• Select the cell for which you want to delete the contents.
• Select Clear on the context menu or press the Del key on the keyboard.
The Clear dialog box appears.
• Select the Values option to delete only the values. Select All to delete
the formats as well, e.g., the frames assigned to the cell. Click OK to
clear the cell.
• Check whether the clear action was successful. Click a different cell and
then click the cell again, for which you wanted to delete the contents.
The edit line is updated for this cell. If the clear action was successful,
the edit line does not contain any information.
Actions in the Printer Layout
477
Notes:
Cells containing an underlying variable can be recognized by the formula
name that is displayed in the edit line whenever the cell is selected, for
example, peak.height. If the selected cell contains only text, the edit line is
empty.
You cannot insert new variables by entering the corresponding formula, for
example, smp.name for the sample name. Chromeleon interprets formulas
that are entered in this way as pure text. Thus, only the text 'smp.name'
appears in the edit line and not the value of the formula, i.e., the name of
the corresponding sample.
Inserting Text
You can insert text in any empty text cell of the worksheet. However, the
size of the cell limits the length of the text that is displayed in the cell. Note
the following:
•
When you type longer texts into a cell than permitted by the cell size,
the entire text is displayed using the neighboring cells to the right as
long as these cells are not used. If the neighboring cells are used, the
visible text is 'cut' to the cell size; the entire text is displayed only in the
edit line.
•
Text cells are not marked by a red triangle because they are no
Chromeleon objects.
•
To insert a line break, select the Wrap Text check box in the Format
Cells dialog box. (To open this dialog box, select Alignment on the
Format menu.)
•
If graphics, such as chromatograms, are inserted, they will always hide
the text.
Saving or Loading a Report Definition File
The ¾Printer Layout of a report template (⇒Report Definition File) can
contain one or several worksheets. If you save or load a Report Definition
File, all worksheets included in the Printer Layout are saved or loaded.
•
Select Save Report Definition on the context menu to save the Report
Definition File and its worksheets under either an existing or a new
name.
•
Select Load Report Definition on the context menu to open an
existing Report Definition File.
478
Actions in the Printer Layout
Tip:
In addition, a Report Definition File contains various settings of the onscreen report, such as the window size, the type, and number of the
columns in a report, axis captions, etc. When you save the Printer Layout,
any modifications made to the on-screen report are saved as well.
Specifying the Pages to be printed
To specify which pages of the ¾Printer Layout shall be printed, enable
Layout Mode on the Edit menu. Select Batch Report Setup on the File
menu to open the Batch Report Setup dialog box:
Click Conditions... to specify the print conditions for the corresponding
worksheet. For example, it would be sensible to print the Summary page
only for the last sample of a sequence:
Actions in the Printer Layout
479
However, you could print the Calibration (Batch) sheet for the Last
Sample in a List of Standards.
Tip:
These settings are saved in the report definition file (RDF). They are used
as default for Batch Report printing. The same defaults are used for
electronically signing sequences (see ¾Electronic Signature).
Specifying the Printout
Before you can print your data, define the printer to be used. In addition,
define the headers and footers, margins, etc. on the Print dialog box and
the Page Setup dialog box. Select Print Setup and Page Setup on the
File menu.
The Print Setup and Page Setup settings are stored separately for each
worksheet in a ¾Report Definition file (Integration, Calibration, Peak
Analysis, Summary, etc.). Therefore, select the desired settings for each
worksheet separately.
480
•
Actions in the Printer Layout
Select Print Setup on the File menu to determine the printer, the paper
size, and the format (portrait or landscape). Having made these
settings, a message box appears. Determine whether these settings
shall be used only for the current worksheet or for all worksheets of the
Printer Layout of the current Report Definition File (RDF).
Tip:
The Print Setup settings selected here only apply to the report
definition file of the current sequence. They do not affect the default
settings for Windows. On the other hand, changing the default printer,
paper size, and format in Windows will not affect the settings specified
here.
•
Select Page Setup on the File menu to determine the appearance of
the headers and footers, the margins, the alignment, etc. You can also
set grid lines, determine whether column and/or row headers are
displayed, and whether the printout should be in black and white or in
color. For more information, refer to How to ...: Actions in the Printer
Layout
Notes on the Page Setup.
Tip:
For technical reasons, direct help information for the Print Setup dialog
box is not available.
Also, refer to: How to ...: Actions in the Printer Layout
Saving/Loading a Report Definition File
Saving the Contents of a Printer Layout Page
Printing the Results of a Single Sample
You can print the results of single samples from either the Printer Layout or
the on-screen report (integration plot).
For each printout, the ¾Printer Layout pages saved in the ⇒Report
Definition File are used as a template. The results of a single sample or of a
sequence are output in the defined way (see
Printing the Results of a
Sequence or a Sample Batch). Please note that the Report Definition File
and the worksheets define only the appearance of the printout, but not the
contents.
Actions in the Printer Layout
481
Unless you have already specified the worksheets to be printed, specify
them now:
In the Printer Layout
Select Batch Report Setup on the File menu to specify which worksheets
shall be printed. The Batch Report Setup dialog box is opened:
Select the corresponding settings on the Print Sheets tab page. The
available worksheets are listed in the Sheets field. Define which pages
shall be printed and under which conditions. Click Conditions… to define
the print conditions.
For example, you can specify for which sample type a page shall be
printed. For instance, it would be sensible to print a summary report only for
the last sample of a sequence.
482
Actions in the Printer Layout
In the Report (Screen Report)
Select Print on the File menu to print the results of the currently open
sample from the on-screen report:
On the Print dialog box, select the worksheet to be printed from the Using
Sheet drop-down list box. The worksheet is always printed with the onscreen results if they are part of the defined view. For example, the current
¾Mass Spectrum is only printed if a mass spectrum is part of the selected
worksheet.
Tip:
In the Printer Layout, you can only select peak spectra but no retention time
spectra. If you want to print a retention time spectrum, open the Report,
select the desired spectrum on-screen, and then print a worksheet that
includes the spectrum.
Printing the Results of a Sequence or a Sample Batch
You may also print the results of an entire sequence, a ¾Query or a
¾Batch. Unless you have already specified which of the worksheets
created in the ¾Printer Layout should be printed, specify the worksheets
now.
In the Browser
Click a ¾Sequence or a sample, and then select Batch Report on the File
menu. The Batch Report dialog box appears. Select the desired Report
Definition File from the Use Report Definition drop-down box. Or else,
click the "…" button and navigate to the desired file.
Actions in the Printer Layout
483
The worksheets defined in the Printer Layout of the Report Definition File
are listed in the Select sheets to be printed field.
Specify which pages shall be printed. To print the pages under certain
conditions only, select the Print under certain conditions only option and
then click Conditions....
Define the print conditions in the Print Conditions dialog box. Click OK to
print the entire sequence or the selected samples of a query. To print the
entire query, select all samples of a query by clicking the No. field at the top
left of the sample list.
On the Control Panel: Printing the Results Directly after Data
Acquisition
To print the results directly after data acquisition, select Reporting on the
Batch menu. The Reporting tab page of the Batch dialog box appears:
484
Actions in the Printer Layout
Select the Print/Export Report check box to reopen the Batch Report
dialog box (see the picture in the Browser section above). Follow the
description in the Browser section.
The tab dialog box indicates the printer, the report definition file (RDF), and
the channel selected for the printout. Click OK to return to the Reporting
tab page. Specify whether each sample shall be printed separately and
immediately after data acquisition or whether all samples shall be printed
when the batch is finished.
Tip:
The settings made in the Browser or on the control panel are saved in the
report definition file of the current sequence. They apply to all sequences
that use this report definition file. The same defaults are used for
electronically signing the worksheets of these sequences (see ¾Electronic
Signature).
For more information, refer to
Sample.
Printing the Results of a Single
Actions in the Printer Layout
485
Setting Print Area and Print Title(s)
To print a defined area of a ¾Printer Layout page:
•
Press the left mouse button and drag the mouse to select the desired
area.
•
Select Printing > Set Print Area on the Format menu.
•
Select Printing > Release Print Area on the Format menu to deselect
the defined print area. The defined print area is indicated in
parentheses behind the command.
When printing a table that exceeds one page, you can print the title on
every page:
•
Select the entire line(s) for the print title with the left mouse button.
•
Select Printing > Set Print Titles on the Format menu.
•
Select Printing > Release Print Titles to deselect the set print titles.
The set print title area is indicated in parentheses after the command.
Automatically Repeating the Output for the Selected
Objects
It is often useful to repeat the output of reports for other channels, samples,
and/or peaks. In Chromeleon, you can automatically repeat the output of
reports for:
•
Each sample of a sequence or selection
•
Each peak in a peak table or the current chromatogram
•
Each channel of a sample
Follow the steps below:
•
Select the desired lines completely, i.e., in the grey column at the
utmost left that indicates the line numbers.
Tip:
Verify for graphic objects that you have selected all lines covered by
the object.
•
To select more than one area, hold the CTRL key down, and then
select the next area by the lines as described above.
486
Actions in the Printer Layout
•
Select Printing > Set Autorepeat Area on the Format menu to specify
the selected area as the area for which the output is automatically
repeated.
•
The Autorepeat Area Properties dialog box is opened. Use this dialog
box to define for which channels, samples, and/or peaks the selected
area shall be output.
Automatically Repeating the Output for the
For an example, refer to
Selected Objects: Example.
It depends on the objects in the selected area whether it makes sense to
repeat the output for other samples and/or peaks. Therefore, please note:
•
The Repeat Samples tab page is disabled if at least one Peak
Summary table and/or one SST Summary table is selected.
•
The Print and Repeat Peaks tab page is disabled if at least one
Integration table is selected.
You can repeat the output for other channels, samples, and/or peaks when
you
•
Print a batch
•
Sign sequences
•
Print interactively from the Printer Layout
Note:
When you repeat the output for channels and peaks, only those objects are
replaced for which no fixed channel/peak is specified. A warning appears if
the Fixed Channel or Fixed Peak option is selected for objects that shall
be output for different channels or peaks.
For example, this applies to chromatograms for which the Fixed Channel
option is selected on the Channel tab page. (To access the Channel tab
page, select Chromatogram Properties on the chromatogram's context
menu).
Actions in the Printer Layout
487
Automatically Repeating the Output for the Selected
Objects: Example
To repeat the output for the header and the table below the chromatogram,
select the following lines:
488
Actions in the Printer Layout
If your sequence contains only two standards of interest, you can specify
that the repetition include all standards; refer to the image below.
In addition, select the Page break after each sample check box to output
the second sample on a separate page:
With these settings, the following two pages are output via the Print
command (File menu):
Actions in the Printer Layout
489
490
Actions in the Printer Layout
For general information, refer to
for the Selected Objects.
Automatically Repeating the Output
Saving the Contents of a Report Definition File
Chromeleon provides various formats for saving the report data of the
current sample:
•
To save the active page of the Printer Layout, select Save as... on the
File menu. The Save As dialog box is opened. Under Save as type:
•
Select Excel ... (*.xls) to convert your data into the Excel format. Enter
the file name and click Save to save your data as a separate file. In
addition to the values of the table, any graphics and diagrams included
in the table are also saved to the new file. If the ¾Printer Layout of the
⇒Report Definition File contains several worksheets, the Excel file has
the same structure. Please note that different Excel file types are
available, depending on the Excel version (Excel V4 or Excel V5 or
V7).
•
Select HTML (*.htm) to save your data as an HTML page. Graphics
and diagrams cannot be converted and saved in HTML format.
•
Select Tabbed Text (*.txt) to save your data as pure text. The content
of the individual report columns is separated by a tab stop. Graphics
and diagrams cannot be converted and saved in Tabbed Text format.
Actions in the Printer Layout
491
Entering User-defined Formulas
Tip:
In order to use the options described below, the ¾Report Publisher add-on
product must be installed on your computer.
Creating user-defined formulas is analogous to the Microsoft Excel
spreadsheet. The following description is intended for users who are not
familiar with entering formulas:
Example 1:
Let's assume that a table containing two columns (A and B) and three lines
(1 to 3) is extended by one column (C), for which there is no Chromeleon
report variable. Thus, for example, the quotient of the cell contents of
columns A and B can be included in column C.
To enter a formula in a cell, select the corresponding cell first. In this
example, select the cell C1. Enter the equals sign. The entire input is
displayed in the edit line (here indicated in blue print).
C1
=
A
B
1
12
5
2
17
10
3
13
20
C
4
Select the cell A1, enter a division sign (slash), select the cell B1, and
complete your input by pressing <Enter>. The formula (C1=A1/B1) is
displayed in the edit line; the cell C1 displays the result of the operation
(2.4). The cursor moves to cell C2.
C1
=A1/B1
A
B
C
1
12
5
2.4
2
17
10
3
13
20
4
Follow the description to output the corresponding results in the cells C2
and C3. To facilitate the procedure, click the cell C1 again, grab the
selection frame on the lower right corner, and drag it to the required cells
492
Actions in the Printer Layout
(C2, C3). For calculating the cell values, Chromeleon uses the previously
entered formula and automatically creates the correct reference, line by
line.
C1
=A1/B1
A
B
C
1
12
5
2.4
2
17
10
1.7
3
13
20
0.65
4
Example 1 (continued)
How to sum the cells of column C in the field C4:
Select the cell C4 and enter an equals sign. Enter the SUM command
required for adding cell values (see SUM in the Report Publisher online
Help). Then enter an opening bracket.
C4
=SUM(
A
B
C
1
12
5
2.4
2
17
10
1.7
3
13
20
0.65
4
Select all cells (C1, C2, C3) that you want to sum up. Close the bracket
after the last cell. Press <Enter> to complete your input.
C4
=SUM(C1:C3)
A
B
C
1
12
5
2.4
2
17
10
1.7
3
13
20
0.65
4
4.75
In addition to the SUM command, many other functions are entered in the
same way. Thus, you can form the average (see AVERAGE), express
conditions (see IF, True, FALSE), create logical operations (see AND,
OR), or enter time values (see TIME, DATE, DAY, YEAR; all in the Report
Publisher online Help).
Actions in the Printer Layout
493
Tip:
For an alphabetical list of the available formulas, refer to the ¾Additional
Functions topic.
Example 1 (continued)
Besides, it is also possible to use "fixed references". Contrary to the
variable value pairs described above (A1/B1, A2/B2, A3/B3), form the
quotient of a variable and a fixed value (A1/C4, A2/C4, A3/C4). In this
example, the result is displayed in column D.
Follow the description for Example 1 above to create the first cell reference
(D1).
D1
=A1/C4
A
B
C
D
1
12
5
2.4
2.5263
2
17
10
1.7
3
13
20
0.65
4
4.75
Extend the formula by adding two $ signs. They convert a variable
reference into a fixed cell reference.
D1
=A1/$C$4
A
B
C
D
1
12
5
2.4
2.5263
2
17
10
1.7
3
13
20
0.65
4
4.75
When the formula is copied, the reference to cell C4 will be retained. The
fields to be calculated (D2 and D3) can then be calculated by
simultaneously selecting the cells D1, D2, and D3.
D1
=A1/$C$4
A
B
C
D
1
12
5
2.4
2.5263
2
17
10
1.7
3.5790
3
13
20
0.65
2.3768
4
4.75
494
Actions in the Printer Layout
For practical examples, refer to:
Calculating the Percentage Value for the Amount
(for Identified Peaks)
Calculating the Percentage Value for the Concentration
(in Relation to the Total Concentration)
Creating Dynamic Columns That Contain Flexible Formulas
Creating Dynamic Links to Lines Other than the Current Line
Calculating the Amount Percentage
(for Identified Peaks)
If you use samples with ¾Internal Standard but wish to calculate the
percentage values of the different substances that have been identified in
the sample (without internal standard):
1. First, disable the display of unidentified peaks in the report table. Select
Table Properties on the context menu. On the General tab page of the
Report Properties dialog box, clear the Including unidentified peaks
check box.
2. Select a column that you do not need (in the example below: column D).
Select Add Column or Insert Column on the context menu. From the
Categories list, select Peak Table, and then select Standard Method from
the Variables list.
3. a) Select a different column (here: column G) that you do not need.
Press the F8 key to open the dialog box. Delete the entry in the Formula
field. Afterward, delete the entries in the column (except the last line, i.e.,
the line named Total).
b) In the field G29, enter the formula
=IF(OR(D29="ISTD Internal ";D29="ISTD Int/Ext ");"ISTD";F29)
(Observe the space following Internal!) Copy the formula to the following
lines.
Tip:
Do not omit the space in the formula behind Internal and Int/Ext,
respectively.
Actions in the Printer Layout
495
4. a) Select another column which you do not need either (here: column H).
Press F8 to open the dialog box. Delete the entry in the Formula field.
Afterward, delete the entries in the column (except the last line).
b) Enter the formula =100*G29/G$36 in the field H29 and copy it to the
following lines
Also, refer to How to ...: Actions in the Printer Layout
Calculating
the Concentration Percentage (in Relation to the Total Concentration)
Calculating the Concentration Percentage (in Relation to
the Total Concentration)
Entering and Calculating the Total Concentration
To calculate the percentage concentration in relation to the total
concentration, calculate the total concentration using your exact sample
weight (without any added ¾Internal Standards) and the liquid volume.
Enter the concentration, for example, in the Weight column of the sample
list. However, it may be better to create a ¾User-defined Column, name the
column concentration, and assign Floating Point as the Value Type.
496
Actions in the Printer Layout
(For more information, refer to How to ...: Actions in the Browser
Creating User-defined Columns.) Enter the corresponding total
concentrations of the single samples in this column.
Creating the Report Table
1. Double-click a variable in the page header that is not necessarily
required (here: H6). For this variable, define the concentration column
created before:
Actions in the Printer Layout
497
2. Follow the description in the previous topic (see How to ...: Actions in
Calculating the Percentage Amount (for
the Printer Layout
Identified peaks)). Instead of the formula described there in 4b, enter the
following
formula
(here:
in
the
field
H13):
=IF(G13="ISTD";"ISTD";100*F13/$H$6).
Tip:
Unless otherwise defined, enter no spaces before and after ISTD.
The value in the Total line of the %-Amount column indicates the
percentage of the substances defined by you in relation of the total
concentration:
Creating Dynamic Columns That Contain Flexible
Formulas
Follow the description below to create a table column that contains flexible
formulas and dynamically adapts to the number of existing peaks. (This
procedure is only important if the table contains columns with report
variables and if these columns are to the right of the newly added column.)
See the following example for information about how to create a column
that allows you to check automatically whether the signal noise is below
0.005 mAU:
498
Actions in the Printer Layout
•
Add a new column to the table. In the Printer Layout, new columns are
always inserted to the left of the selected column. Select the first field
below the header in the column that shall appear to the right of the new
column:
•
On the context menu, select Insert and then select Insert Chromeleon
Report Column.
•
The Insert Report Column dialog box opens. Select the desired
variable.
•
Delete any existing entry in the Formula field.
•
Click OK to exit the dialog. This action automatically selects the first
field in the new column.
Actions in the Printer Layout
499
•
Enter the desired formula in the layout line without changing the
selection in the report table. Press <Enter> to confirm your entry.
•
Do not change the selection, i.e., the first field below the header is
selected in the new column. Place the cursor on the black rectangle on
the bottom right corner of the selection frame. The cursor now displays
as a solid black cross. Left-click and draw the formula into all lines of
the selected report table column.
•
Double-click any field in the new column to open the Report Column
Properties dialog box for the new report column. Edit the Header and
Dimension fields as appropriate:
500
Actions in the Printer Layout
Creating Dynamic Links to Lines Other than the Current
Line
To reference lines in a report table other than the current line, use indirect
references instead of direct references such as G6. For indirect references,
use the following ¾Report Publisher variables:
INDIRECT and ADDRESS
The following formula references the cell G6 from the cell E2:
=INDIRECT(ADDRESS(ROW()+4;COLUMN()+2)
Note:
Use the ROW() and COLUMN() formulas to return the current line and
column numbers.
Creating Charts
Tip:
In order to perform the operations described below, verify that the ¾Report
Publisher add-on product is licensed.
The Chart Wizard assists you in creating charts:
•
Enable ¾Layout Mode on the Edit menu.
•
Select the columns and rows that shall be represented in a chart.
•
Select Insert ... Charts on the context menu. The mouse cursor will
change its shape to a small + character.
•
Hold down the left mouse button down and draw a rectangle in the size
required for the chart.
•
The Wizard guides you through the chart creation process. Select the
chart type and style and then enter the desired layout settings and axis
titles.
•
Click Finish to exit the Wizard and insert the chart.
The new chart is displayed in the previously drawn frame. If required, you
can edit the chart later. For example, you can change the width, color, and
pattern of lines, areas, or captions, smooth curves, or select a different
chart type.
Actions in the Printer Layout
•
501
Select a single chart element by (double-) clicking and then doubleclick the selected element to change it. The Chart Designer is opened.
After you have made the desired changes and click OK to apply the
changes to the chart.
You can also change the size and position of the entire chart:
•
Click inside the chart. Hold down the left mouse button and move the
chart to the desired position. Resize the chart by dragging the
corresponding size markers.
Changing the Page Format
In the ¾Printer Layout, the page format can be changed as follows:
Define the printer settings
•
Select Print Setup on the File menu. The Print Setup dialog box
appears.
•
In the Paper section, specify the paper size. Click the arrow then select
the desired size from the list. In the Orientation section, select Portrait
or Landscape. The size and orientation is used for all pages of the
respective printout but is not accepted as default.
Tip:
The settings are only valid for the report definition file of the current
sequence. They do not overwrite the default Windows settings. In addition,
any change of the default printer, paper size, or format that is made under
Windows does not affect the settings made here.
Directly printing in the desired format:
•
Select Print on the File menu.
•
Click Properties to open the Properties dialog box.
•
Select the desired paper size and orientation.
502
Actions in the Printer Layout
Notes on the Page Setup
Headers and footers are printed at the upper and lower page margins.
Select Page Setup on the File menu. Make the desired settings in the
Page Setup dialog box.
Headers and footers can contain text and specific format codes. The syntax
is compatible with Microsoft Excel. Observe the order of the single entries.
First, define the alignment of the single header and/or footer, using one of
the following commands:
&L
Left-aligns the characters that follow
&C
Centers the characters that follow (default)
&R
Right-aligns the characters that follow
The default font is Arial 10. You can only define a different font after you
have defined the header and/or footer alignment. Define the new font as
follows:
&B
Bold
&I
Italic
&U
Underline
&S
Strikeout
&"fontname"
Uses the specified font
&nn
Uses the specified font size
Tip:
If you do not enter these font definitions after the header and/or footer
alignment, they will be ignored. You can change the font after each
alignment code (&l/&C/&R).
Finally, enter the expression to be printed:
&A
Prints the current sheet name
&D
Prints the current date
&T
Prints the current time
&F
Prints the ⇒Report Definition File
&P
Prints the page number
&P+Number
Prints the following page number: current page + entered number.
For example: &P+4 prints page 16 if the current page is page 12
and you entered 4.
&&
Prints an ampersand
&N
Prints the total number of pages in the document
Actions in the Printer Layout
503
When entering headers and footers longer than one line, note that the
alignment codes (&L,&C,&R) must be column-oriented. Enter all left-aligned
rows first, then enter all centered rows, and finally enter all right-aligned
rows. To separate rows, press <Ctrl> <Enter>.
In addition to the pre-defined variables above, you can enter variables
pertaining to Chromeleon. Enclose these variables in braces.
Example:
&LOperator: {gen.operator} , Timebase: {seq.timebase} ,
Sequence:{seq.name} &RPage &P of &N
&D
This example produces the following header or footer
Operator: cmadmin, Timebase: HPLC, Sequence: Calibration
Page 1 of 12
4/6/2002
You can include any Chromeleon variable in the header. However,
including sample and sequence variables makes the most sense. The
Chromeleon formula variables are identical with the ones generated in the
dialog box for entering report variables. Enter the decimal places for
numerical variables after the formula. Separate them by ";" (default: 0).
Example: {smp.inject_volume;2}. Time entries cannot be formatted.
Note:
All worksheets of the ¾Printer Layout that are selected for printing are
considered one single print job; that is, page numbering is consecutive for
all worksheets.
Answering Frequently Asked Questions
Question: How can I have the results printed out automatically after the
analysis?
Answer: Mark the sequence of interest in the Browser. Select Reporting on
the Batch menu and select Print/Export Report by the box. Afterward
select the desired option: Print each sample immediately or Print when
the entire batch is finished.
504
Actions in the Printer Layout
Question: The view in the ¾Printer Layout window and the printed report
have a different layout. Why?
Answer: This may happen. There are different ways how to print a sample
report. Usually, you will print the report via the Batch Report dialog
window. When you print the report in this way, the report definition defined
in this dialog is used. When you have the results printed automatically via
Reporting as described above, it may happen that the report definition
used to display the data in the Printer Layout is different from the report
definition used for the printout.
Notes:
When you either select Print on the File menu in the Printer Layout or click
the Print icon on the standard toolbar, it is always the current view that is
printed.
Question: Where is the used ⇒Report Definition File saved?
Answer: Some predefined Report Definition Files (RDFs) are available in
the Dionex Templates > Reports folder. However, it is not imperative that
the used RDF is stored in this folder. To find out where the used RDF is
stored, proceed as follows: Mark the sequence of interest in the Browser.
Right-click and select Properties on the context menu.
In the Preferred Report & Channel section, the entry in the Preferred
RDF File field indicates the path of the current report definition file. To
determine a different report definition file as preferred RDF, click the
Browse button ("…") and navigate to the desired report definition.
The settings of this RDF are then always used when you open a sample of
this sequence afterward. However, you can select a different RDF in the
on-screen report or in the Printer Layout, using the Load Report Definition
command on the Workspace menu.
Notes:
If you select the RDF via Load Report Definition, this does only apply to the
current view. The Preferred RDF File of the sequence is not changed.
Actions in the Printer Layout
505
Question: What do the terms Printer Layout, Report Template, and Report
Definition File refer to?
Answer: The Printer Layout is an editor window that allows you to define
the content and the layout of the pages to be printed. This information is
saved to the Report Definition File (RDF) together with the layout of the onscreen report. Sometimes, the Report Definition File is also referred to as
report template.
Question: How can I save the scaling?
Answer: The scaling, too, is saved to the Report Definition File. Select
Save Report Definition on the Workspace menu. Afterward, select the
previously saved RDF as Preferred RDF File in the properties of the
sequence (see above). (Unlike the peak parameters, the scaling is not
saved to the QNT File.)
Question: How can I save comments in the report? Where can I enter
information about the column I am using?
Answer: Chromeleon provides different possibilities for doing this. For
example, you can use the Title field to enter information about the column:
Mark the sequence in the Browser and select Properties on the context
menu. Afterward, add the Title variable of the ⇒Sequence category to the
Printer Layout.
Tip:
Keep in mind to update the Title field entry whenever you change the
column.
For tips to solve similar questions, refer to How to …:
Printer Layout and the corresponding subtopics.
Actions in the
506
Actions in the Printer Layout
Actions in the Peak Purity Analysis (PPA) Window
507
Actions in the Peak Purity Analysis
(PPA) Window
The PPA (Peak Purity Analysis) plot (see Data Reprocessing
PPA
(Peak Purity Analysis)) displays the ¾3D Field of a sample if there is a
corresponding raw data file. This is the case if the 3DFIELD channel was
selected for ¾Data Acquisition. The 3D field in the lower right-hand window
section has cross-wires. With the mouse, the axes can be moved
separately or together. The spectrum or chromatogram extracted along the
current y and x-axis will be displayed on the left or above the 3D field.
•
Move the y-axis of the cross-wires to successively display all spectra of
the active chromatogram.
•
Move the x-axis of the cross-wires to display the appearance of a
chromatogram at different wavelengths.
•
The status bar shows the retention time, wavelength value, and signal
height of the current cross wire position.
Apart from this simple spectra and chromatogram selection, use this
method to perform the following operations:
Analyzing Peak Purity
Selecting the Optimum Integration Path
Extracting and Exporting Spectra, Chromatograms, and 3D Fields
Analyzing Peak Purity
Peak purity can be evaluated using the following methods:
Visual Check of Iso Line Plot
PPI and PPI Match Factor
Normalized Spectra Overlay
Peak Inhibition via Virtual Channels
Multiple Ratio
508
Actions in the Peak Purity Analysis (PPA) Window
Visual Check of Iso Line Plot
The visual check of the Iso line plot is a method that allows peak purity
evaluation. High peak purity is indicated by:
•
All recognizable absorption
maxima
are
located
vertically
below
one
another in direction of the
wavelength axis.
•
The maxima are separated
so that the surrounding iso
lines do not touch (no
overlapping).
•
Pure peaks have (local)
symmetry or an idealized
ellipse shape in the
maximum.
PPI and PPI Match Factor
In the chromatogram window of the PPA method, curves for the ¾PPI
(Peak Purity Index) and the ¾Peak Purity Match Factor can be displayed:
•
Double-click within the window and select the options Draw PPI and
Draw Match from the Chromatogram Plot tab page.
The peak purity index is represented by a purple curve, and the match
factor is indicated by a green curve. A distinctive rectangular shape is one
criterion (of many) for peak purity. The exact match value at the current
mouse cursor position is shown on the status bar. The ideal value is 1000
and requires approximately 80% of the available window height.
PPI, match value, and the corresponding standard deviation (RSD) can be
included in a report. The report variables are in the category Peak Purity.
The smaller the standard deviation, the more critical the quality of the
rectangular curve should be judged; that is, the better the two spectra will
match in various points of a peak.
Actions in the Peak Purity Analysis (PPA) Window
509
Normalized Spectra Overlay
In a normalized spectra overlay, single spectra of the spectra recorded for
the peak are extracted and are compared with each other. Usually,
extraction is at the run time of the peak and at a specific peak height in the
leading and trailing edges of the peak; for example, at 10% and 50% of the
peak height. Comparing spectra that are ¾normalized by the same method
is a means of evaluating the peak purity.
The more closely the spectra match, the higher the possibility that the peak
is pure. The following are indications for the impurity of a peak:
•
The number of relative maxima and minima of two spectra does not
match
•
The relative maxima are clearly offset against each other
•
The height of the relative maxima strongly deviates
However, please note the following:
•
Especially near peak limits, spectra have an increased noise level due
to the normalization procedure. This applies in particular to the UV
range.
•
In the case of very long peaks, baseline correction cannot completely
compensate for detector drift.
•
The spectra shape depends on the solution; that is, the spectra of a
peak can only be compared if the mixing ratio was constant during
detection.
•
It is possible only to compare spectra with the (complete) absorption in
the dynamic validity range of the Lambert Beer Law. If the peak
maximum, including the baseline spectrum, is considerably above 1
AU, caution is called for.
Tip:
The normalized spectra overlay via peak height is currently possible only
The Spectra Plot). Within the
on the spectra plot (also see Integration
PPA method, the "animated" extraction of spectra is possible. Pressing the
CTRL key in the 3D field extracts the current spectrum. If the y-axis of the
cross-wires is moved simultaneously, all spectra existing for this range are
displayed in the spectra window. A different color indicates the extraction
period in the chromatogram. This process can be repeated; for example, for
different retention times within the peak width. If you release the CTRL key
between the individual extractions, the spectra extracted per range are
displayed in a separate color.
510
Actions in the Peak Purity Analysis (PPA) Window
Peak Inhibition via Virtual Channels
If two overlapping peaks have different spectra s1(λ) and s2(λ) the
following equitation is true when the area below the baseline is ignored:
A(λ, t) = s1(λ)*c1(t) + s2(λ)*c2(t)
(1).
c1 and c2 stand for the time-dependant concentrations of the
corresponding components in the flow cell. Then, two wavelengths λ1 and
λ2 are selected and the following signal is created:
d(t) = A(λ1, t) - K*A(λ2, t) = c1(t)[s1(λ1) - K*s1(λ2)] + c2(t)[s2(λ1) - K*s2(λ2)].
(2),
The c2 term disappears provided the appropriate expression was selected
for K, that is:
K = s2(λ1) / s2(λ2) (3)
This is the channel ratio of the second peak. It can be read off the height
ratio of the two peaks if they do not overlap completely. In order to be able
to use this approach, the channel ratio of the first peak
K’ = s1(λ1) / s1(λ2) (4)
must be different from K. If not, the first term in equation (2) will disappear
as well. Select the λ1 and λ2 wavelength in such a way that the difference
between K and K’, that is:
∆K = ⏐K - K’⏐
(5)
is the maximum difference. The best way to determine the appropriate
wavelengths is to do so in the PPA Window. After this, the virtual channel
d(t) can be defined. Of course, it is possible to inhibit the first peak in the
same way.
If you know the wavelengths for which ∆K is the maximum before you
record the chromatogram, the two channels, for example, UV_VIS_1 and
UV_VIS_2 can be adjusted to those wavelengths.
Actions in the Peak Purity Analysis (PPA) Window
511
You can then use the following ¾Program:
As already mentioned in the comment line of the program, the following
equation is true for this example: K = 0.3.
Multiple Ratio
Each channel extracted from a 3D field can be used for performing the ratio
test. Especially suited are chromatograms in the range of spectral minima
or maxima. If the ratio condition is met, this can indicate, but does not prove
peak purity. Performing the test with a larger number of channels does not
change this fact.
Selecting the Optimum Integration Path
Chromeleon is capable of calculating the ¾Optimum Integration Path within
a 3D field. The calculation is performed automatically. The result can be
displayed in the 3D field window of the PPA method.
•
Double-click within the window and select the Draw Opt-Int-Path
option on the Iso/3D Plot tab page.
Tip:
You may also define extraction of the optimum integration path in the
The
Post-acquisition steps view of the PGM Editor (see Control
PGM Editor.) Open the PGM File in which you want to define
extraction of the optimum integration path and then, select the Postacquisition steps view.
512
Actions in the Peak Purity Analysis (PPA) Window
A green line in the 3D field indicates the integration path. Similar to
chromatograms, it can be saved as a separate channel with variable
wavelength.
•
Select Extract > Opt. Int. Path to file on the View or context menu
and determine the name under which the path is saved as a separate
channel or accept the default name (OPTINT).
Tip:
In the PGM Editor, follow the steps below: Select Insert line on the
context menu to add a new post-acquisition step. The New postacquisition step dialog box is opened. Select Extract optimum
integration path to open the Extract optimum integration path
dialog box.
•
Select Extract from all samples of current sequence or query
option, if the path should be extracted for all samples of the underlying
sequence or query. The shape of the path is identical for all samples!
Tip:
This option is not provided in the dialog box of the PGM Editor.
Note:
The channel extracted in this way may have baseline jumps. For distinctly
absorbing solvents, they may be due to changes in the wavelength.
If the path should serve as a basis for a ¾Wavelength Switch in future
samples, the switch times and the selected wavelength values must be
entered in a program with exact time specifications. This can also be
performed by automatically inserting the data.
•
Select Extract > Opt.Int.Path to clipboard on the View or context
menu to copy the data to the clipboard.
•
Select the Name of the channel, for which the wavelength is
automatically switched in the future.
•
Enter a value for the Bandwidth if several chromatograms should be
averaged to one. The bandwidth determines the range of the path. All
sections of a chromatogram within this range are averaged to one
chromatogram.
•
Open a ¾PGM File and insert the data at the beginning of the program
(Commands view) via the Paste command.
Actions in the Peak Purity Analysis (PPA) Window
513
The resulting PGM File could have the following appearance:
0.000
3.320
4.830
6.100
8.100
9.660
10.480
•
UV_VIS_1.Bandwidth = 0
UV_VIS_1.Wavelength = 210
UV_VIS_1.Wavelength = 210
UV_VIS_1.Wavelength = 206
UV_VIS_1.Wavelength = 272
UV_VIS_1.Wavelength = 262
UV_VIS_1.Wavelength = 278
UV_VIS_1.Wavelength = 250
Enter more commands in the program to complete it. Sort it according
to ascending retention times.
Extracting and Exporting Spectra,
Chromatograms, and 3D Fields
Spectra
You can copy each spectrum extracted from a 3D field in the PPA method
The Spectra Plot) to the
or on the spectra plot (see Integration
Windows clipboard (Copy command). From the clipboard, you can then
paste the spectrum in a spectra library (Paste command). (For information
Spectra Libraries).
about spectra libraries, refer to Data Reprocessing
Follow the steps below:
•
Select Extract > Spectrum to clipboard on the View or context menu
to copy the current spectrum from the 3D field of the method PPA to
the Windows clipboard.
•
Open an existing Spectra Library. Select the command by opening the
corresponding LIB file from the Browser, or
•
Create a new library via the File> New > Spectra Library commands.
•
Select Paste Spectra to save the spectrum and the data in the library.
514
Actions in the Peak Purity Analysis (PPA) Window
Chromatograms
From an open 3D field in the method PPA, you can extract a chromatogram
of any wavelength:
•
Select Extract > Chromatogram to file on the View or context menu
to save the active chromatogram as a separate channel.
•
Select the wavelength and the bandwidth at which to extract the
chromatogram.
Chromeleon will suggest a name for the extracted chromatogram,
considering the wavelength. However, you may also enter any other name
of your choice. The chromatogram is saved in addition to the raw data of
the existing 3D field. Simultaneously, the extracted chromatogram is
opened via the Integration method to give the user an overview of the
saved data.
In addition, the Extract > Chromatogram to file command provides a
special option. Instead of saving one single chromatogram, another
chromatogram of the same wavelength can be extracted and saved from all
samples of the underlying sequence or query. Enable Extract from all
samples of current sequence or query. Especially in this case,
automatically naming the extracted chromatogram (see above) is very
useful.
3D Field Data
Select Export > 3DFIELD to include and display the current 3D field raw
data in other applications, such as Microsoft Excel. Data is converted into a
general ASCII format.
•
After you have executed the Export 3DFIELD command, change to the
other application and insert the data, using the Paste command. In
addition to the pure raw data, additional sample and sequence
information is transferred to the application.
Actions in the Peak Purity Analysis (PPA) Window
515
Answering Frequently Asked Questions
Question: How do I perform spectra scanning?
Answer: In the Program Wizard, select the 3DField check box on the UV
Options page to record the 3D channel for the corresponding samples.
Question: How do I change the ⇒Detection Parameters, such as
⇒Minimum Area or ⇒Inhibit Integration?
Answer: Enter the parameters on the Detection tab page of the ⇒QNT
Editor.
Question: How do I save the modifications to the layout of the on-screen
report?
Answer: Modifications to the layout of the on-screen report are saved in the
workspace. To save any modifications, select Save Workspace on the
Workspace menu. The workspace contains the arrangement of the
individual windows. Which information is displayed in the single windows is
defined by the information saved in the corresponding file. For example, for
the Printer Layout window, the information is saved in the Report Definition
File (RDF).
516
Actions in the Peak Purity Analysis (PPA) Window
Actions Related to Mass Spectrometers
517
Actions Related to Mass
Spectrometers
For MS data acquisition, use the Thermo Finnigan aQa or MSQ ¾Mass
Spectrometer with Chromeleon. However, before you can record and
process mass spectra, several conditions must be fulfilled. For more
information, refer to:
Creating an MS Program and Sequence
Creating a PGM File for the aQa MS
Creating aQa MS Channels with the aQa PGM File
Creating a PGM File for the MSQ
Creating MSQ Channels with the MSQ PGM File
Acquiring MS Data in MCA Mode
Extracting Mass Traces Online
Extracting Mass Traces Afterward
Extracting a Temporary Mass Trace
Showing Mass Spectra
Minimizing the Noise of Mass Spectra
Defining Further QNT Settings for MS
Creating an MS Program and Sequence
Manual data acquisition is not supported for ¾Mass Spectrometers.
Therefore, you must prepare a sequence before you can start data
acquisition. This requires a control file (¾PGM File).
Program Wizard (PGM Editor: Commands)
Create a new PGM File, using the Program Wizard (see Programmed
The Program Wizard). If a mass spectrometer is installed in
Control
the current timebase, select the data acquisition, using the MS channel
option on the Acquisition Options tab page. This action opens the page
for MS-specific parameters.
518
Actions Related to Mass Spectrometers
Tip:
Log on to the PC as a main user or as administrator if you wish to create a
program for the MSQ mass spectrometer. If you do not log on this way, a
No MS device configured... error message appears.
Set the parameters required for ¾Mass Trace processing (= MS
chromatogram). The Range parameter is the scaling factor for the online
display of mass traces on the control panel; for example, Range = 5 allows
a maximum value of 100.000 = (1 E+5) counts in the online display.
However, the stored data are not affected. The ¾Smoothing parameter
determines which type of smoothing filter is used for smoothing MS
chromatograms.
In the Points field, define the number of data points to be used for
smoothing. Select the number of data points such that the width of the
smoothing filter approximately equals the peak's half width. For example,
the following program will be created:
Actions Related to Mass Spectrometers
519
The PGM File that was generated using the PGM Wizard does not include
⇒AcqOn/Off (Data Acquisition On/Off) commands for the MS channels.
The channels that are needed for mass trace acquisition depend on the
contents of the MS method.
The Program Wizard automatically generates the wait MS.ready command
before the inject command. This synchronization is required between the
mass spectrometer and Chromeleon. If you want to create your own PGM
Files manually, always add the wait MS.ready command before the inject
command.
If you want to use ¾Blank Run Samples, verify that the inject mode is set to
Inject. The aQa mass spectrometer will not start data acquisition unless an
injection signal is received.
Example:
Wait MS.Ready
Inject Blank=Inject
On the last Wizard page, select the Review the program in a new
window option. This action automatically opens the PGM Editor as soon as
the new PGM File is saved. Use the PGM Editor to define the mass
spectrometer settings for the PGM File. Select the Mass Spectrometer
view by clicking the corresponding icon in the left editor section. For
information about mass spectrometer methods, refer to
Creating a
Creating a PGM File for the MSQ.
PGM File for the aQa MS or
Sequence
Then, create a sequence for your HPLC or IC system.
a) Using the Sequence Wizard: Enter the created PGM File in step 4.
b) From a previous sequence: Copy the PGM File to a new sequence and
enter the PGM File in the sample list.
Tip:
Verify that the Operation property of the aQa mass spectrometer is in the
On state. Otherwise, the gas flow and the probe heating are turned off and
data acquisition cannot be started.
Then, start data acquisition as usual (see How to ...: Device Control
Starting Data Acquisition). The current mass spectrum can be
displayed on the ¾Control Panel.
520
Actions Related to Mass Spectrometers
Creating a PGM File for the aQa MS
Use the Mass Spectrometer view of the PGM Editor (see Control
The
PGM Editor) to create a new instrument method for the aQa ¾Mass
Spectrometer as part of the PGM File. Open this view by clicking this icon
on the shortcut bar in the left PGM Editor section:
Tip:
If the PGM File was not created for a timebase that includes a mass
spectrometer, neither the MS method nor the Thermo Finnigan aQa symbol
will be available. To use such a PGM File for a timebase that includes a
mass spectrometer, select the Add MS Instrument Method command to
the Edit or context menu. This creates a standard MS method and adds the
Mass Spectrometer view. In addition, enter the following command in the
¾Program (Commands view) at the time t = 0.000 min:
0.000 Wait MS.Ready
The Mass Spectrometer view is part of the ¾Xcalibur software and allows
you to specify the method used by the aQa mass spectrometer.
On the Ionization Mode tab page, select Electrospray (Electrospray
Ionization) or APCI (Atmospheric Pressure Chemical Ionization) as the
ionization mode.
On the Analysis tab page, set the sensitivity of the mass spectrometer via
the detector voltage. Via Advanced (from tune file), load a tune file that
has been previously defined. To fine-tune the mass spectrometer directly,
click the Tune… button.
Caution:
When you use the Xcalibur method editor of Chromeleon, the Other
detectors section is irrelevant. Do not use this section for data acquisition
with other detectors, such as UV detectors! In this case, perform data
acquisition as usual.
Actions Related to Mass Spectrometers
521
On the Acquisition tab page, enter the aQa-specific signal parameters for
¾Mass Spectra acquisition:
Select the data acquisition mode first: Select ¾Full-Scan to acquire the
entire mass spectrum for each analyte or ¾SIM to obtain MS
chromatogram at a defined mass.
Note:
For a list of SIM masses for anions, cations, and amines in water, refer to
How to ...: Actions Related to Mass Spectrometers
SIM Mass Lists
for IC-MS.
522
Actions Related to Mass Spectrometers
In Full-Scan mode, use the Simultaneous acquisitions tab page to set the
polarity and maximum voltage on the aQa MS for four single channels
(¾TICF channels). In the Acquisition rate field, specify the rate for data
acquisition; in the Mass spectrum field, specify the mass range for which
to perform data acquisition.
Tip:
This view of the PGM Editor is part of the Xcalibur software. Thus, you can
open the Xcalibur help either via the Help menu or by clicking Help. The
Xcalibur help provides detailed information about mass spectra acquisition.
Tip:
When saving the PGM File, only use ASCII characters to name the entire
path, i.e., including the datasource and the directories. If you use other
characters, too, it may be impossible to start data acquisition.
For more information, refer to
aQa PGM File.
Creating aQa MS Channels with the
Actions Related to Mass Spectrometers
523
Creating aQa MS Channels with the aQa PGM File
The examples below describe which channels Chromeleon creates for the
different types of data acquisition with the aQa MS.
Note:
For a list of SIM masses for anions, cations, and amines in water, refer to
SIM Mass Lists
How to ...: Actions Related to Mass Spectrometers
for IC-MS.
The first example creates two SIM channels plus the TIC channel:
The SIM_01 channel records the chromatogram at a mass of 100 amu,
while the SIM_02 channel records the corresponding chromatograms at a
mass of 200 amu.
524
Actions Related to Mass Spectrometers
The second example creates six SIM channels and the TIC channel:
The channel assignment is as follows:
SIM_01: Mass 100 amu, positive voltage, 100V,
SIM_02: Mass 100 amu, negative voltage, -80V,
SIM_03: Mass 200 amu, positive voltage, 100V,
SIM_04: Mass 200 amu, negative voltage, -100V,
SIM_05: Mass 300 amu, positive voltage, 100V,
SIM_06: Mass 300 amu, positive voltage, 80V.
Actions Related to Mass Spectrometers
525
The third example creates three TICF channels and the TIC channel:
The channel assignment is as follows:
TICF1: positive voltage, 100V
TICF2: positive voltage, 200V
TICF3: negative voltage, -100V.
Tip:
When saving the PGM File, only use ASCII characters to name the entire
path, i.e., including the datasource and the directories. If you use other
characters, too, it may be impossible to start data acquisition.
If you notice after data acquisition that a channel is missing, you can extract
separate ¾Mass Traces (or mass ranges) from the ¾Mass Spectrum and
save them as new channels. (For more information, refer to How
to ...: Actions Related to Mass Spectrometers
Extracting Mass
Traces Afterward.)
526
Actions Related to Mass Spectrometers
Creating a PGM File for the MSQ
Use the Surveyor MSQ view of the PGM Editor (see Control
The PGM
Editor) to create a new instrument method for the MSQ ¾Mass
Spectrometer as part of a PGM File. Open this view by clicking this icon on
the shortcut bar in the left PGM Editor section:
Tip:
If the PGM File was not created for a timebase that includes a mass
spectrometer, neither an MS method nor the MSQ symbol will be available.
To use such a PGM File for a timebase that includes a mass spectrometer,
select Add MS Instrument Method on the Edit or context menu. This adds
the Surveyor MSQ view. From the standard MS methods (Surveyor MSQ
Templates) in the dialog box, select the desired MS method. The
appropriate MS method depends on the ionization mode, the MS mode
(¾Full Scan or ¾SIM), and the polarity of the recorded ions. In addition,
enter the following command in the ¾Program (Commands view) at the
time t = 0.000 min:
0.000 Wait MS.Ready
The Surveyor MSQ view is part of the ¾Xcalibur software and allows you
to specify the method used by the MSQ mass spectrometer.
The upper left section shows a runtime preview of the single channels to
record. In the Preview section, select:
•
All Scans to display all channels.
•
Full Scans to display the ¾Full Scan channels (orange) only.
•
SIM to display the ¾SIM channels (green) only.
For an overview of the defined channels, including their measurement
parameters, select the Parameter option.
If a chromatogram was recorded with one MS channel only, you can also
have the chromatogram displayed in the preview. Select Show Method
Options to open the method options, and then select the desired sample
by clicking the "..." button under Display.
Actions Related to Mass Spectrometers
527
On the top right, in the Per Method Parameters section, select ESI
(Electrospray Ionization) or APCI (Atmospheric Pressure Chemical
Ionization) as the ionization mode, depending on your MSQ installation. In
addition, enter the nominal temperature on the ion source (range: 0 to
655°C).
In the Full/SIM Scan Events section, enter the MSQ-specific signal
parameters for ¾Mass Spectra acquisition:
In the Mass Range field, define the desired mass range (¾Full Scan). In
the Mass field, define the mass. Define the corresponding bandwidth
(¾SIM) in the Span field. In addition, define the duration of the data
acquisition (Time Range), the data acquisition rate (Dwell Time (SIM) or
Scan Time (Full Scan)), the Polarity, and the ionization voltage (Cone
[V]).
Note:
For a list of SIM masses for anions, cations, and amines in water, refer to
SIM Mass Lists
How to ...: Actions Related to Mass Spectrometers
for IC-MS.
In the Scans section, select the Add Full option to add a new full-scan
channel or Add SIM to add a new SIM channel. The settings from the
preceding channel are adopted. Use the Create Group option to group all
528
Actions Related to Mass Spectrometers
SIM channels that are recorded in the same retention time window. (To do
so, hold down the Ctrl key and select the desired SIM channels by clicking.)
Select Ungroup to undo this action. To delete a channel, select the
channel, and then click Delete.
Tips:
This view of the PGM Editor is part of the Xcalibur software. The Xcalibur
Help system provides detailed information about mass spectra acquisition.
First, click the question mark (at the top right) and then click the option of
interest to open the corresponding Help topic.
Tip:
When saving the PGM File, only use ASCII characters to name the entire
path, i.e., including the datasource and the directories. If you use other
characters, too, it may be impossible to start data acquisition.
For more information, refer to
PGM File.
Creating MSQ Channels with the MSQ
Creating MSQ Channels with the MSQ PGM File
Chromeleon creates a channel for each scan event of the MSQ instrument
method. In the case of ¾SIM groups, this also applies to each sub scan
event. The examples below describe which channels Chromeleon creates
automatically for the different types of data acquisition with the MSQ.
Note:
For a list of SIM masses for anions, cations, and amines in water, refer to
SIM Mass Lists
How to ...: Actions Related to Mass Spectrometers
for IC-MS.
The first example creates four SIM channels plus the TIC channel:
Actions Related to Mass Spectrometers
529
The SIM1 channel records the chromatogram at a mass of 80.00 amu,
while the other channels record the same chromatogram at a mass of
85.00 amu (SIM2), 90.00 amu (SIM3), and 100.00 amu (SIM 4).
Data acquisition is performed for all four channels at the same time (0 to 1
min). That is why you can group all four channels. In this case, only SIM
channels and the TIC channel would be created.
The next example creates 11 SIM channels (SIM 01 to SIM 11) and the TIC
channel:
The channel assignment is as follows:
Xcalibur (CM program: MSQ)
Name
Mass [amu]
Chromeleon
Span [amu]
Name
Mass Range [amu]
GROUP 1: Retention time 0.00 to 2.00 min
SIM 1
100
1
SIM_01
99.5 - 100.5
SIM 2
200
1
SIM_02
199.5 - 200.5
SIM 3
300
1
SIM_03
299.5 - 300.5
SIM 4
400
1
SIM_04
399.5 - 400.5
530
Actions Related to Mass Spectrometers
Xcalibur (CM program: MSQ)
Name
Mass [amu]
Chromeleon
Span [amu]
Name
Mass Range [amu]
GROUP 2: Retention time 2.00 to 3.00 min
SIM 1
100
1
SIM_05
99.5 - 100.5
SIM 2
100
3
SIM_06
98.5 - 101.5
SIM 3
100
5
SIM_07
97.5 - 102.5
SIM 4
150
1
SIM_08
149.5 - 150.5
SIM 5
200
SIM_09
199.5 - 200.5
Single channels:
SIM 1
99
1
SIM_10
98.5 - 99.5
SIM 2
110
1
SIM_11
109.5 - 110.5
The next examples creates 3 TICF channels and the TIC channel:
The channel assignment is as follows:
TICF_1:
FS 1, Mass range 30.00 to 500.00 amu; retention time 0.00 to 1.00 min,
TICF_2:
FS 2, Mass range 60.00 to 400.00 amu; retention time 2.00 to 4.00 min,
TICF_3:
FS 3, Mass range 600.00 to 800.00 amu; retention time 0.00 to 2.00 min.
Tip:
When saving the PGM File, only use ASCII characters to name the entire
path, i.e., including the datasource and the directories. If you use other
characters, too, it may be impossible to start data acquisition.
If you notice after data acquisition that a channel is missing, you can extract
separate ¾Mass Traces (or mass ranges) from the ¾Mass Spectrum and
save them as new channels. (For more information, refer to How
to ...: Actions Related to Mass Spectrometers
Extracting Mass
Traces Afterward.)
Actions Related to Mass Spectrometers
531
SIM Mass Lists for IC-MS
¾SIM (Selected Ion Monitoring) is the ¾Mass Spectrometer method used
for recording an MS chromatogram at a specific mass-to-charge ratio.
The tables below list the SIM masses for anions, cations, and amines in
water. Refer to the appropriate table when entering SIM masses in aQa or
MSQ ¾PGM Files, or to identify found masses.
Anion SIM Mass List for IC-MS (in water)
m/z
Anion
Detected As
19
Fluoride
F−
35
Chloride
Cl−
45
Formate
HCOO−
46
Nitrite
NO2−
58
Thiocyanate
SCN−
59
Acetate
CH3COO−
61
Bicarbonate
HCO3
62
Nitrate
NO3
67
Chlorite
ClO2 −
73
Glyoxylate
CHOCOO−
73
Propionate
CH3CH2COO−
75
Glycolate
HOCH2COO−
79
Bromide
Br −
83
Chlorate
ClO3
87
Butyrate
CH3CH2CH2COO−
87
Pyruvate
CH3COCOO−
89
Lactate
CH3CH(OH)COO−
89
Oxalate
COOHCOO−
93
Chloroacetate
CICH2COO−
Isotopes
(decreasing
frequency)
−
−
67/69
79/81
−
83/85
93/95
532
Actions Related to Mass Spectrometers
Anion SIM Mass List for IC-MS (in water)
m/z
Anion
Detected As
Isotopes
(decreasing
frequency)
95
Methanesulfonate
CH3SO3
96
Sulfamate
NH2 SO 3
97
Sulfate
HSO4 −
97
Phosphate
H2PO4 −
99
Perchlorate
ClO4 −
101
Valerate
CH3CH2CH2 CH2COO−
103
Hydroxybutyrate
CH3CHOHCH2COO−
103
Malonate
COOHCH2 COO−
113
Thiosulfate
HS2O3
113
Trifluoroacetate
F3CCOO−
115
Maleate
COOHCHCHCOO−
115
Fumarate
COOHCHCHCOO−
117
Succinate
COOHCH2CH2COO−
127
Bromate
BrO3
127
Dichloroacetate
Cl2CHCOO−
127
Iodide
I−
128
Selenite
SeO3
131
Glutarate
COOH(CH2 )3 COO−
133
Malate
COOHCH2CHOHCOO−
133
Tartrate
COOH(CHOH)2 COO−
137
Bromoacetate
BrCH2COO−
137/139
144
Selenate
SeO 4 −
144/142
145
Adipate
COOH(CH2 )4 COO−
173
Bromochloroacetate
BrClCHCOO−
183
Styrenesulfonate
CH2CHC6H4SO3
−
95/97
−
96/98
97/99
99/101
−
113/115
−
127/129
127/129
−
128/126
173/171/175
−
Actions Related to Mass Spectrometers
533
Anion SIM Mass List for IC-MS (in water)
m/z
Anion
Detected As
Isotopes
(decreasing
frequency)
191
Citrate
HOOCCH2COH(COOH)CH2COO−
191
Quinate
C6H7 (OH)4 COO−
191
Isocitrate
HOOCCHOHCH(COOH)CH2COO−
207
Dichlorobromoacetate
Cl2BrCCOO−
207/205/209/211
217
Dibromoacetate
Br2CHCOO−
297/295/293/299
251
Dibromochloroacetate
Br2ClCCOO−
251/253/249/255
297
Tribromoacetate
Br3CCOO−
297/295/293/299
Cation and Amine SIM Mass List for IC-MS (in water)
m/z
Cation
Detected As
18
Ammonium
NH4 +
19
Hydronium
H3O +
20
Calcium
1 / 2Ca2 +
23
Sodium
Na +
39
Potassium
K+
46
Ethylamine
CH3CH2NH3
46
Dimethylamine
(CH3 )2 NH2
60
Trimethylamine
(CH3 )3 NH+
60
Guanidine
(NH2 )2 CNH2+
61
Ethanediamine
NH2CH2CH2NH3
62
Monoethanolamine
OHCH2CH2NH3
69
Imidazole
C3H4N2H+
74
Diethylamine
(CH3CH2 )2 NH2
74
Dimethylethylamine
(CH3 )2 (C 2H5 )NH+
74
Butylamine
C4H9NH3
+
+
+
+
+
+
534
Actions Related to Mass Spectrometers
Cation and Amine SIM Mass List for IC-MS (in water)
m/z
Cation
Detected As
75
Propanediamine
NH2CH2CH2CH2NH3
76
Methylethanolamine
HOCH2CH(CH3 )NH3
76
Dimethylmethanolamine
HOC(CH3 )2 NH3
76
Propanolamine
C3H6OHNH3
80
Pyridine
C5H5NH+
85
Lysidine
C 4 H8 N2H +
88
Morpholine
C4H8ONH2
88
Methyldiethylamine
CH3 (C2H5 )2 NH+
89
Butanediamine (Putrescine)
H2NC4H8NH3
90
Dimethylethanolamine
C5H13NH+
90
Methylpropanolamine
C4H8 (OH)NH3
100
Cyclohexylamine
C6H11NH3
102
Triethylamine
(CH3CH2 )3 NH+
103
Pentanediamine (Cadaverine)
H2NC5H10NH3
104
Dimethylamino-2-propanol
(C3H6OH)(CH3 )2 NH+
106
Diethanolamine
(OHCH2CH2 )2 NH2 +
118
Diethylethanolamine
C 6H15ONH+
120
Methyldiethanolamine
C5H13O2NH+
132
Diethylaminopropanol
C7H17ONH+
146
Spermidine
H2N(CH2 )3 NH(CH2 )4 NH3
150
Triethanolamine
(OHCH2CH2 )3 NH+
203
Spermine
C10 H 26 N 4 H +
+
+
+
+
+
+
+
+
+
+
Actions Related to Mass Spectrometers
535
Acquiring MS Data in MCA Mode
Use the MCA (= Multi-Channel Analysis) mode to calibrate the aQa ¾Mass
Spectrometer and analyze pure, low-concentration solutions of substances.
Usually, the solution in question is provided to the mass spectrometer via
infusion.
Tip:
The ¾MCA mode is not available for the MSQ.
The MCA mode summarizes all ¾Mass Spectra of the single scans. Only
the resulting averaged mass spectrum from each of the scan filters (up to
four) is saved when the analysis is finished.
Caution:
The MCA mode does not allow recording and showing mass spectra at a
defined time of the chromatogram. Therefore, the MCA mode is not
suitable for chromatographic analyses!
Data acquisition in MCA mode is as follows:
•
Create a PGM File, using the Program Wizard (see Programmed
Control
The Program Wizard).
•
Verify that the PGM File includes an Inject command even if you do
not use an autosampler. Otherwise, data acquisition cannot be started.
•
The PGM Editor), open the
In the PGM Editor (see Control
Acquisition tab page of the Finnigan aQa window. In the Mass
spectrum field, select MCA as Peak format.
•
Save the PGM File and close the PGM Editor.
•
Enter the PGM File in your sequence and start the sequence in a
batch.
Tip:
¾MCA data acquisition is not possible in demo mode!
536
Actions Related to Mass Spectrometers
The ¾Xcalibur window below opens automatically when data acquisition is
started:
The status bar indicates the time that passed since the data acquisition was
started. At the top left and right, the results of the last two scans are given.
As soon as the data acquisition is finished, you can display the results in
the Chromeleon report. To display the results click the line at the highest
retention time on the chromatogram plot using the Spectra Tool. This action
opens the following view:
Actions Related to Mass Spectrometers
537
The chromatogram in the left pane contains one data point only that is at
the highest retention time. To illustrate the summing up of the entire
acquisition period, it shows one line in the height of the entire counts of all
summed up mass spectra. In addition, only one single (entire) mass
spectrum is displayed in the right window section.
Tip:
As MCA mass spectra are not spectra from chromatographic peaks, but are
formally retention time spectra, they cannot be inserted in the ¾Printer
Layout. Therefore, select Print on the File menu to print mass spectra. A
dialog box appears. Select the Printer Layout page that contains the mass
spectrum.
Extracting Mass Traces Online
It is possible to extract ¾Mass Traces (MS chromatograms) and save them
as additional channels. This is possible online during data acquisition
Extracting Mass Traces
(online) but can be done later, as well (see
Afterward).
In the ¾Server Configuration Program, define the required number of
Online Mass Extract Channels on the Installed Channels tab page. (Up to
32 channels can be defined.) These channels are automatically named
MS_01 to MS_32.
Create a ¾PGM File for data acquisition in ¾Full Scan mode using the
Program Wizard (see Programmed Control
The Program Wizard).
Each single MS channel needs its own ⇒AcqOn/Off command. Define as
well the following parameters for each single MS channel:
Parameter
Min.
FilterIndex 0: NoFilter
MinMass
0.00 amu
(MSQ)
2.0 amu
(aQa)
Max.
Default
n: TICF_0n (MSQ) or
0
TICF_n (aQa)
(n = number of the
TICF channel
configured in the Server
Configuration)
2000.00 amu (MSQ)
1636.0 amu (aQa)
Usage
Selects the filter for extraction.
The filter indexes correspond to
the nine MSQ filters or the four
aQa filters that can be defined
in the MS method.
0 = NoFilter means that the
¾TIC channel is used.
Minimum mass of interval that
will be extracted.
538
Actions Related to Mass Spectrometers
Parameter
Min.
Max.
MaxMass
0.00 amu
(MSQ)
2.0 amu
(aQa)
2000.00 amu (MSQ)
1636.0 amu (aQa)
Default
Usage
Maximum mass of interval that
will be extracted.
These parameters cannot be set using the Program Wizard. Therefore,
follow the steps below:
•
Open the PGM File.
•
Select Command on the Control menu to open the Commands dialog
box.
•
Select your ¾Mass Spectrometer (listed by the name defined in the
Server Configuration).
•
Open the mass channel to be extracted; for example, MS_01.
•
Specify the individual parameters:
Use the Upper/Lower Limit parameters to specify the signal limits.
You can also extract the trace of the ¾Base Peak. In this case, verify that
the BasePeakMode property is set to Yes.
Actions Related to Mass Spectrometers
539
Tip:
Do not change the mass trace settings during a run. This might result in
confusion. Therefore, we recommend not entering a retention time.
The corresponding section in the ¾Program could look as follows:
MS_01.MinMass =
149.5
MS_01.MaxMass =
150.5
MS_01.FilterIndex = 1
Tip:
To start data acquisition, manually enter the following command for the
MS_01 channel:
MS_01.AcqOn
To stop data acquisition for the MS_01 channel, enter:
MS_01.AcqOff
You can display these channels on the ¾Control Panel online during data
acquisition.
Extracting Mass Traces Afterward
If you did not extract a ¾Mass Trace online during data acquisition (see
Extracting Mass Traces Online), you can do this afterward as well:
Tip:
To view a mass trace before finally extracting it, extract a temporary mass
trace first (see
Extracting a Temporary Mass Trace).
1. To extract a mass trace, open the ¾Mass Spectrum from the
Integration plot or the ⇒QNT Editor.
Tip:
You may also define mass trace extraction in the Post-acquisition
steps view of the PGM Editor (see Control
The PGM Editor). Open
the PGM File in which you want to define mass trace extraction and
then, select the Post-acquisition steps view.
540
Actions Related to Mass Spectrometers
2. Select Extract Mass Trace… on the context menu to open the Extract
Mass Trace dialog box:
Tip:
In the PGM Editor, follow the steps below: Select Insert line on the
context menu to add a new post-acquisition step. The New postacquisition step dialog box is opened. Select Extract MS channel to
open the Extract Mass Trace dialog box.
3. Select the desired filter and mass range, as well as the ¾Smoothing
type for the MS chromatograms and the number of data points to be
used and the type of the mass trace to be extracted.
4. In the Channel Name box, enter the name for the new channel or
accept the default name. Chromeleon creates the suggested name from
the Filter Index, the Mass, if indicated, the Smoothing information, and
the Trace Type for the mass trace to be extracted.
Note:
If the Trace Type is ¾TIC, Chromeleon does not consider the mass (or
the mass range if < 1.00 amu) for the channel name.
Actions Related to Mass Spectrometers
541
5. To extract the mass trace for all samples in the sequence or ¾Query,
select the Apply to all samples in the current sequence or query
check box.
Tip:
This option is not provided in the Extract Mass Trace dialog box of the
PGM Editor.
6. Click Extract to make the new channel available for chromatogram
representation.
If you know prior to data acquisition which channels you will need, you can
omit this step and record the required channels right from the beginning
(see the Administrator Manual: How to ...: Actions Related to Mass
Spectrometers
Defining the Number of MS Channels) or extract
them as described above.
Note:
If you need help identifying found masses, refer to How to ...: Actions
Related to Mass Spectrometers
SIM Mass Lists for IC-MS.
Extracting a Temporary Mass Trace
Before you extract a ¾Mass Trace (see
Extracting Mass Traces
Afterward), you can create it temporarily by just clicking the mouse. The
following options are available:
Using the mouse cursor
Cursor
Activated by
What it does
Placing the cursor near a
mass data point or a mass
needle.
A temporary MS channel is extracted for the
current mass. An existing temporary MS
channel will be overwritten.
Placing the cursor near a
mass data point or a mass
needle and simultaneously
pressing the Shift key.
A temporary MS channel is extracted for the
current mass. The new channel will overlay
existing temporary MS channels.
Placing the cursor between
two mass range delimiters.
This action moves the mass range as desired.
Placing the cursor on or near
a mass range delimiter.
This action moves the left or right delimiter in
the desired direction.
542
Actions Related to Mass Spectrometers
On the context menu
To extract a mass trace, click the corresponding mass or right-click the
mass range. On the context menu, select Extract Temporary Mass Trace
and define the corresponding parameters in the dialog box:
Showing Mass Spectra
The ¾Mass Spectra view can be added to almost all Chromeleon plots
(Integration, ⇒QNT Editor, Printer Layout) if ¾Xcalibur is installed. To
enable the mass spectra view click the following icon:
Or else, select Show Mass Spectra on the View menu:
Actions Related to Mass Spectrometers
543
To add a mass spectrum to the ¾Printer Layout, enable Layout Mode on
the Edit menu. Select Insert on the View or context menu and then, select
Mass Spectra Plot. The appearance of the displayed mass spectra may
be quite different, depending on which the MS instrument method was
used. For more information, refer to How to …: Actions Related to Mass
Spectrometers
Creating a PGM File for the aQa MS or
Creating a
PGM File for the MSQ. For information about which the parameters should
be used in this method to receive a certain mass spectrum, refer to the
Xcalibur help.
In the captions of the single mass spectra plots, the peak name (if the mass
spectrum of a peak is given) plus the retention time of the mass spectrum is
given on the left. On the right, the acquisition mode is given: Full ms
indicates ¾Full-Scan mode; SIM ms indicates ¾SIM mode. In the caption
of full-scan mass spectra, the entire mass range is given in parentheses on
the right. The fragmentation voltage that is given in front of the mode is
important, as well.
With mass spectra that were acquired in SIM mode, no entire mass spectra
are available. These SIM mass spectra are extracted from single mass
traces, so the resulting SIM mass spectrum usually shows gaps between
the single traces. In the caption, the single mass ranges are given together
with the corresponding fragmentation voltage behind the respective mass
range (following a @ sign) on the right.
How to set the MS specific parameters of the view
Place the cursor on the mass spectrum and right-click to open the context
menu. Select Decoration, go to the MS Filter tab page, and then make the
desired settings:
544
Actions Related to Mass Spectrometers
Minimizing the Noise of Mass Spectra
¾Mass Spectra usually include more details than UV spectra. However,
they often have an increase noise level. Especially with low signal intensity,
they are considerably affected by the background spectrum. To use the
information of mass spectra in the best possible way, we recommend that
you reprocess them as described below. There a two ways:
Spectra Bunching
To reduce mass spectra noise, you can bunch several single mass spectra
to one entire mass spectrum. Spectra bunching can be performed for both
peak spectra and retention time spectra.
Use the MS tab page of the ⇒QNT Editor to bunch several single spectra
to the left and the right of the chromatogram peak together with the peak
maximum spectrum to one entire peak spectrum.
In the chromatogram, define the range for which to display the entire
retention time spectrum. Use the Spectra Tool to select the desired
range while left clicking. You can perform this in a UV channel as well.
Subtracting Background Mass Spectra
Background subtraction of mass spectra eliminates the influence of the
background on the mass spectra. The background mass spectrum to be
subtracted can be defined either for the entire chromatogram or
automatically by Chromeleon for each single peak.
The corresponding setting is made on the MS tab page of the ⇒QNT Editor
or in the chromatogram. The settings made in the chromatogram are saved
to the QNT File of the current sample as well. Thus, your input affects all
samples that are evaluated using this QNT File.
For more information, refer to How to …:
Actions in the QNT Editor
Processing Mass Traces
Actions in the Chromatogram
Subtracting MS Background Spectra
Actions Related to Mass Spectrometers
545
Tracking the Effects of Background Subtraction
You can track the effects of spectra subtraction directly on the mass
spectra plot:
•
Select Decoration on the context menu to open the mass spectrum
decoration and then the select the Peak Spectra tab page.
•
In the Background Subtraction Overlay section, select the
Background Spectrum to display the subtracted background mass
spectrum in addition to the peak and retention time spectra.
•
In addition, select the Original Spectra to display the respective mass
spectrum without subtraction.
Defining Further QNT Settings for MS
Handling the Retention Time Delay as against a second detector
Use the ¾Delay Time option on the General tab page to take the retention
time difference into account that is due to the time needed by the
substances to travel from the first detector, for example, to the MSQ
¾Mass Spectrometer.
For more information, refer to How to ...: Actions in the QNT Editor
Defining the QNT Method for Several Detectors in the Creating a
Peak Table section.
Defining Peaks via Mass Spectra
For peak identification via mass spectra, use the six MS columns:
•
Mass Peak 1 (as well as Mass Peak 2 and Mass Peak 3)
•
MS threshold
•
MS filter conditions
•
Check MS retention times
For more information, refer to How to ...: Actions in the QNT Editor
Identifying Peaks via Their Mass Spectra (MS Tracking) Detectors.
546
Actions Related to Mass Spectrometers
Actions Related to Fraction Collection
547
Actions Related to Fraction Collection
¾Fraction Collectors are often used for fraction collection after the detector.
Fractionation can be automated by the online interpretation of the signal.
On the one hand, fractions can be collected for preparative use. However,
often it is also intended to analyze the single fractions more exactly after
they have been collected. In both cases, you can collect the fractions based
on the signal. Signal-based fraction collection is quite complex, but the
Fraction Collection driver provided by Chromeleon facilitates the process.
In the Server Configuration program, install the Fraction Collection driver in
addition to the device driver for your fraction collector.
Tip:
The Fraction Collection driver also allows you to collect fractions,
independent of the signal, with a fixed volume at predefined retention
times.
For more information about fraction collection, refer to:
Setting up Fraction Collection
PGM Wizard: Fraction Collection - General Options
Setting the Peak Detection Parameters
Checking the Fraction Collection Status on the Control Panel
Recognizing the Peak Start, Peak Maximum, and Peak End
Defining the Reactions to Certain Events
Tracking Fraction Collection in the Chromatogram
Tracking Fraction Collection in the Report
Program Example (One Detection Channel)
Program Example (Two Detection Channels)
Fraction Collection Control via an MS
Fraction Collection Control via an MS for Different Samples
548
Actions Related to Fraction Collection
Tip:
The Fraction Collection Automation driver from earlier Chromeleon
versions is obsolete and included in the distribution for compatibility
reasons only. We recommend that you use the new Fraction Collection
driver instead.
Setting up Fraction Collection
Before you can collect fractions, you have to set up fraction collection as
follows:
Server Configuration
Install the Fraction Collection driver in the desired timebase. (Select Add
Device on the context menu, select General from the left list box, and then
select Fraction Collection from the right list box.) In addition, install the
¾Device Driver for the respective fraction collector.
Determine how to collect the fractions, either in the Program Wizard (see
Programmed Control
The Program Wizard) or on a ¾Control Panel.
On a control panel, select Command... on the Control menu and then
select the required settings under Fraction Collection.
Actions Related to Fraction Collection
549
Program Wizard
The Program Wizard assists you in creating a ¾Program for fraction
collection control:
•
On the Fraction Collection - General Options page, determine the
PGM Wizard: Fraction Collection fraction collection period (see
General Options).
•
On the Fraction Collection Options page, define the fraction
collection control parameters.
•
On the Fraction Collection - Channel Selection Options page,
determine the channel(s) for peak recognition during fraction collection.
•
On the Peak Detection Options page, specify the peak detection
Setting the Peak Detection
algorithm for fraction collection (see
Parameters).
Standard Program Example
If you have not yet saved a FractionCollectionTemplate.pgm, the PGM
Wizard creates the following standard program. (The program only includes
the default fraction collection commands for a single detection channel plus
the standard commands.)
Collect =
On
CollectOutsidePeaks =
No
;**************************************************************
;* Definition of triggers for fraction collection starts here.
;**************************************************************
;
Definitions
copied
from
template
<Timebase>
\FractionCollectionTemplate!
Trigger
FracStart
FracStartDetected
EndTrigger
Trigger
TubeChange
FracTubeChange
EndTrigger
Trigger
FracEnd
FracEndDetected
EndTrigger
;**************************************************************
;* Definition of triggers for fraction collection ends here.
;**************************************************************
PumpDevice =
"Pump"
TubeMaxVolume =
Unlimited
TotalNumberInstalled =
100
MaxTubesPerFraction =
Unlimited
TubeWrapping =
No
DelayTime =
0.0
OffsetTime =
0.0
DetectionChannel1.Name =
"UV_VIS_1"
550
Actions Related to Fraction Collection
DetectionChannel1.PeakStartSlope =
DetectionChannel1.PeakStartThreshold =
DetectionChannel1.PeakMaxSlope =
DetectionChannel1.PeakEndSlope =
DetectionChannel1.PeakEndThreshold =
DetectionChannel1.ThresholdNoPeakEnd =
DetectionChannel1.BaselineOffset =
DetectionChannel1.BaselineDrift =
Flow = 1.00
%B = 0.0
%C = 0.0
%D = 0.0
0.500
10.00
0.000
-1.000
10.00
2000.000
0.000
0.000
0.000
Wait Frac.Ready and Sampler.Ready
Inject
3DFIELD.AcqOn
UV_VIS_1.AcqOn
10.000
3DFIELD.AcqOff
UV_VIS_1.AcqOff
10.100
End
;wait for all fraction events
Note:
To make sure that the fractions are collected exactly, you have to enter
and/or consider the actual delay time or the actual delay volume.
For more complex program examples, refer to
Program Example (One
Program Example (Two Detection Channels),
Detection Channel),
Fraction Collection via an MS.
and
For an overview of the fraction collection topics, refer to How to …:
Actions Related to Fraction Collection.
Actions Related to Fraction Collection
551
PGM Wizard: Fraction Collection - General Options
Use the Program Wizard: Fraction Collection - General Options page
The Program Wizard) to determine the
(see Programmed Control
time range for fraction collection:
When you click Next>, more fraction collection Wizard pages are
displayed. It depends on the settings made on this page, which pages are
displayed and which program commands are generated:
Never
Select this option to disable fraction collection. In this case, no other
fraction collection Wizard pages are displayed. The program includes the
following commands:
Collect = Off
CollectOutsidePeaks = No
Always
Select this option to collect fractions over the entire chromatogram. The
program looks as follows:
Collect = On
CollectOutsidePeaks = No ;(or Yes - see Note below)
;*******************************************************************
;* Definition of triggers for fraction collection starts here.
;*******************************************************************
; Definitions copied from <Timebase>\FractionCollectionTemplate!
Trigger FracStart
FracStartDetected
EndTrigger
Trigger TubeChange FracTubeChange
EndTrigger
Trigger FracEnd FracEndDetected
EndTrigger
;*******************************************************************
;* Definition of triggers for fraction collection ends here.
;*******************************************************************
552
Actions Related to Fraction Collection
Note:
It is also possible to collect fractions outside peaks (CollectOutsidePeaks =
Yes). To do so, select the Collect outside peaks check box.
Restricted time range
Select this option to collect fractions during a specified period only. For
example, for a time range from 2.000 to 8.000 min, the following commands
are included in the program:
CollectFractions = No
CollectOutsidePeaks = No
;*******************************************************************
;* Definition of triggers for fraction collection starts here.
;*******************************************************************
; Definitions copied from <Timebase>\FractionCollectionTemplate!
Trigger FracStart
FracStartDetected
EndTrigger
Trigger TubeChange FracTubeChange
EndTrigger
Trigger FracEnd FracEndDetected
EndTrigger
;********************************************************************
;* Definition of triggers for fraction collection ends here.
;*******************************************************************
2.000
Collect = On
CollectOutsidePeaks = Yes ;(or No - see Note below)
8.000
Collect = Off
CollectOutsidePeaks = No
Note:
It is also possible to collect fractions outside peaks (CollectOutsidePeaks =
Yes). To do so, select the Collect outside peaks check box.
Restricted time range & user-defined columns
You can use ¾User-defined Columns in the sample list, to define when
fraction collection shall start and when it shall stop:
•
Create two user-defined columns (see How to ...: Actions in the
Browser
Creating User-defined Columns) in the ¾Standard
Datasource of the server.
Actions Related to Fraction Collection
553
•
Name the two columns, e.g., FractionStart and FractionEnd:
•
Restart the server.
•
On the Fraction Collection - General Options pages, select the
Restricted time range from option. In the from field, select
FractionStart; in the to field, select FractionEnd. The following
triggers are generated:
Trigger FractCollectionOn Sample.FractionStart < System.Retention,
True=0.0,Hysteresis=0.0
Collect=
On
CollectOutsidePeaks =
Yes
EndTrigger
Trigger FractCollectionOff Sample.FractionEnd
True=0.0,Hysteresis=0.0
Collect=
On
CollectOutsidePeaks =
No
EndTrigger
<
System.Retention,
This program part starts fraction collection at the retention time entered in
the *FractionStart column for the corresponding sample. Fraction
collection is stopped at the retention time from the *FractionEnd column.
554
Actions Related to Fraction Collection
Setting the Peak Detection Parameters
The Fraction Collection driver supports the parameters described in the
tables below. They are available independently of the installed ¾Fraction
Collector. Use these parameters to start fraction collection in the ¾PGM
File or on the ¾Control Panel.
Press the F8 key in the PGM File or on the control panel to open the
Commands dialog box, and then enter the desired parameters. You can
also establish controls with the desired functionality. (For more information,
refer to How to ...: Actions on the Control Panel
Modifying a Control
Panel.)
The tables below list the parameters in the order in which they appear in
the Commands dialog box. (For information about the read-only
parameters, refer to
Checking the Fraction Collection Status on the
Control Panel.)
Signal-Based Parameters (Min., Max., and Default depend on the UV
detector; here for a UV detector)
Parameter
Min.
Max.
Default
Description
Name
N/a
n/a
UV_VIS_1
Name of the signal channel
used for peak detection (this
may be a ¾Virtual Signal, also).
OffsetTime
0.0 s
9999.9 s
0.0 s
Retention time offsets between
the first detector and other
detectors
OffsetVolume
0.0 µl
5000.0 µl
0.0 µl
Offset volume between the first
detector and other detectors.
(Based on the flow, the offset
volume is converted into the
corresponding OffsetTime.)
PeakStartThreshold
-1e10
1e10
10.00
The actual signal value must
exceed the PeakStartThreshold
value for the peak start to be
recognized.
PeakStartSlope
0.000
1e10
0.500
The actual slope value must
exceed the PeakStartSlope
value for the peak start to be
recognized.
PeakMaxSlope
-1e10
1e10
0.000
The actual slope value must be
below the PeakMaxSlope value
for the peak maximum to be
recognized.
Actions Related to Fraction Collection
555
Parameter
Min.
Max.
Default
Description
PeakEndThreshold
-1e10
1e10
10.00
The actual signal value must be
below the PeakEndThreshold
value for the peak end to be
recognized.
PeakEndSlope
-1e10
0.000
-1.000
The slope value must exceed
the PeakEndSlope value for the
peak end to be recognized.
ThresholdNoPeakEnd
-1e10
1e10
2000
The actual signal threshold
value must be below the
ThresholdNoPeakEnd value for
the peak end to be recognized.
(This parameter supports the
PeakEndSlope parameter for
peak detection.)
BaselineOffset
-1e10
1e10
0.0000
Offset of the baseline used to
correct the signal value (see
BaselineDrift).
BaselineDrift
-1e10
1e10
0.0000
Drift of the baseline used to
correct the signal value. The
specified thresholds
(PeakStartThreshold,
PeakEndThreshold, and
ThresholdNoPeakEnd) are
compared to:
signal value - (BaselineOffset +
∆t*BaselineDrift)
∆t is set to 0 whenever
BaselineDrift changes.
Baseline Offset is then set to
the current value of the
correction term. Thus, the
BaselineDrift value always
matches the actual drift.
Basic Parameters (directly under Fraction Collection)
Parameter
Min.
Max.
Default
Description
Collect
Off
On
No
Peak detection and thus,
fraction collection is
performed only if the
variable is set to Yes.
Note: Detected fractions
will trigger related events
after the delay time, even if
Collect is Off at that time.
CollectOutsidePeaks
No
Yes
No
Determine whether
fractions are collected
outside peaks, also.
556
Actions Related to Fraction Collection
Parameter
Min.
Max.
Default
Description
¾DelayTime
0.0 s
9999.9 s
0.0 s
Delay time between the
first detector and the
¾Fraction Collector
DelayVolume
0.0 µl
5000.0 µl
0.0 µl
Delay volume between the
first detector and the
fraction collector. (Based
on the flow, the delay
volume is converted into
the
corresponding
DelayTime.)
Pump
Pump whose flow is used
to convert between time
and volume.
PumpDevice
TubePosition
1
9999
1
Position of the tube in the
current fraction.
TubeMaxVolume
0.000000
1000.000000
Unlimited
Maximum filling volume of
the tube in [ml]. If the
specified
volume
is
reached, the next tube is
filled.
Tip:
Set the TubeMaxVolume
parameter to Unlimited to
prevent the next tube from
being filled automatically.
MaxTubesPerFraction
0
99
Unlimited
Maximum number of tubes
per fraction. If the specified
number is reached, no
additional tubes are used
to collect the current peak.
Set this parameter to
Unlimited to collect all
peaks completely.
TotalNumberInstalled
0
9999
100
Number of tubes installed
in the rack. If this number
is
exceeded,
the
TubePosition parameter is
reset
to
1
if
the
TubeWrapping option has
been selected. Otherwise,
the batch is terminated.
Tip:
A warning appears in the
Audit Trail when the
TubePosition parameter is
reset to 1.
Actions Related to Fraction Collection
557
Parameter
Min.
Max.
Default
Description
TubeWrapping
No
Yes
No
If this option is selected,
the
TubePosition
parameter is reset to 1
when
TotalNumberInstalled
is
reached.
ChannelEvaluation
All
Any
All
Indicates how the results
of the single detection
channels are used for peak
detection.
These properties cannot be changed during a peak, that is, while PeakOn
is set to Yes. Any attempted change will produce the following warning:
"Parameters cannot be changed until the current peak has ended. New
value will be assigned at peak end." The change will become effective as
soon as the current peak ends.
In addition to these parameters, various read-only variables are available.
These variables are only displayed if you open the Commands dialog box
on a control panel. They allow you to check the peak detection status (see
Checking the Fraction Collection Status on the Control Panel).
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
Checking the Fraction Collection Status on the Control
Panel
The Fraction Collection driver supports the various status variables that
allow you to check the fraction collection status on a ¾Control Panel. The
variables are read-only. They are available independently of the installed
¾Fraction Collector.
On the control panel, create display elements indicating the status of the
desired parameter. You can use a Color Box, String Display, Gauge
Indicator, or a Lamp. (For more information, refer to How to ...: Action on
the Control Panel
Modifying a Control Panel and the following
topics.)
The tables below list the parameters in the order in which they appear in
the dialog box. (For information about the parameters for active fraction
collection control, refer to
Setting the Peak Detection Parameters.)
558
Actions Related to Fraction Collection
Parameters for the Single Channels
Status Variable
Description
PeakStartDetected
Yes when the peak start is detected. When the peak end is detected,
the variable is reset to No.
The Delay Time does not delay a change.
PeakMaxDetected
Yes when the peak maximum is detected. When the peak end is
detected, the variable is reset to No.
(Not delayed)
PeakEndDetected
Yes when the peak end is detected. When a new peak start is
detected, the variable is reset to No.
(Not delayed)
PeakOn
Yes when a peak start or a peak maximum is detected.
(Not delayed)
PeakOnDelayed
Yes when a peak start or a peak maximum is detected.
(Delayed)
Parameters for the Fraction Collection Driver
Parameter
Description
FracStartDetected
Set to Yes when a fraction start is detected. When the fraction end
is detected, the variable is reset to No.
(Delayed)
FracEndDetected
Set to Yes when a fraction start is detected. When the fraction end
is detected, the variable is reset to No.
(Delayed)
FracTubeChange
Set to Yes when a filled tube is detected. The variable is
automatically reset to No.
(Delayed)
FractionOn
Set to Yes during fraction collection.
(Corresponds to PeakOn, but delayed)
FractionOnUndelayed
Set to Yes during fraction collection.
(Corresponds to FractionOn, but not delayed)
TubeCntCurrentFracD
Number of filled tubes in the current fraction.
(Delayed)
TubeFilling
Volume collected in the current tube.
(Delayed)
In addition to these status variables, various parameters are provided for
fraction collection control. For more information about these parameters,
Setting the Peak Detection Parameters.
refer to
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
Actions Related to Fraction Collection
559
Recognizing the Peak Start, Peak Maximum, and Peak
End
To ensure that the ¾Fraction Collector fills the desired tube, the peak start,
peak maximum, and peak end all must be correctly recognized.
Peak Start
The prerequisites for recognizing the peak start (and hence, the beginning
of a fraction) are as follows:
•
No peak start has been recognized so far.
•
Fraction collection, and thus peak detection, is enabled via the Collect
property.
•
The signal of the detection channel is greater than the signal defined by
PeakStartThreshold.
•
The signal slope is greater than the slope defined by PeakStartSlope.
•
Data acquisition on the selected channel is not yet completed.
•
The run itself is not completed yet
•
The condition is fulfilled for at least 1 second.
The PeakStartThreshold and PeakStartSlope variables influence the
peak recognition sensitivity.
The PeakStartSlope variable can be changed within broad limits. The
higher the value, the later the peak start is recognized. If the detector signal
exceeds the PeakStartThreshold, the peak is recognized only if the slope
threshold value is also exceeded.
Chromeleon remembers a peak start so that a peak maximum can be
recognized next. Thus, a peak maximum can be recognized only if a peak
start has been detected before.
Peak Maximum
The peak maximum is recognized only if
•
A peak start has been detected before.
•
No peak maximum has been detected yet.
•
The signal slope is greater than the signal slope defined by
PeakMaxSlope.
•
The condition is fulfilled for at least 1 second.
560
Actions Related to Fraction Collection
A peak maximum can be recognized only if a peak start has been detected
before. Thus, if no peak start has been detected, no maximum will be
recognized either. The PeakMaxSlope variable is defined as negative
slope value as it applies to the tailing side of the peak. The closer the value
is to zero, the closer to the peak maximum the PeakMaxRecognition
⇒Trigger will be executed.
Chromeleon remembers a peak maximum so that a peak end can be
recognized next. Thus, a peak end can be recognized only if a peak
maximum has been detected before (unless one of the end-run conditions
apply).
Peak End
A peak end (and hence the end of a fraction) is recognized if:
•
The signal slope is less than the signal slope defined by
PeakEndSlope and
•
The signal of the detection channel is less than the signal defined by
TresholdNoPeakEnd and
•
A peak maximum has been recognized
OR:
•
The signal of the detection channel is less than the signal defined by
PeakEndThreshold and
•
A peak maximum has been recognized
OR:
•
A peak start has been recognized and
•
Data acquisition on the signal channel has been finished
AND:
•
The condition is fulfilled for at least 1 second.
The first group of conditions checks whether the signal is below the signal
maximum, which is defined by the ThresholdNoPeakEnd variable. With
heavily overloaded detector signals, there is a lot of signal noise so that a
peak end and/or start would be recognized several times near the top of the
peak. The top of the peak could also be formed like a plateau. To inhibit
this set the ThresholdNoPeakEnd variable to a value below this level. If
the value is set to the maximum for the detector signal, this part of the
Actions Related to Fraction Collection
561
condition will always be true so that this check will be disabled.
PeakEndSlope delays the peak end.
The second group of conditions uses the signal height criterion. If the signal
value falls below PeakEndThreshold, the peak is completed in any case.
The third group of conditions completes the current peak in case the data
acquisition is disabled.
For more information, refer to
Events.
Defining the Reactions to Certain
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
Defining the Reactions to Certain Events
A peak start, peak maximum, and peak end are recognized if certain
Recognizing Peak Start, Peak Maximum,
conditions are fulfilled (see
and Peak End). If a peak start or peak end has been recognized on a
channel, the system does not necessarily start or stop collecting a fraction.
Whether the system starts or stops collecting, depends on the setting of the
Channel Evaluation parameter:
Option
Description
All
(Default) The system starts fraction collection when a peak start is detected in all
detection channels. And:
The system stops fraction collection when the end of the first peak is detected.
Any
The system starts fraction collection when a peak start is detected in at least one
detection channel. The system stops fraction collection when the end of the last
peak is detected.
If one of these events occurs, Chromeleon issues certain commands to the
¾Fraction Collector. These commands can be defined in the ¾Program. In
the respective ⇒Trigger block, you can define device-specific actions for
your fraction collector.
Event
Condition
Action
FracStartDetected
A peak start, which means
the start of a new fraction,
"arrives" at the switching
valve/ tube.
Switch to next fraction.*
Switch to collect.
562
Actions Related to Fraction Collection
Event
Condition
Action
FracEndDetected
A peak end, which means the
end of a fraction, "arrives" at
the switching valve/ tube.
Switch to waste.
Switch to next fraction.*
FracTubeChange
The tube is filled to the limit.
Switch to next tube.
*
Switching to the next fraction is needed only once. It depends on the
type of hardware whether this is better to switch to the first tube of the
next fraction at the peak start or at the peak end.
The events are generated with a delay that takes the volume between the
detector and the switching valve or tube into account. The following
parameters are available:
Parameter
Min.
Max.
Default
Function
DelayTime
0s
9999 s
0
Time delay between the detector and the
switching valve or tube.
This parameter cannot be used for flow
gradients.
The driver is capable of tracking several peaks in the capillary between the
detector and the fraction collector as it internally maintains a queue of peak
recognition events.
Tip:
Do not change the DelayTime and/or PumpDevice while collecting
fractions. Usually this does not make sense either. Performing such a
change in the program, for example, when a part of the peak is in the
capillary between the detector and the fraction collector, will lead to
unexpected results.
For more information, refer to
Maximum, and Peak End.
Recognizing the Peak Start, Peak
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
Actions Related to Fraction Collection
563
Tracking Fraction Collection in the Chromatogram
After you have collected fractions during the analysis, it is important that
you can later track which substances have been collected in which tube.
This is possible in Chromeleon:
•
In the chromatogram
•
In the report (see
Tracking Fraction Collection in the Report.)
In the chromatogram, you can display which fractions have been collected
at which time:
You can adapt the appearance of the display according to your
requirements. Select Decoration... on the context menu. The
Chromatogram Decoration dialog box appears.
564
Actions Related to Fraction Collection
On the Fractions tab page, select the desired options and thus, determine
how the fractions are displayed in the chromatogram:
Note:
The settings in the picture correspond to the settings used for the above
chromatogram.
In the Fraction Visualization section, determine how the fractions shall be
displayed. Usually, it makes sense to select the Vertical stripes and Fill
stripes options. In this way, you can display the exact time assignment of
the single peaks to the corresponding fractions.
In the lower section, determine how the fractions shall be labeled. In order
to label fractions, select the Label Fractions check box first, and then
determine the label in the Formula input field. The following formula has
proved a good choice:
F{frac.number} ({frac.tube(1).position}-{frac.tube(frac.nTubes).position})
The corresponding label could then read, e.g., "F2 (37-38)". This means
that the second fraction was collected in the tubes 37 and 38. (Note: To
mark the single tubes in the chromatogram by dotted lines, select the Mark
tubes inside fraction or Mark tubes outside fraction check box.) The 'F'
in the above formula serves to distinguish fraction labels from peak labels.
Actions Related to Fraction Collection
565
Tip:
It is also possible to track fraction collection in the chromatogram during
data acquisition. However, in this case, several complex settings are
required that must be tailored to the individual installation. A description of
these settings goes beyond the scope of this online Help. Therefore, if you
need more information, please contact your local Dionex representative.
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
Tracking Fraction Collection in the Report
You can display fraction collection information in the report at any time. The
easiest way is to insert one of the two predefined report tables:
•
Double-click to open the desired sequence, and then display the report.
•
On the Table menu, select Insert Report.
•
The Insert Report Table dialog box appears. Click the '+' characters in
front of Result Tables and Fraction Collection Results to display the
reports underneath.
•
Select one of the two reports:
•
Select the Fraction Report to display an overview of the fractions.
566
•
Actions Related to Fraction Collection
Select Tube Report to add an overview of the single tubes to the
report:
The picture shows a standard tube report that was created with the default
settings. To insert additional columns:
•
Select the column in front of which you want to insert the new column.
•
Select Insert Column on the context menu. The Insert Report
Column dialog box appears.
•
Select one of the variables provided in the Fraction and Fraction
Tube report categories. These categories provide the variables shown
in the first picture plus some more.
•
To access the variables of the Fraction Detection Parameter
category, select the Channel Parameter variable of the Fraction
category.
For a list of all available variables, refer to the following topics in the
Reference Manual:
⇒'Fraction' Category
⇒'Fraction Tube' Category
⇒'Fraction Detection Parameter' Category
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
Actions Related to Fraction Collection
567
Program Example (One Detection Channel)
A ¾Program for a timebase including a ¾Fraction Collector and one
detection channel might look as follows:
-0.300 Flow =
%B =
UV_VIS_1.Step =
UV_VIS_1.Average =
UV_VIS_1.MaxAutoStep =
Pressure.LowerLimit =
Pressure.UpperLimit =
20.000
10
0.50
On
1.0
10.00
200.00
;Basic fraction collection parameters:
Collect =
CollectOutsidePeaks =
Off
No
;********************************************************************
;*Definition of triggers for fraction collection starts here
;********************************************************************
;
Definition
copied
from
template
<Timebase>\FractionCollectionTemplate!
Trigger FracStart FracStartDetected
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
Collect =
On
;Switches the fraction collector valve to "Collect" (= on)
EndTrigger
Trigger TubeChange FracTubeChange
Collect =
Off
;Turns off the fraction collector valve
Tube =
TubePosition
;Selects the next tube
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
Collect =
On
;Switches the fraction collector valve to "Collect" (= on)
EndTrigger
Trigger FracEnd FracEndDetcted
Collect =
Off
;Turns off the fraction collector valve
Tube =
TubePosition
;Selects the next tube
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
EndTrigger
;********************************************************************
;*Definition of triggers for fraction collection ends here
;********************************************************************
568
Actions Related to Fraction Collection
;Maximum filling volume of a tube in ml:
PumpDevice =
TubeMaxVolume =
"Pump"
10
;Maximum number of installed fraction tubes:
TotalNumberInstalled =
240
;Basic fraction collection parameters:
MaxTubesPerFraction =
TubeWrapping =
Unlimited
No
;Delay time between detector output and switching valve or tube:
DelayTime =
0.4
OffsetTime =
0.0
;Conditions for online peak recognition at program start:
Name =
"UV_VIS_1"
;Slope at peak start:
PeakStartSlope =
2.000
;Minimum signal height at peak start:
PeakStartThreshold =
10.00
;Slope after the peak maximum:
PeakMaxSlope =
-4.000
;Slope at peak end:
PeakEndSlope =
-4.000
;Maximum signal height at peak end:
PeakEndThreshold =
5.00
;Signal must be less than this threshold value
;before a new peak start can be recognized:
ThresholdNoPeakEnd =
200
;Baseline drift correction:
BaselineOffset =
0.000
BaselineDrift =
0.000
;Separation start with injection:
-0.100 UV.Autozero
0.000 Wait
Sampler.Ready
Flow =
20.000
%B =
10
Sampler.Inject
UV_VIS_1.AcqOn
;Gradient program:
Flow =
%B =
10.000 %B =
13.000 %B =
13.000
20.000
10.0
90
90
UV_VIS_1.AcqOff
;Regeneration and equilibration phase of the gradient program:
15.000 Flow =
20.000
%B =
90
16.000 %B =
End
10
Actions Related to Fraction Collection
569
Note:
The trigger block used in this program example refers to a preparative
application of the Gilson 215 Liquid Handler whose relay has been named
Collect. Besides, the trigger block is intended for a preparative pump.
For more program examples, refer to:
Program Example (Two Detection Channels)
Fraction Collection Control via an MS
Fraction Collection Control via an MS for Different Samples
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
Program Example (Two Detection Channels)
A ¾Program for a timebase including a ¾Fraction Collector and two
detection channels might look as follows:
-0.300 Flow =
%B =
UV_VIS_1.Step =
UV_VIS_1.Average =
UV_VIS_1.MaxAutoStep =
Pressure.LowerLimit =
Pressure.UpperLimit =
20.000
10
0.50
On
1.0
10.00
200.00
;Basic fraction collection parameters:
Collect =
ChannelEvaluation =
CollectOutsidePeaks =
Off
Any
No
;********************************************************************
;*Definition of triggers for fraction collection starts here
;********************************************************************
;Definition
copied
from
template
<Timebase>\FractionCollectionTemplate!
Trigger FracStart FracStartDetected
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
Collect =
On
;Switches the fraction collector valve to "Collect" (= on)
EndTrigger
Trigger TubeChange FracTubeChange
Collect =
;Turns off the fraction collector valve
Off
570
Actions Related to Fraction Collection
Tube =
TubePosition
;Selects the next tube
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
Collect =
Off
;Turns on the fraction collector valve
EndTrigger
Trigger FracEnd FracEndDetected
Collect =
On
;Turns off the fraction collector valve
Tube =
TubePosition
;Selects the next tube
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
EndTrigger
;********************************************************************
;*Definition of triggers for fraction collection ends here
;********************************************************************
;Maximum filling volume of a tube in ml:
PumpDevice =
TubeMaxVolume =
"Pump"
10
;Maximum number of installed tubes:
TotalNumberInstalled =
240
;Basic fraction collection parameters:
MaxTubesPerFraction =
TubeWrapping =
Unlimited
No
;Delay time between detector output and switching valve or tube:
DelayTime =
0.8
DetectionChannel2.OffsetTime =
0.0
DetectionChannel3.OffsetTime =
0.0
;Conditions for online peak recognition at program start:
DetectionChannel2.Name =
"UV_VIS_1"
;Slope at peak start:
DetectionChannel2.PeakStartSlope =
2.000
;Minimum signal height at peak start:
DetectionChannel2.PeakStartThreshold =
10.00
;Slope after peak maximum:
DetectionChannel2.PeakMaxSlope =
-4.000
;Slope at peak end:
DetectionChannel2.PeakEndSlope =
-4.000
;Maximum signal height at peak end:
DetectionChannel2.PeakEndThreshold =
5.00
;Signal must be less than this threshold value
;before a new peak start can be recognized
DetectionChannel2.ThresholdNoPeakEnd =
200
;Baseline drift correction:
DetectionChannel2.BaselineOffset =
0.000
DetectionChannel2.BaselineDrift =
0.000
;Conditions for online peak recognition for the 2. detection channel:
Actions Related to Fraction Collection
DetectionChannel3.Name =
DetectionChannel3.PeakStartSlope =
DetectionChannel3.PeakStartThreshold =
DetectionChannel3.PeakMaxSlope =
DetectionChannel3.PeakEndSlope =
DetectionChannel3.PeakEndThreshold =
DetectionChannel3.ThresholdNoPeakEnd =
DetectionChannel3.BaselineOffset =
DetectionChannel3.BaselineDrift =
571
"UV_VIS_2"
2.000
10.00
-5.000
-4.000
5.00
200
0.000
0.000
;Separation start with injection:
-0.100 UV.Autozero
0.000 Wait
Ready
Flow =
20.000
%B =
10
Sampler.Inject
UV_VIS_1.AcqOn
;Gradient Program:
Flow =
%B =
10.000 %B =
13.000 %B =
13.000
20.000
10.0
90
90
UV_VIS_1.AcqOff
;Regeneration and equilibration phase of the gradient program:
15.000 Flow =
20.000
%B =
90
16.000 %B =
End
10
Note:
The trigger block used in this program example refers to a preparative
application of the Gilson 215 Liquid Handler whose relay has been named
Collect. Besides, the trigger block is intended for a preparative pump. In
the example, DetectionChannel1 has not been used as the first detection
channel because '1' and 'I' look identical when Courier is the selected font.
For more program examples, refer to:
Program Example (One Detection Channel)
Fraction Collection Control via an MS
Fraction Collection Control via an MS for Different Samples
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
572
Actions Related to Fraction Collection
Fraction Collection Control via an MS
¾Mass Traces can be used to selectively collect certain substances in
fractions. This selective collection is possible since the occurrence of peaks
with data acquisition via ¾Mass Spectrometers depends much more
selectively on the corresponding masses than it depends on the
wavelength with ¾UV Detectors.
It is possible to use certain mass traces at specific times to ¾trigger the
fraction collection. For this purpose, online extraction of mass traces is
required (see How to ...: Actions Related to Mass Spectrometers
Extracting Mass Traces Online). The following program requires five
channels for online extraction of mass traces:
;Definition of the minimum/maximum mass and of the filter index
;for the channels MS_01 to MS_05:
MS_01.MinMass =
243.5
MS_01.MaxMass =
244.5
MS_01.FilterIndex =
2
MS_02.MinMass =
MS_02.MaxMass =
MS_02.FilterIndex =
145.5
146.5
2
MS_03.MinMass =
MS_03.MaxMass =
MS_03.FilterIndex =
164.5
165.5
2
MS_04.MinMass =
MS_04.MaxMass =
MS_04.FilterIndex =
178.5
179.5
2
MS_05.MinMass =
MS_05.MaxMass =
MS_05.FilterIndex =
192.5
193.5
2
;Basic fraction collection parameters:
Collect =
ChannelEvaluation =
CollectOutsidePeaks =
Off
Any
No
;********************************************************************
;*Definition of triggers for fraction collection starts here
;********************************************************************
;Definition
copied
from
template
<Timebase>\FractionCollectionTemplate!
Trigger PeakStart FracStartDetected
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
Collect =
On
;Switches the collection valve to "Collect" (= on)
EndTrigger
Actions Related to Fraction Collection
573
Trigger TubeChange FracTubeChange
Collect =
Off
;Turns off the fraction collector valve
Tube =
TubePosition
;Selects the next tube
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
Collect =
On
;Switches the collection valve to "Collect" (= on)
EndTrigger
Trigger PeakEnd FracEndDetected
Collect =
Off
;Turns off the collection valve
Tube =
TubePosition
;Selects the next tube
Z.Nominal =
Z.Nominal
;Moves the needle to the upper zero position
EndTrigger
;********************************************************************
;*Definition of triggers for fraction collection ends here
;********************************************************************
;Maximum filling volume of a tube in ml:
PumpDevice =
TubeMaxVolume =
"Pump"
10
;Maximum number of installed fraction tubes:
TotalNumberInstalled =
240
;Basic fraction collection parameters:
MaxTubesPerFraction =
TubeWrapping =
Unlimited
No
;Delay time between detector output and switching valve or tube:
DelayTime =
0.8
DetectionChannel2.OffsetTime =
0.0
DetectionChannel3.OffsetTime =
0.4
;Conditions for online peak recognition at program start:
Collect =
On
DetectionChannelN2.Name =
"UV_VIS_01"
;Slope at peak start [mAU/s]:
DetectionChannelN2.Name PeakStartSlope =
2.000
;Minimum signal height at peak start [mAU]
DetectionChannelN2.Name PeakStartThreshold =
5000.00
;Slope after peak maximum [mAU/s]
DetectionChannelN2.Name PeakMaxSlope =
-5.000
;Slope at peak end [mAU/s]
DetectionChannelN2.Name PeakEndSlope =
-4.000
;Maximum signal height at peak end [mAU]
DetectionChannelN2.Name PeakEndThreshold =
5000.00
;Signal mst be less than this threshold value
;before a new peak start can be recognized [mAU]
574
Actions Related to Fraction Collection
DetectionChannelN2.Name ThresholdNoPeakEnd =
;Baseline drift correction:
DetectionChannel2.BaselineOffset =
DetectionChannel2.BaselineDrift =
10000
0.000
0.000
;Conditions for online peak recognition for the MS detection channel:
DetectionChannel3.Name =
"MS_01"
DetectionChannel3.PeakStartSlope =
2.000
DetectionChannel3.PeakStartThreshold =
10.00
DetectionChannel3.PeakMaxSlope =
-5.000
DetectionChannel3.PeakEndSlope =
-4.000
DetectionChannel3.PeakEndThreshold =
5.00
DetectionChannel3.ThresholdNoPeakEnd =
200
DetectionChannel3.BaselineOffset =
0.000
DetectionChannel3.BaselineDrift =
0.000
;Separation start with injection and acqusition start
0.000 UV.Autozero
Wait Sampler.Ready and MS.Ready
Inject
3DFIELD.AcqOn
UV_VIS_1.AcqOn
MS_01.AcqOn
MS_02.AcqOn
MS_03.AcqOn
MS_04.AcqOn
MS_05.AcqOn
;Change of the detection channel according to the expected substances
3.20
DetectionChannel3 =
"MS_02"
3.7
DetectionChannel3 =
"MS_03"
4.3
DetectionChannel3 =
"MS_04"
5.5
DetectionChannel3 =
"MS_05"
;End of data acquisition
10.000
3DFIELD.AcqOff
UV_VIS_1.AcqOff
MS_01.AcqOff
MS_02.AcqOff
MS_03.AcqOff
MS_04.AcqOff
MS_05.AcqOff
End
Note:
The trigger block used in this program example refers to the Gilson 215
Liquid Handler. In the example, DetectionChannel1 has not been used as
the first detection channel because '1' and 'I' look identical when Courier is
the selected font.
Actions Related to Fraction Collection
575
If the ¾Program added below is used, fractions would be collected at the
following retention times provided a peak occurs within the following mass
traces:
Retention timeinterval [min]
Channel
Mass
[amu]
Positions of the
collection vials
0.000 - 3.200
MS_01
244
1
3.200 - 3.700
MS_02
146
2
3.700 - 4.200
MS_03
165
3
4.200 - 5.500
MS_04
179
4
5.500 - 10.000
MS_05
193
5
(Usually, the desired mass peak will occur only once within the
corresponding time interval - this is assumed in the above table. Otherwise,
the positions of the collection vials will change accordingly).
This program example for mass-controlled fraction collection presumes that
the same substances shall be collected in all samples. If there are different
substances in the samples, fraction collection via a mass channel needs to
be adapted accordingly.
For more information, refer to
Fraction Collection Control via an MS for Different Samples.
For more program examples, refer to:
Program Example (One Detection Channel)
Program Example (Two Detection Channels)
For an overview of the fraction collection topics, refer to
Actions Related to Fraction Collection.
How to …:
576
Actions Related to Fraction Collection
Fraction Collection Control via an MS for Different
Samples
If there are samples with different substances, the procedure described in
Fraction Collection Control via an MS has to be changed accordingly.
In this case, ¾Mass Traces have to be extracted online, as well (see How
Extracting Mass
to ...: Actions Related to Mass Spectrometers
Traces Online).
1. Define User-defined Columns
Define the ¾User-defined Columns Mass1 to Mass5 and FilterIndex to
FilterIndex5 as Integer columns (see How to ...: Actions in the Browser
Creating User-defined Columns). With the program part quoted
below, you read out the content of the corresponding user-defined columns
during data acquisition and thereby control fraction collection ¾Triggering.
2. Modify the Program
In the example described in Fraction Collection via an MS (see above),
replace the corresponding paragraph in the ¾Program with the following
program part:
;Definition of the minimum/maximum mass and of the filter index
;for the channels MS_01 to MS_05:
MS_01.MinMass =
sample.mass1-0.5
MS_01.MaxMass =
sample.mass1+0.5
MS_01.FilterIndex = sample.filterindex
MS_02.MinMass =
sample.mass2-0.5
MS_02.MaxMass =
sample.mass2+0.5
MS_02.FilterIndex = sample.filterindex2
MS_03.MinMass =
sample.mass3-0.5
MS_03.MaxMass =
sample.mass3+0.5
MS_03.FilterIndex = sample.filterindex3
MS_04.MinMass =
sample.mass4-0.5
MS_04.MaxMass =
sample.mass4+0.5
MS_04.FilterIndex = sample.filterindex4
MS_05.MinMass =
sample.mass5-0.5
MS_05.MaxMass =
sample.mass5+0.5
MS_05.FilterIndex = sample.filterindex5
Actions Related to Fraction Collection
577
3. Enter the Values in Sample List
Finally, enter the corresponding values of the single samples into each
column of the sample table:
In this example, the following mass traces are relevant for controlling the
¾Fraction Collector at the corresponding samples:
Retention time
interval [min]
Channel
Standards
Mass [amu]
Sample 1 /2
Mass [amu]
Sample3/4
Mass [amu]
0.000 - 3.200
MS_01
149 ± 0,5
176 ± 0,5
163 ± 0,5
3.200 - 3.700
MS_02
197 ± 0,5
153 ± 0,5
215 ± 0,5
3.700 - 4.200
MS_03
275 ± 0,5
235 ± 0,5
179 ± 0,5
Note:
The retention times refer to the program example described in
Collection Control via an MS (see above).
Fraction
For an overview of the fraction collection topics, refer to
Related to Fraction Collection.
Actions
578
Actions Related to Fraction Collection
Actions in the Virtual Column Separation Simulator
579
Actions in the Virtual Column
Separation Simulator
To use ¾Virtual Column to simulate retention data and chromatograms,
you first select the analysis parameters (analytes of interest, methodology,
column, etc.). Virtual Column then calculates retention data for the selected
parameters, and displays the resulting Resolution Response Surface and
Virtual Chromatogram.
Note:
Results obtained with Virtual Column are intended only to represent typical
results for a particular column type. Because no two columns or systems
are identical, the results you obtain in an actual analysis may differ
somewhat from the Virtual Column predictions.
Starting Virtual Column
In the Chromeleon Browser, select Virtual Column on the Tools menu.
Tip:
You can open more than one Virtual Column window.
Using Virtual Column (Overview)
1. Select an analyte category.
2. Select two or more analytes.
3. Select a column.
Resolution
4. View
the
Response Surface (a plot of
the lowest resolution values
found at each eluent
condition).
5. View
the
Chromatogram.
Virtual
Tip:
Right-click on the Resolution
Response Surface to select a
different resolution criterion.
580
Actions in the Virtual Column Separation Simulator
For details about how to use Virtual Column, refer to:
Selecting the Analysis Parameters
Viewing the Results Table
Viewing the Resolution Response Surface
Viewing the Virtual Chromatogram
Selecting a Resolution Criterion
Finding the Fastest Chromatogram
Finding the Global Optimum
Selecting Product Test Conditions
Manually Selecting an Eluent Condition
Saving and Reloading Virtual Column Settings
Note:
Parts of the Virtual Column software were developed jointly by the
Australian Centre for Research on Separation Science (ACROSS) at the
University of Tasmania, Australia and Dionex Corporation.
Selecting the Analysis Parameters
The left side of the ¾Virtual Column window provides controls for selecting
the desired analysis parameters.
Tip:
To clear selections and return the Virtual Column window to the initial blank
state, click Reset All.
Actions in the Virtual Column Separation Simulator
1. Select an Analyte Category.
To begin a Virtual Column
simulation,
first
select
the
Analyte Category. The table on
the Select Analytes tab lists all
analytes in the selected category
for which embedded data are
available. The example shows
the list when Anions is the
selected category.
2. Select the analytes.
Select the check box for each
analyte to be included in the
Virtual Chromatogram.
Tip:
If you want Virtual Column to
calculate retention data for the
void dip, or if you want to change
its Peak Area, Asymmetry, or
Plates values, select the Void
Dip check box. If the check box is
not selected, the void dip is
displayed
on
the
Virtual
Chromatogram, but retention and
resolution data are not calculated
for it.
3. (Optional) Select a methodology.
After you select two or more
analytes, the methodologies and
columns
available
for
the
selection are displayed. If more
than one methodology is shown,
you can restrict the column
choices by clearing the highlight
from the methodologies you do
not want to use.
581
582
Actions in the Virtual Column Separation Simulator
4. (Optional) Select a column diameter.
To restrict the column choices to
a particular diameter category,
clear the check box for the
diameter of column (Standard
Bore or Micro Bore) you do not
want to use.
5. Select a column.
Select a column from the list.
Virtual
Column
calculates
retention and resolution data for
the selected parameters and
displays the resulting Resolution
Response Surface and Virtual
Chromatogram.
The following example shows the plot and chromatogram for Fluoride,
Chloride, Nitrite, Sulfate, Bromide, and Nitrate, when the AS18 column
is selected.
Actions in the Virtual Column Separation Simulator
583
6. (Optional) Select a Temperature from the list.
For some columns, you can select a different temperature to view the
effect changing the temperature has on the Virtual Chromatogram.
7. (Optional) Enter Peak Area, Asymmetry, and Theoretical Plate
Values
After you select the analytes and choose a column, the Select Analytes
table displays the default ¾Peak Area, ¾Asymmetry, and ¾Theoretical
Plates values for each selected analyte. The values displayed were
obtained from the experimental data embedded in Virtual Column. If you
have data specific for your system, you can enter those values in the
table. Virtual Column uses the new values in retention and resolution
calculations.
After entering the values, you can save them to a file for later use (see
How to ...:
Saving and Reloading Virtual Column Settings).
584
Actions in the Virtual Column Separation Simulator
8. (Optional) Change the Flow Rate, Void Volume, and Void Time
After you select the analytes and choose a column, a default flow rate,
void volume (also called ¾Dead Volume), and void time (also called
¾Dead Time) for the selected column is displayed. You can change the
flow rate and/or void volume values to more accurately model your
specific system. Changing either value affects the void time (void time =
void volume/flow rate).
Note:
If you change the flow rate in Virtual Column, the peak shapes on the
virtual chromatogram are not affected. However, under actual operating
conditions, peak shapes are affected by changes in the flow rate.
9. (Optional) View the retention data.
To view the calculated retention data, click the Display Results tab.
See How to ...: Actions in the Virtual Column Tool
Viewing the
Results Table for details.
Viewing the Results Table
After you select the analytes and choose a column, click the Display
Results tab to view the calculated retention data. The results table lists the
calculated results (Retention Time, Retention Factor, and Resolution)
for each selected analyte. The analytes are listed in order of ascending
retention time. Results are calculated based on the analyte data (peak
area, asymmetry, and theoretical plates) and on the selected column,
temperature, void time, and eluent conditions.
Actions in the Virtual Column Separation Simulator
585
•
¾Retention Time is the time (in minutes) since injection.
•
¾Retention Factor (also called Capacity Factor) is the ratio of the net
retention time to the void time (also called ¾Dead Time).
•
¾Resolution is the degree of separation between the current peak and
the next peak in the chromatogram.
Viewing the Resolution Response Surface
The Resolution Response Surface is a plot of the lowest resolution values
found for the Virtual Chromatogram at each possible eluent condition.
Specific features of the plot vary depending on which resolution criterion is
Selecting a Resolution Criterion) and on
selected (see How to ...:
whether the eluent is a single- or dual-species type.
Single-Species Eluents
When a single-species eluent is used, the Resolution Response Surface is
represented by a line plot. In this example, the Minimum Resolution
criterion is selected.
586
Actions in the Virtual Column Separation Simulator
Dual-Species Eluents
When a dual-species eluent is used, the Resolution Response Surface is
represented by a contour plot. In this example, the Minimum Resolution
criterion is selected.
Viewing the Virtual Chromatogram
The Virtual Chromatogram simulates an actual analysis using the currently
selected analytes, column, void time, temperature, resolution criterion, and
eluent condition. The Virtual Chromatogram is updated whenever the
selected analytes or other parameters are changed.
The figure below describes the features of the Virtual Chromatogram. In
this example, the Minimum Resolution criterion is selected.
Actions in the Virtual Column Separation Simulator
587
Zooming/Unzooming
To zoom into an area of the chromatogram, press the left-mouse button
and drag to form a box around the area.
To return to the previous view, right-click and select Unzoom, or select Full
Size to display the full chromatogram in the pane.
588
Actions in the Virtual Column Separation Simulator
Selecting a Resolution Criterion
¾Virtual Column provides three options for determining the optimal eluent
condition for peak resolution:
•
Minimum Resolution
•
Normalized Resolution Product
•
Resolution Optimized for Analyte
To select a resolution criterion, right-click on the Resolution Response
Surface and select a criterion on the menu, or select on the Criterion
menu on the Chromeleon menu bar.
The sections below describe each resolution criterion. Examples illustrate
how each criterion affects the same Virtual Chromatogram. For the
examples, the AS18 column was selected and the following list of anions
was used: Bromide, Carbonate, Chloride, Fluoride, Nitrate, Nitrite, and
Sulfate. Note: Carbonate was added to the list to better illustrate the
differences among the criteria.
Actions in the Virtual Column Separation Simulator
589
Minimum Resolution
When Minimum Resolution is selected, Virtual Column finds the least
resolved peak pair for the selected eluent condition. The resolution of the
entire chromatogram for that eluent condition is defined as the resolution of
the least resolved peak pair. All other peak pairs of higher resolution are
ignored. Minimum Resolution is the default criterion. This option is useful
for difficult separations because it optimizes the resolution of any peak
pairs that are hard to separate.
In general, a resolution value of at least 1.5 (peak areas overlap less than
0.2%) is regarded as good baseline separation. For many applications, a
value of 1.2 (peak areas overlap less than 2%) is considered an acceptable
separation. A value of 0 indicates that at least two peaks are eluting at the
same retention time.
Normalized Resolution Product
When Normalized Resolution Product is selected, Virtual Column finds
the eluent condition that provides the most evenly spaced peaks across the
entire chromatogram. A normalized resolution product value of 1 indicates
that all peaks are evenly resolved across the chromatogram. A value of 0
indicates that at least one peak pair is co-eluting. Normalized Resolution
Product is useful for easy separations, as it optimizes the resolution of all
peak pairs. However, for more difficult separations, Normalized Resolution
590
Actions in the Virtual Column Separation Simulator
Product may find a chromatogram with evenly spaced peaks, but the peaks
may not all be resolved.
The normalized resolution product (r) is defined by the following equation:
⎛
⎞
⎜
⎟
R
si ,i +1
⎜
⎟
r =∏
n −1
⎜
⎟
1
i =1
Rsi ,i +1 ⎟
⎜
∑
⎝ n − 1 i =1
⎠
n −1
where n is the number of peaks and
i+1.
R s i , i +1
is the resolution of peaks i and
Notice that with this example, when the Normalized Resolution Product
option is selected, the Carbonate/Nitrite peak pair is no longer resolved.
Resolution Optimized for Analyte
When Resolution Optimized for Analyte is selected, Virtual Column finds
the eluent condition that optimizes the resolution of a selected peak. Virtual
Column finds the best resolution for the selected analyte. The resolution of
the other peaks is not taken into account. Resolution Optimized for
Analyte is useful if resolving a particular analyte's peak is more critical than
resolving all other peak pairs.
Actions in the Virtual Column Separation Simulator
591
In this example, Chloride is the selected analyte (indicated by the asterisk
next to the Resolution value in the Display Results table and the blue
markers on the peak). Notice that with this option, the Carbonate/Bromide
and Nitrate/Sulfate peaks are not resolved.
Tip:
When Resolution Optimized for Analyte is selected, you can select a
different analyte to be optimized by double-clicking the analyte peak on the
Virtual Chromatogram.
592
Actions in the Virtual Column Separation Simulator
Finding the Fastest Chromatogram
¾Virtual Column provides an option that lets you optimize a separation for
speed rather than for peak resolution. To select this option, right-click on
the Resolution Response Surface and select Find Fastest
Chromatogram on the menu, or select the option on the Criterion menu
on the Chromeleon menu bar. Enter the minimum acceptable resolution
and click OK.
Virtual Column finds the eluent condition that gives the fastest
chromatogram that satisfies the currently selected resolution criterion.
The example below shows the results of a Find Fastest Chromatogram
command. The minimum acceptable resolution entered was 1.5. Notice that
the eluent concentration selection bar was moved from 24.912 to 29.459
mM and the chromatogram time was reduced from 11.8 to 10.5 minutes.
Actions in the Virtual Column Separation Simulator
593
Finding the Global Optimum
The ¾Virtual Column Find Global Optimum option finds the eluent
composition that gives the maximum value for the selected resolution
Selecting a Resolution Criterion).. To select
criterion (see How to ...:
this option, right-click on the Resolution Response Surface and select
Find Global Optimum on the menu, or select the option on the Criterion
menu on the Chromeleon menu bar. This option is the default when you
select a column or resolution criterion.
594
Actions in the Virtual Column Separation Simulator
Selecting Product Test Conditions
The ¾Virtual Column Product Test Conditions option sets the eluent
composition to that specified in the production control test performed on
every column before it is shipped. For details about the production test
conditions, refer to the column manual. To select this option, right-click on
the Resolution Response Surface and select Product Test Conditions
on the menu, or select the option on the Criterion menu.
Note:
The Product Test Conditions option only changes the eluent composition;
it does not affect the analyte selections. If the production test
chromatogram includes more analytes than those selected for display in
Virtual Column, the missing analytes are not automatically added to the
Virtual Chromatogram.
Manually Selecting an Eluent Condition
To determine the optimum eluent condition, you can select an option (Find
Fastest Chromatogram, Global Optimum, or Product Test Conditions)
on the ¾Virtual Column Criterion menu, or you can manually select the
eluent condition on the Resolution Response Surface.
To manually select the eluent condition if you are working with a singlespecies eluent, drag the single vertical bar to the desired eluent
concentration (see the example below), or click on the plot where you want
to move the bar. The virtual chromatogram is updated to reflect the new
concentration.
Actions in the Virtual Column Separation Simulator
595
If you are working with a dual-species eluent, two eluent selection bars are
provided: one for concentration and one for percent carbonate. You can
move both bars together or drag each individually. To move both bars, click
on the Resolution Response Surface at the desired eluent composition, or
point to the intersection of the two bars, wait for the cursor to change to a
four-directional arrow and then drag the bars. See the following example.
To move only one of the bars, point to the bar, wait for the pointer to
change to a two-directional arrow and drag the bar to the desired location.
Drag the horizontal bar to change the percentage of carbonate (see the
example below) or drag the vertical bar to change the total concentration.
596
Actions in the Virtual Column Separation Simulator
Saving and Reloading Virtual Column Settings
After you have selected analytes and a column, you can save the selected
settings to a file to be available for later use. The following information is
saved and can be reloaded from the saved file:
•
Each analyte's name, peak area, asymmetry, and theoretical plates
•
Column name and size
•
Temperature
•
System void time
When you reload the saved file, the settings are restored and the
corresponding Resolution Response Surface and Virtual Chromatogram
are displayed.
To save settings:
Select Save on the File menu. The saved file is assigned a .vcol extension.
Tip:
Virtual Column settings files are saved in the ..\My Documents\Virtual
Column Custom Files folder.
To reload a saved file:
1. Select Open on the File menu.
2. Select a .vcol file and click OK.
The Load Selected File dialog box opens. This dialog box displays the
analyte data saved in the selected .vcol file and gives you the
opportunity to choose the data from the saved file that you want loaded
into the Virtual Column analyte table.
3. Clear the check box above any column (Peak Area, Asymmetry, or
Plates) that you do not want loaded.
4. Clear the Void Time check box if you do not want the void time loaded.
Note:
If you clear a check box in the Load Selected File dialog box, Virtual
Column loads data for the item from the embedded Virtual Column
database instead of from the saved file.
5. Click OK.
Device Control
597
Device Control
Device Control (Overview)
In addition to the standard commands of the ¾Program, which are easily
The
created with the Program Wizard (see Programmed Control
Program Wizard), Chromeleon supports various additional ⇒Control
Commands.
For more information about these commands and the command syntax,
refer to:
Control: Pumps
Control: Autosamplers
Control: Detectors
Control: IC
Control: GC and Temperature
Control: Component Controller
Control: Special Commands, Relays, and Others
•
Transfer the structure and syntax of the commands that you want to
use to your program file.
•
Use Cut & Paste to install the command directly from online Help at
the appropriate position in the standard program.
•
Specify the time when to execute each command.
•
Save the result of your input as a PGM File.
598
Device Control
Control: Pumps
The following commands are available for pump control. For the "simple"
Pump Commands.
commands, refer to
For more information, refer to:
Setting the Flow Rate
Determining the Solvent Composition
Determining a Gradient
Determining Pressure and Pressure Limits
Starting/Stopping the Pump Flow
Holding the Pump Flow
In addition, refer to the following information about
Recording the Pump Pressure
Setting Automatic Pre-Compression Control (P580)
Viewing Leak Sensor and Workload Status.
Parking Peaks
Pump Commands
The following commands are supported for pump control:
Hold/Continue Gradient
0.000
HoldMode
= On / Off
Alternatively, the following short command is valid:
0.000
⇒Hold
0.000
⇒Continue
or
Device Control
599
Stop Pump Flow/Gradient
0.000
0.000
StopMode
= On
or
⇒StopFlow
Determining Pressure Limits (see ⇒Pressure.Lower/UpperLimit)
0.000
0.000
Pressure.LowerLimit
Pressure.UpperLimit
= Value[bar, MPa, psi]
= Value[bar, MPa, psi]
Note:
The pressure unit (bar, MPa, or psi) depends on the pump type.
Generating a Flow or % Gradient
The flow or solvent value determined for the time t is continually adjusted to
the following flow or solvent command. If the two values coincide, the flow
or solvent value is kept at a specific level (see ramp profile 0 to 1), if they
differ from each other, the value is modified. The difference between the
two time values corresponds to the length of the ramp (see ramp profile 1
to 2min).
0.000
1.000
2.000
3.000
%B.Value
%B.Value
%B.Value
%B.Value
=
=
=
=
20
20
50
50
Ramp Profile
Tip:
Dionex GP40/GP50, IP20/IP25, IC20/IC25/IC25A, GS50, and IS25 pumps
do not deliver flow gradient ramps. Instead, changing the flow rate between
one time value and the next results in an immediate change in the flow rate
(step change).
600
Device Control
To determine a sharp increase, two different values must be defined at the
same time. The start and end values must be entered at the exact times
(see rectangle profile at the time t=1min).
0.000
1.000
1.000
2.000
2.000
3.000
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
=
=
=
=
=
=
20
20
40
40
20
20
Rectangle Profile
By combining the just mentioned possibilities, any multi-step gradient
profile can be realized. For example:
0.000
0.500
1.500
2.000
2.000
2.500
2.500
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
=
=
=
=
=
=
=
20
20
40
40
30
30
20
Multi-Step Profile
Instead of the arbitrarily selected quantity %B used in these examples, any
other solvent (see ⇒%B, %C, %D) or the flow rate can be changed.
Creating a Non-Linear Gradient Ramp (Dionex GP40/GP50/GS50
pumps only)
A Curve command (also, refer to ¾Gradient Curves) instructs the pump to
apply the selected curve number when adjusting the solvent composition
between two retention times. In the example below, the Curve = 8
command at 2 min creates a concave ramp between 1 and 2 min.
Consecutive commands with identical solvent compositions generate an
isocratic segment, regardless of the curve number selected (see 0 to 1 min
and 2 to 3 min below).
0.000
1.000
2.000
3.000
%B.Value
%B.Value
%B.Value
Curve
%B.Value
=
=
=
=
=
20
20
50
8
50
Non-Linear Ramp Profile
%B
50
20
t
0
1
2
3
Device Control
601
Setting the Flow Rate
For non-controlled pumps, the ⇒Flow rate is set directly on the instrument
(also, refer to the Operating Instructions of the instrument). For controlled
pumps, the flow rate can be set via the corresponding controls on the
control panel (slider, edit field, or button). For more information about the
The Control Panel.
control panel, see Control
•
To set a higher or lower flow rate, use the mouse to move the gauge
slider in the desired direction.
•
Enter the desired flow rate value in the edit field via the keyboard.
Confirm your input by pressing <Enter> (Return).
•
Click a button to execute the assigned function.
•
Alternatively, select Flow on the Control menu.
It is also possible to include the Flow command in the ¾Program (see How
Creating a Program). Example:
to ...: Actions in the PGM Editor
0.000
0.000
Pump.Flow
InjectorB.flow
= Value[ml/min%]
= Value[ml/min%]
;HPLC/IC pump
;GC HP5890
Several Flow commands result in a ¾Flow Gradient. For a description on
how to enter gradients, refer to How to ...: Device Control
Determining a Gradient.
Determining the Solvent Composition
Manually
For non-controlled pumps, solvent composition (see ⇒%B, %C, %D) is set
directly on the instrument (also, refer to the Operating Instructions of the
instrument). For controlled pumps, Chromeleon features the appropriate
controls (sliders, edit fields, switches, etc.). Depending on the control panel
layout, you can set the values for %B, %C, and %D via either a slider or an
edit field.
•
Use the mouse to move the slider of a gauge in the desired direction.
•
Type a value in an edit field and press <Enter>.
•
To determine a gradient, select ⇒Flow on the Control menu (also, see
Determining a Gradient).
How to ...: Device Control
602
Device Control
Programmed
To change the solvent composition via the program, enter the following
commands in the ¾Program:
t=....
t=....
t=....
%B.Value
%C.Value
%D.Value
= Value[%]
= Value[%]
= Value[%]
If you enter the command several times for at least one component, this
input results in a ¾% Gradient.
For more information, refer to How to ...: Actions in the PGM Editor
Creating a Program.
Determining a Gradient
There are two ways how to determine a gradient. You can do this either
manually on a control panel or automatically via a program:
Manual Input
Different dialog boxes are provided for each device for which a gradient can
be determined. Use these dialog boxes to enter the device-specific
settings. You can determine:
•
⇒Flow and ¾% gradients for HPLC and IC pumps.
•
Temperature, pressure, and flow gradients for gas chromatographs.
A gradient can be run either as a ramp or as a ¾Step Gradients (in IC, also
as a ¾Gradient Curve).
•
Select Flow on the Control menu or press <Ctrl> + <F>. Make the
necessary settings in the dialog box.
•
For HPLC/IC, determine the desired gradient on the Gradient tab
page. For gas chromatographs, select the desired gradient on the GC
and Column1 and Column2 tab pages.
Device Control
603
Programmed Input
The easiest way to create a new program is to use the Program Wizard
(see Programmed Control
The Program Wizard). To open the wizard,
select New on the File menu, and then select Program File.
HPLC/IC
On the Pump Options page, select Multi-Step Gradient. The
Flow Gradient Options dialog box appears. This dialog box
corresponds to the Gradient page during manual input.
GC
On the GC Options page (or the Front/Back Inlet Options
tabs), click the arrow of the Type field, and then select Ramped
Temperature from the list. This setting allows you to program a
temperature gradient. In the same way, you can program flow
and pressure gradients. Select Ramped Flow or Ramped
Pressure on the Column1 Options and Column2 Options
pages.
Entering "Basic points"
HPLC/IC
•
In the Retention column, enter the time (relative to the injection time)
of the desired modification.
•
In the Flow column, enter the new value for the flow rate.
•
In the %B, %C, and/or %D columns, enter the new value for the
delivered solvent composition. %A is the difference between the total of
the entered values and 100%.
•
For Dionex GP40, GP50, and GS50 pumps only: Enter a value in the
Curve column to determine whether the pump delivers a linear or
¾Curved Gradient. Curve 5 (the default) is linear; curves 1 - 4 are
convex upward and curves 6 - 9 are concave upward.
•
Click Insert Line to append another line to the table. After appending
the first new line, further lines are appended automatically, as
necessary. Enable or disable this option by selecting or deselecting
Autom. Append New Lines on the context menu.
•
Click Delete Line to remove unnecessary lines from the table.
•
If an entry is wrong, the input in the corresponding cell is displayed in
red color. Invalid entries must be corrected immediately.
604
Device Control
•
Click Clean Up to delete superfluous information from a previously
selected area.
•
To calculate missing values between two or more time values can, click
Interpolate. The values are automatically added to the table.
•
Select Fill Column on the context menu to fill the cells in the column
with the currently selected value. Only the cells below the selected
value are filled.
In a graphical representation, each flow and solvent value of the value table
is represented as a point. By interpolation between the basic points of a
column, an area gradient profile is created. The solvent areas are
represented in the color of the corresponding caption; the flow rate is
displayed as a thin line.
Direct Input into a Program
If you know the ¾Program syntax, you can directly determine the gradient
in the program. Entering the ⇒%B, %C, %D, ⇒Flow, and Temp.
commands in the program allows you to change the solvent composition,
flow rate, and temperature at a precise time. The gradient profile results
from the change in value of a certain quantity at the time t.
Tip:
The Dionex GP40/GP50, IP20/IP25, IC20/IC25/IC25A, GS50, and IS25
pumps do not deliver flow gradient ramps. Instead, changing the flow rate
between one time value and the next, results in an immediate change in
the flow rate (step change).
Example:
The three different profiles are realized via the commands
listed below the profiles.
Rectangle Profile
0.000
1.000
1.000
2.000
2.000
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
=
=
=
=
=
Ramp Profile
20
20
40
40
20
0.000 %B.Value = 20
1.000 %B.Value = 20
2.000 %B.Value = 50
Multi-Step Profile
0.000
0.500
1.500
2.000
2.000
2.500
2.500
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
%B.Value
=
=
=
=
=
=
=
20
20
40
40
30
30
20
Device Control
605
For a rectangle profile, indicate exactly how long the specific solvent
composition is valid. If the composition is changed at the same time as
another percent command is defined, the change in solvent composition is
executed immediately. The solvent percentage achieved is then maintained
until replaced by another command.
To realize an increase over a longer period, the start value and end value
must be specified with the precise time. The difference between the two
time values corresponds to the duration of the increase (see ramp profile).
You can realize any multi-step gradient profile by combining the abovementioned possibilities.
For more information about how to enter basic points in GC, refer to
Determining a Gradient (GC).
Determining Pressure and Pressure Limits
Pressure
The operating pressure can only be determined for gas chromatographs. It
depends on the layout of the used control panel which control realizes this
setting.
•
Determine the pressure via the corresponding slider or type the value
in the input field.
Pressure Limits
Some device drivers provide the possibility to determine the upper and
lower pressure limits.
•
Type the corresponding value in the input field.
Or
•
Position the slider for the upper and lower pressure limits with the
mouse cursor. Assign different colors to the controlling sliders so that
they indicate whether a pressure limit is exceeded or whether it is
within the selected limits (see How to ...: Actions on the Control
Modifying a Control).
Panel
606
Device Control
If defined ⇒Pressure Limits, for example, of an HPLC pump, are exceeded,
Chromeleon automatically turns off the flow, issues an error message, and
stops the sample batch, as necessary. In addition, all operations are logged
in the ¾Audit Trail.
It is also possible to determine the pressure limits by entering the following
commands in the ¾Program:
0.000
0.000
X.Pressure.LowerLimit
X.Pressure.UpperLimit
=Value[bar, MPa, psi]
=Value[bar, MPa, psi]
where X refers to the name of the instrument defined in the Server
Configuration. The pressure unit depends on the pump type.
Starting and Stopping the Pump Flow
•
Select ⇒StopFlow to stop the pump flow and the ¾Gradient formation.
During a running ¾Batch, batch processing is stopped.
•
Select ⇒Continue to resume the pump flow, a running gradient
program, or batch processing.
Tip:
The ⇒Hold command interrupts gradient formation and automatic batch
processing, but not the pump flow.
Holding the Pump Flow
•
Select ⇒Hold to interrupt ¾Gradient formation or a running batch.
During a running ¾Batch, batch processing is stopped.
In Hold mode, the pump delivers a constant flow rate and solvent
composition until a stop or a ⇒Continue command is entered.
Device Control
607
Recording the Pump Pressure
To determine whether signal variations are related to pressure variations,
that is, whether the pump causes those variations, we recommend that you
record the pump pressure as an additional signal.
Hardware Configuration
To display the pump pressure, an analog pressure output must be available
on your pump. In addition, a ¾UCI-100 Universal Chromatography
Interface is required. Connect the pressure output of your pump to one of
the free channels of the UCI-100.
Tip:
If you do not have a UCI-100 installed, a virtual channel can be used to
record the pump pressure. If you use a virtual channel, note the greater
distance between the measured values (⇒Step). For an example, refer to
Program Examples for Virtual Channels.
How to …: Device Control
Server Configuration
P680:
On the General tab page, select the Pressure Signal check box to record
the pump pressure. Chromeleon generates the Pump_Pressure channel for
data acquisition.
Other Pumps:
Add an ¾Integrator Driver to the devices of the corresponding timebase.
Name the unassigned signal, for example, Pump_Pressure, and click
Change.
608
Device Control
Select the channel to which the pressure output of your pump is connected
as AD Port. Enter under Factor how many bar correspond to an output
voltage of 1 mV. For example, enter 0.5 bar/mV (0.5 bar = 50 kPa = 7.25
psi) for the Dionex P580 pump or 5 bar/mV (5 bar = 500 kPa = 72.5 psi) for
the Dionex M480 pump.
You can now record data for the Pump_Pressure channel. Data
acquisition can also be defined in the ¾PGM File or by selecting ⇒AcqOn
on the Control menu on the ¾Control Panel.
Panel
•
When you are on the signal plot of your panel, select Signals on the
context menu.
•
Select the desired signal in the Available Signals field (that is, for the
above example, select Pressure).
•
When starting the data acquisition, select the Pump_Pressure channel
Starting
and define the ⇒Step (see How to ...: Device Control
Data Acquisition).
Device Control
609
Setting Automatic Pre-Compression Control (P580)
The better the pump is set to the varying compressibility of the different
components of the solvents, the lower the pump's pulsation. Automatic precompression control of the Dionex P580 pump considers the varying
compressibility of different solvents. Automatic pre-compression control can
also be used for unknown solvents.
With low-pressure gradients and isocratic pumps, pre-compression control
is fully automatic. On the ¾Control Panel, select Commands on the
Control menu. Select the pump and the solvent components one after the
other and assign the component type Automatic.
The high-pressure gradient pumps must "learn" the automatic precompression control. Select Commands on the Control menu. Select the
pump and the solvent component. Assign the solvent type Custom. Deliver
100% of this solvent at 1 ml/min and a backpressure of approximately
100 bar (= 10 MPa = 1450 psi). Select Commands on the Control menu
and issue the ⇒Learn command. Observe the pressure signal for at least
10 minutes. Issue the corresponding pump ⇒Freeze command to save the
optimum pre-compression setting when the pressure fluctuations from the
pump are minimal.
Tip:
During the Learn phase, the backpressure should correspond to the
maximum pressure in normal operation. If the backpressure is less than
25°bar (= 2.5 MPa = 362.5 psi) during this phase, pre-compression control
cannot be set correctly! Increase the backpressure, for example, by
installing a second column or a longer capillary before the detector. Again,
wait for approximately 10 minutes before saving the pre-compression value
using the Freeze command.
Change the flow to 100% of the next solvent and set the pre-compression
control as described above.
For a detailed description, refer to the P580 Operating Instructions.
610
Device Control
Viewing Leak Sensor and Workload Status
The Dionex P580 pump allows you to display of the status of the leak
sensor. In addition, you can display the ¾Cumulated Workload.
To create the corresponding controls, follow the description in How to ...:
Actions on the Control Panel:
Modifying a Control Panel
Modifying a Control
Linking a Control to a Device
Select Commands... on the Control menu. Under Pump, select Leak to
display the status of the leak sensor. Click Cumulated WorkLoad to
display the total workload [in mega joule, MJ] of the pump.
Peak Parking
The UltiMate system pump allows peak parking, for example, to increase
the ¾Mass Spectrometer acquisition time while the peak elutes. The
⇒ParkPercentage enables peak parking. Select the PeakParked property
to display the peak parking state.
Peak parking is similar to the behavior of the ⇒StopFlow command:
•
The ¾Gradient program is interrupted.
•
Usually, the pump flow is reduced (but not turned off).
However, unlike the behavior of the StopFlow command:
•
Data Acquisition (see ¾Acquisition On/Off) is not interrupted.
•
A running ¾Batch is not stopped.
Select ParkPercentage to enable and disable peak parking. The following
value disables peak parking:
0.000
ParkPercentage
= Disabled
ParkPercentage > 0.00 sets the flow to the following relative value during
PeakParked state:
current flow * ParkPercentage.
Device Control
611
The PeakParked state is entered whenever the signal at the pump’s
START IN input changes from Open to Closed (edge trigger) and continues
until the signal changes from Open to Closed again:
At each sample start, the PeakParked state is reset.
Tip:
Peak parking freezes the gradient with its current composition and reduced
flow while data acquisition continues. Make sure that data acquisition is
long enough (that is, the expected running time of the chromatogram +
expected time for peak parking). Therefore, with peak parking the retention
times do not correspond to the expected times. In addition, ¾Audit Trail
entries and gradient plots are no longer synchronized.
Control: Autosamplers
Depending on the ¾Autosampler type, different commands are available.
For an overview of the individual commands that are available for the
different Dionex autosamplers, refer to:
Autosampler Commands (GINA 50)
Autosampler Commands (ASI-100 Series)
Autosampler Commands (AS50)
Examples for User-Defined Programs for the FAMOS Autosampler
(LC Packings)
612
Device Control
The following pages provide detailed information about:
Injecting a Sample
Setting Up Remote Injection
Priming the Syringe (ASI-100 Series)
Defining Sample Preparation Steps (AS50)
Overlapping Samples
Injecting Two Samples Simultaneously
Opening the AS50 Door during Operation
Monitoring the Status of the AS50
Autosampler Commands (GINA 50)
Suck (see ⇒Draw)/⇒Dispense Sample
0.000
Suck
0.000
Dispense
Position
Duration
Position
Duration
=
=
=
=
Value, Volume = Value [µl],
Value [s]
Value, Volume = Value [µl],
Value [s]
Note:
In the case of the Dionex Autosampler GINA 50, the corresponding
operations can be synchronized with Chromeleon via a remote input and
the Suck and Dispense commands. The device automatically reports the
completion of each operation to Chromeleon. In the ¾Program, the wait
command Sucked must be inserted. All following commands are executed
only after the autosampler confirmed the execution of the Suck command,
that is, after the sample has been drawn. The program performs this
independently of the time required by the autosampler for the suck and
dispenses processes. This is shown in the following example:
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Suck
Wait
Suck
Wait
Dispense
Wait
⇒Inject
Position = 20, Volume = 30, Duration = 0
Sucked
Position = 21, Volume = 30, Duration = 0
Sucked
Position = , Volume = 60, Duration = 0
Sucked
Device Control
613
Description of the program part: First, the Autosampler GINA 50 draws
30 µl solution from ⇒Position 20 and afterward draws 30 µl solution from
position 21. The entire drawn volume is dispensed in the current vial
(current = last position if no other position is specified). Then, the injection
volume, which has been specified in the sample list, is drawn and injected
from there.
Short command syntax:
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Suck
Wait
Suck
Wait
Dispense
Wait
Inject
20, 30, 0
Sucked
21, 30, 0
Sucked
Position = , Volume = 60, Duration = 0
Sucked
Relative Sample Location
The Dionex Autosampler GINA 50 is capable of executing the suck and the
dispense commands relative to a certain sample position. The
Position+Location is entered in the field Position of the Suck or
Dispense commands.
In the program this is expressed as follows:
0.000
0.000
0.000
0.000
0.000
0.000
Suck
Wait
Suck
Wait
Dispense
Wait
Position = 20, Volume = 30, Duration = 0
Sucked
Position = Position+10, Volume = 30, Duration = 0
Sucked
Position = Position+20, Volume = 60, Duration = 0
Sucked
Description of the program part: The autosampler takes 30 µl of solution
from position 20, moves 10 positions from the current position in the
sample list, and takes 30 µl of solution from there. Then, the 60-µl solution
is dispensed into a vial that is located 20 positions from the current sample
vial.
614
Device Control
Air Segment
For the Dionex GINA 50 Autosampler, the input position = 100 is an
imaginary Air Vial from which a certain air volume can be drawn as a
separating segment; for example:
0.000
0.000
0.000
0.000
0.000
0.000
Suck
Wait
Suck
Wait
Suck
Wait
Position = 20, Volume = 30, Duration = 0
Sucked
Position = 100, Volume = 30, Duration = 0
Sucked
Position = 21, Volume = 30, Duration = 0
Sucked
Description of the program part: After 30 µl of solution is sucked from
position 20, 30 µl of air is sucked before another 30 µl of solution is sucked
from position 21. Thus, the two solutions do not encounter each other.
Note:
Only use the Segment command in combination with the Dispense
command, as otherwise the sucked air volume is also injected.
Dispense in Needle Seat
Entering position 101 in a Dispense command enables dispensing the
volume contained in the needle into the needle seat of the Autosampler
and thus into the waste container.
0.000
Dispense
Position = 101, Volume = 60, Duration = 0
Wash Injection Loop
Select the ⇒Wash command to rinse the injection loop of the Gina 50
autosampler with solvent. This corresponds to the normal solvent flow
during the Inject command.
0.000
0.000
DEVICENAME.Wash
Wash
Or
Device Control
615
Lift/Lower Sample Needle
Select the ⇒NeedleUp command to lift the sample needle. When lifting the
needle, an active Wash process is automatically interrupted, i.e., solvent
flow is not through the injection loop any longer but directly from the pump
to the column.
0.000
NeedleUp
Execute the Wash command to lower the needle again and direct the
solvent flow through the sample loop again (for more information see
¾Autosampler).
0.000
Wash
The combination of the two commands prevents crystallization of
substances in the sample loop.
For an overview of the individual commands for the GINA 50 or GINA 160
samplers, refer to Control
Dionex GINA 50 and GINA 160 AutoAutosampler
samplers. Also, refer to How to: Device Control
Commands (ASI-100 Series ).
Autosampler Commands (ASI-100 Series)
Many commands of the Dionex ASI-100 ¾Autosamplers (ASI-100 T and
ASI-100 PT= with temperature control) are similar to those of the GINA 50
Autosampler Commands (GINA 50)).
(see How to: Device Control
However, there are some important differences, which are described below:
Sample Positions
The sample vials are situated in three different segments, which are
distinguished by color. Within the segments, the vials are situated in
different rows. Thus, the sample ⇒Positions are indicated as follows:
Letters according to their color describe the individual segments: R, G, or B
(indicating the red, green, and blue segment, respectively). The different
rows are described from the outer to the inner row: A, B, C, or D. The
individual positions within the respective rows are numbered
counterclockwise. For example, the RA1 position is located in the outer row
of the red segment (also, refer to the Operating Instructions for the ASI-100
Series).
616
Device Control
Autosampler Configuration
Specify the device configuration before starting the actual program. It is
important to define the sample positions for the reagents and the wash
liquid. These positions are valid throughout the entire running time of the
program:
0.000
0.000
0.000
0.000
0.000
Sampler.ReagentAVial
Sampler.ReagentBVial
Sampler.ReagentCVial
Sampler.WashVial
Sampler.PrepVial
BB1
BC1
BA1
G99
R99
The following commands define the number of draw and dispense actions
(for the Mix command):
0.000
Sampler.MixRepeat
3
⇒Draw, ⇒Dispense Sample, ⇒Mix
Before issuing the Draw, Dispense, or Mix command, specify the vial
(depending on the selected option - see below) from which to draw and/or
dispense and the volume to draw and/or dispense (the latter for the Mix
command):
0.000
0.000
0.000
PrepSubject
PrepVolume
Draw
Option (see below)
Value [µl]
0.000
0.000
0.000
PrepSubject
PrepVolume
Dispense
Option (see below)
Value [µl]
The following options are available for the PrepSubject command:
Option
Description
PrepVial
Actual mixing vial position (current PrepVial value)
Sample_Vial
Vial for the actual sample in the sample list (current
Sampler.Position value)
WashVial
Vial containing the wash liquid (current
Sampler.WashVial value)
Air
Air (with the Draw command) and needle port (with the
Dispense command), respectively
ReagentA (B, C or D)
Reagents A (B, C, or D) (actual Sampler.ReagentAVial
value)
Some commands need to be synchronized with Chromeleon, that is, the
autosampler automatically reports completion of the respective operation to
Chromeleon. In addition, the Sampler.Ready wait condition must be part of
Device Control
617
the ¾Program. The following commands are executed only after the
autosampler has confirmed that the command has been executed. This
applies to the Draw, Dispense, Mix, Test, and ⇒Wash commands and is
illustrated below:
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
PrepSubject
PrepVolume
Draw
Wait
PrepSubject
PrepVolume
Draw
Wait
PrepSubject
PrepVolume
Dispense
Wait
⇒Inject
Sample_Vial
30
Sampler.Ready
ReagentA
30
Sampler.Ready
MixVial
60
Sampler.Ready
Position = PrepVial
Description of the program part: First, the autosampler draws 30 µl of
solution from the current sample vial, and, upon completion, 30 µl of
solution from the reagent A vial. The entire volume drawn is dispensed into
the MixVial. The injection volume, which has been specified in the sample
list, is then drawn and injected from this position.
Air Segment
To draw an air segment, specify that the PrepSubject be Air. For example,
use the following program part to draw two samples separated by an air
segment. The air segment prevents early mixing in the needle and makes
sure that an exactly defined reaction time is met.
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
PrepSubject
PrepVolume
Draw
Wait
PrepSubject
PrepVolume
Draw
Wait
PrepSubject
PrepVolume
Draw
Wait
PrepSubject
PrepVolume
Dispense
Wait
ReagentA
100
Sampler.Ready
Air
5
Sampler.Ready
Sample_Vial
100
Sampler.Ready
MixVial
205
Sampler.Ready
618
Device Control
Description of the program part: Having drawn 100 µl of reagent A, 5 µl of
air is drawn first before another 100 µl of sample is drawn from the vial.
Thus, the two solutions encounter each other in the mixing vial only.
Relative Sample Location
1. Different rings: The autosampler can execute commands relative to a
given sample position. Type the description "Position + relative entry" in the
Position field of the Draw or Dispense commands. In the program, this is
expressed as follows (if the current position is in the RA ring):
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
PrepSubject
PrepVolume
Draw
Wait
Position
PrepSubject
PrepVolume
Draw
Wait
Position
PrepSubject
PrepVolume
Dispense
Wait
Inject
Sample_Vial
30
Sampler.Ready
Position + RB1 - RA1
Sample_Vial
30
Sampler.Ready
Position + RC1 - RB1
Sample_Vial
60
Sampler.Ready
Volume=30
Description of the program part: The autosampler draws 30 µl of solution
from the actual sample vial, moves to the respective position in the RB ring,
and draws 30 µl of solution from this position as well. Then, the 60 µl
solution is dispensed into the vial at the respective position in the RC ring.
For example, if the current sample is situated at position RA3, another 30 µl
will be drawn from position RB3, and the entire volume of 60 µl will be
dispensed at position RC3. After that, 30 µl are injected from position RC3.
Note:
To complete the program, the PrepSubject command is repeated with the
Sample_Vial argument. However, this command is not required, as
Chromeleon keeps the latest setting. The second command
(= PrepVolume) could also be omitted, as it only repeats the argument
"30."
2. Different segments: You can also place the samples to derivate into the
red segment, for example, and execute the mixing process with a reagent
in the corresponding sample vial in the green segment using the following
commands:
Device Control
0.000
0.000
0.000
Sampler.PrepVial
PrepSubject
Mix
619
Position + GA1 - RA1
PrepVial
The first line specifies that the corresponding vial in the green segment be
used as the mixing vial for the sample in the red segment. For example, if
you wish to process the sample at position RB5, mixing takes place at
position GB5.
3. Incrementing the positions: Another way is to increment the positions.
Use the following example to define a position 5 after the current position
for MixSubject:
0.000
0.000
0.000
Sampler.PrepVial
PrepSubject
Mix
Position + 5
PrepVial
For example, if you wish to process the sample from position RB3, the vial
at position RB8 is the mixing vial.
Wash Needle
Use the Wash command to rinse the autosampler's needle with the wash
liquid. The wash volume is drawn and dispensed into the needle seat.
0.000
0.000
WashVolume
Wash
Value [µl]
For an overview of the individual commands for the ASI-100 Autosamplers,
Dionex ASI-100
refer to Commands for Device Control
Autosampler
Autosamplers. Also, refer to How to: Device Control
Commands (AS50).
Autosampler Commands (AS50)
Sample Loading and Injection
In the AS50, sample loading and injection are two distinct events. The
Load command switches the injection valve to the load position and moves
the sample from the AS50 inject port into the sample loop. The Inject
command switches the injection valve to the inject position, which directs
the pump flow through the loop. The sample is then transported from the
loop to the column.
620
Device Control
Tip:
Clicking the Inject button on the AS50 control panel sends both the Load
and the Inject commands to the autosampler.
Note:
If the AS50 is in simultaneous injection mode, separate Valve.LoadPosition
and Valve.InjectPosition commands are required (in addition to the Load
and Inject commands). See the description of simultaneous injections
below for details.
Flush the Inject Port
The Flush and Wait commands must be in the order shown and they must
occur before any other ¾Autosampler commands, or after all other
autosampler commands.
Flush
Wait
Volume = Value [µl]
FlushState
Autosampler Options
All AS50 autosampler option commands must be grouped together in the
¾Program. Place them at the beginning of the program, before
t = 0.000.You do not need to specify event times for the AS50 option
commands.
Devicename.NeedleHeight
Devicename.CutSegmentVolume
Devicename.SyringeSpeed
Devicename.ColumnTemperature
= Value [mm]
= Value [µl]
= Value
Devicename.TrayTemperature
= Off / Value [°C]
= Value
Devicename.Cycle
= Off / Value [°C]
Sample Prep
All AS50 autosampler sample prep commands (Pipet, Mix, FlushSP,
DelaySP, SetNeedleHeight, Dilute, and Dispense) must be grouped
together in the program. Place them at the beginning of the program, after
the AS50 option commands and before t=0.000. You do not need to specify
event times for the sample prep commands. Include a Wait SampleReady
command after the sample prep commands to allow them to be completed
before injection.
Device Control
621
Note:
The Dilute and Dispense commands are only available if the AS50 is
equipped with the sample preparation option.
In the example below, 20 µl are pipetted from vial 1 and delivered to vial
10. 200 µl are dispensed from reservoir A into vial 10. The contents of vial
10 are then mixed by drawing in and then expelling 100 µl of the vial
contents. The mixing cycle is repeated 5 times.
Note:
Due to limitations here, commands are shown on two lines. In an actual
program, one command must be entered on one line.
Sampler.Pipet
Sampler.Dispense
Sampler.Mix
Wait
Also, refer to
Volume
=
20.0,
SourceVial
=
DestinationVial = 10
Volume
=
200.0,
SourceReservoir
Reservoir_A, DestinationVial = 10
SourceVial = 10, NumberOfTimes =
Volume = 100.0
SamplePrep
1,
=
5,
Defining Sample Preparation Steps.
Relative Vial Location
In the sample preparation commands, a vial location can be specified either
as an absolute position or as a relative position. In relative positioning, the
CurrentVial is the current sample vial position, specified in the sample list in
the Sequence. In the example below, 20 µl of liquid are pipetted from the
current sample vial and then expelled into the vial, 1 position past the
current vial.
Sampler.Pipet
Volume = 20.0, SourceVial = CurrentVial,
DestinationVial = CurrentVial+1
Sample Overlap
If the AS50 Sample Overlap option is enabled in the Server Configuration,
the AS50 performs the following commands for the next sample in a
sequence, while data acquisition is occurring for the currently running
sample in the sequence:
622
Device Control
•
Flush
•
Autosampler
Option
Commands
(ColumnTemperature,
TrayTemperature, WaitForTemperature, SyringeSpeed, CycleTime,
SetNeedleHeight, CutSegmentVolume)
•
Sample Prep Commands (Pipet,
NeedleHeight, Dilute, Dispense)
Mix,
DelaySP,
FlushSP,
When the AS50 is preparing a sample, the Status field in the sequence
displays Preparing and the sample line is highlighted in yellow. A message
is also logged in the Audit Trail.
Also, refer to
Overlapping Samples.
Cycle Time
Cycle time controls the time between injections. When a cycle time is
specified, the autosampler delays sample injection until the specified time
has elapsed since the previous injection. This is accomplished with the
Wait CycleTimeState command. When running a batch, the Wait
CycleTimeState command in the first PGM File in the batch is ignored.
The following example sets a cycle time of 30 minutes.
0.000
Cycle
Sampler.Load
Wait
Sampler.Inject
=30
CycleTimeState
Priming the Liquid Lines
The Prime command is used to prime the flush reservoir line to the
sampling valve and the sample transfer line. In addition, if the sample prep
option is installed, the Prime command is used to prime the lines from each
installed reagent reservoir. The following example uses the prep syringe to
prime the line to reservoir A with 2000 µl.
0.000
Sampler.Prime
Volume=2000,
PrimeReservoir=Reservoir_A,
PrimeSyringe=Prep
Device Control
623
Simultaneous Injection
If the AS50 in equipped with the simultaneous injection mode option, the
program includes additional commands for controlling two injection valves.
The additional commands are used to switch the position of each valve
during sample injection.
In the following example, the AS50 is connected to two ICS-2000 systems.
Each ICS-2000 is equipped with an injection valve, and there are no
injection valves installed in the AS50.
0.000
Pump_InjectValve.LoadPosition
Pump_InjectValve_2.LoadPosition
Load
Wait
CycleTimeState
Pump_InjectValve.InjectPosition
Pump_InjectValve_2.InjectPosition
Pump_ECD.Autozero
Pump_ECD_2.Autozero
Inject
Wait
InjectState
Note:
If the AS50 is not in simultaneous injection mode, separate
Valve.LoadPosition and Valve.InjectPosition commands are not required. In
this case, the valve position is switched automatically at the appropriate
times when the Load and Inject commands are issued.
For an overview of the individual commands for the AS50 autosampler,
refer to Commands for Device Control
Dionex AS50 Autosampler.
Examples for User-Defined Programs for the FAMOS
Autosampler (LC Packings)
The user-defined program is loaded and executed at the autosampler's
start time (Sampler.Inject command) even if the program steps appear
prior to that command in the program file. Make sure that the user-defined
program includes an InjectMarker command at the appropriate position so
that the system recognizes that an injection has been made:
The following programs are typical application examples for the FAMOS
HPLC ¾Autosampler:
624
Device Control
0. User-Defined Standard Program
;User-defined program (UDP) template for FAMOS autosampler
;******
PLEASE DO NOT EDIT ******
Sampler.ReagentAVial= 1
Sampler.ReagentBVial= 2
Sampler.ReagentCVial= 3
Sampler.ReagentDVial= 4
Sampler.PrepVial=
A1
Sampler.Draw
From=SampleVial,
Volume=5,
SyringeSpeed=Low,
SampleHeight=0
Sampler.Dispense To=PrepVial,
Volume=5,
SyringeSpeed=Low,
SampleHeight=0
Sampler.Draw
From=ReagentAVial, Volume=10,
SyringeSpeed=Low, SampleHeight=0
Sampler.Dispense To=PrepVial, Volume=10, SyringeSpeed=Low,
SampleHeight=0
Sampler.MixWait
Duration=10
Sampler.Draw
From=PrepVial, Volume=5, SyringeSpeed=Low,
SampleHeight=0
Sampler.InjectValve Position=Inject
InjectMarker
1. Drawing a Sample Volume of 1 nl
0.000
;User-defined program (UDP) template for FAMOS autosampler
;******
PLEASE DO NOT EDIT ******
InjectMode =
UserProg
InjectValve
Position=Load
SyringeValve
Position=Needle
Draw
From=SampleVial, Volume=1.0
MixWait
Duration=5
Draw
From=SampleVial, Volume=0.0
Draw
From=ReagentAVial, Volume=25
MixWait
Duration=5
InjectValve
Position=Inject
InjectMarker
; creates Inject Response during the program run
MixWait
Duration=5
Draw
From=ReagentAVial, Volume=0.0
SyringeValve
Position=Waste
MoveSyringeHome
SyringeValve
Position=Needle
MixNeedleWash
Volume=50
Inject
; starts the above program and waits for the Inject Response
Acquisition On
...
4.000 Acquisition Off
End
Device Control
625
2. Sample Preparation with 2 Reagents
0.000
;User-defined program (UDP) template for FAMOS autosampler
;******
PLEASE DO NOT EDIT ******
InjectMode =
UserProg
Draw
From=ReagentAVial, Volume=20.0
MixWait
Duration=5
Draw
From=ReagentAVial, Volume=0.0
MixWait
Duration=5
Dispense
To=SampleVial, Volume=20.0
MixWait
Duration=10
Draw
From=ReagentBVial, Volume=10.0
MixWait
Duration=5
Draw
From=ReagentBVial, Volume=0.0
MixWait
Duration=5
Dispense
To=SampleVial, Volume=10.0
MixWait
Duration=10
InjectValve
Position=Inject
SyringeValve
Position=Needle
Draw
From=SampleVial, Volume=3.0
MixWait
Duration=5
InjectValve
Position=Load
Draw
From=SampleVial, Volume=5.0
MixWait
Duration=5
InjectValve
Position=Inject
InjectMarker
; creates Inject Response during the program run
MixWait
Draw
SyringeValve
MoveSyringeHome
SyringeValve
MixNeedleWash
Duration=5
From=SampleVial, Volume=0.0
Position=Waste
Position=Needle
Volume=50
Inject
; starts the above program and waits for the Inject Response
4.000
Acquisition On
...
Acquisition Off
End
626
Device Control
3. Gel Extraction
;User-defined program (UDP) template for FAMOS autosampler
;******
PLEASE DO NOT EDIT ******
InjectMode =
UserProg
Draw
From=SampleVial, Volume=5.0
Dispense
To=PrepVial, Volume=5.0
Draw
From=ReagentAVial, Volume=5.0
Dispense
To=SampleVial, Volume=5.0
MixWait
Duration=10
0.000
Draw
Dispense
Draw
Dispense
MixWait
From=SampleVial, Volume=5.0
To=PrepVial, Volume=5.0
From=ReagentBVial, Volume=5.0
To=PrepVial, Volume=5.0
Duration=10
Draw
From=SampleVial, Volume=5.0
Dispense
To=PrepVial, Volume=5.0
MixWait
Duration=5
InjectValve
Position=Inject
SyringeValve
Position=Needle
Draw
From=SampleVial, Volume=1.0
MixWait
Duration=5
InjectValve
Position=Load
Draw
From=PrepVial, Volume=5.0
MixWait
Duration=5
InjectValve
Position=Inject
InjectMarker
; creates Inject Response during the program run
MixWait
Duration=5
Draw
SyringeValve
MoveSyringeHome
SyringeValve
MixNeedleWash
From=SampleVial, Volume=0.0
Position=Waste
Position=Needle
Volume=50
Inject
; starts the above program and waits for the Inject Response
Acquisition On
...
4.000 Acquisition Off
End
Tips:
If the previous action was Draw or Dispense, the needle slowly returns to
its start position while the MixWait command is executed. For the needle to
remain at its current position, enter another Draw or Dispense command
with "Volume=0.0" as shown in the above program examples.
Device Control
627
Notes:
Use the PGM Wizard to create a user-defined program. You can either
create a new user-defined program or use an existing program as a
template for the new one.
Each user-defined program must include an InjectMarker command. If this
command is not included, a message appears in the PGM Wizard.
For an overview of the individual commands that are available for the
FAMOS autosampler, refer to Commands for Device Control
Dionex/LC Packings FAMOS Autosampler.
For information about the sample preparation commands supported by the
FAMOS autosampler, refer to Commands for Device Control
Dionex/LC Packings FAMOS Autosampler: Sample Preparation.
Injecting a Sample
It depends on the laboratory equipment how the injection is performed:
•
Manually
•
Via an ¾Autosampler
•
Automatically (the injection is programmed).
Click the corresponding control on the control panel or select Inject… on
the Control menu to open the Inject dialog box. Determine how much of a
substance is injected (⇒Volume) and from which Autosampler ⇒Position.
Then, issue the ⇒Inject command.
Reporting the end of the injection process to Chromeleon completes
successful injection. If a hand-operated valve is used, this is via a contact
closure relay. Modern autosamplers automatically send the message via
the serial interface or a ¾DX-LAN. When Chromeleon has received this
message, the retention time is started.
Hand-operated valve (manual injection):
A µl-syringe is used to inject the sample into the needle seat of the handoperated valve. In this way, it reaches the sample loop (Load). By
switching the valve (Inject), the solvent flow is directed to the sample loop,
and the sample enters the high-pressure circuit of the system. If correctly
628
Device Control
connected (via a ¾Remote Input), switching the hand-operated valve
triggers the Inject signal and thus the timer. Very exact operation is
possible if the injected volume corresponds to the sample loop size. In this
case, the sample loop is completely filled. When switching the valve, the
exact volume of the sample loop is injected without any loss.
Autosampler (manual injection):
The autosampler can be operated via the input panel on the instrument or
via the PC. Controlling the autosampler via the PC, is a very convenient
method. However, this is only possible if the autosampler is connected with
Chromeleon via an RS-232 interface, the DX-LAN, or USB (Universal Serial
Bus).
•
Select ⇒Inject on the Control menu and determine the ⇒Volume and
the ¾Autosampler position for the injection.
•
If your control panel provides the corresponding controls, enter the
Volume and Position parameters directly in the corresponding edit
box.
The time required by the autosampler to inject the sample can be indicated
optically. Link a color area or a lamp with the Inject Wait property (for more
Linking
information, refer to How to ...: Actions on the Control Panel
a Control to a Device). In a controlled system, the pump is set to ⇒Hold
during injection.
Automatic injection:
•
Enter the Inject command at the time t = 0.000 in the ¾Program.
If the command is executed by an autosampler that is controlled by
Chromeleon, the autosampler returns a signal to Chromeleon when
injection is completed. Then, the timer is started.
Tip:
With the AS50 autosampler, sample loading and injection are two distinct
events. Therefore, include a Load command in the program at t = 0.000
and then the Inject command. See
Autosampler Commands (AS50)
for details.
When injecting via a hand-operated valve or an autosampler that is not
controlled by Chromeleon, the data system also waits for a signal before it
starts the timer. For example, with a hand-operated valve, the signal is
Device Control
629
returned after the valve has switched from Load to Inject, that is, program
execution is delayed until the injection is actually performed. This type of
automatic injection requires connection of a remote input or remote start
device via remote inputs (TTL or relay) and configuration of a remote inject
Setting Up Remote Injection.
device. See How to ...: Device Control
Tip:
As an Inject signal is not specified, there can only be one injection unit per
timebase, that is, install either a hand-operated valve or an autosampler.
Setting Up Remote Injection
If Chromeleon does not directly control the injection valve, a ¾Remote
Inputs (remote start) device can be set up that communicates to
Chromeleon that injection was performed. The setup procedure is as
follows: Connect the remote input device via TTL or relay, install and
configure a remote inject device in the ¾Timebase, and add an ⇒Inject
command to the ¾Program.
Tip:
For the UI20, remote injection can be triggered by the remote input or by
pressing the Run button on the UI20 front panel. For the DX-120, remote
injection can only be triggered by the remote input. The Load/Inject button
on the DX-120 front panel is disabled during remote control.
Connect the Remote Input Device (TTL or Relay Connections)
The remote input of the remote input (remote start) device must be
connected via TTL or a relay to the injection valve or to another device,
e.g., an ¾Autosampler. Only then, the device can communicate to
Chromeleon that injection was performed. When injection is performed, the
injection valve or autosampler sends a signal to the remote input device
and the remote input device then communicates to Chromeleon that
injection was performed.
630
Device Control
Example Connections: AS40 and DX-120
Tip:
For detailed TTL and relay connection instructions, refer to the operator's
manual for each device.
The following connections allow completely automated control of the AS40
and DX-120.
1. Connect the Relay Control Ready Out pin on the AS40 rear panel to
the Inject TTL Input pin on the DX-120 rear panel.
2. Connect the Ready Out Ground pin on the AS40 to the TTL Inputs
Ground pin on the DX-120.
3. Connect the Load pin on the AS40 to the TTL 1 Out pin on the DX-120.
4. Connect the Load Ground pin on the AS40 to the TTL Outputs
Ground pin on the DX-120.
Configure a Remote Inject Device
1. Open the Server Configuration.
2. Select the timebase in which the remote input device is configured.
3. Select Add Device on the Edit or context menu. The Add device to
timebase dialog box appears.
4. Select General from the Manufacturers list box and then select
¾Remote Inject from the Devices list and click OK.
The Properties dialog box for the Remote Inject device appears.
Device Control
631
5. The default device name is InjectValve. You can accept the default
name or enter a different one.
6. From the Inject Port drop-down list, select the remote input device that
was connected through TTL or relay. Click OK.
Add an Inject Command to the Program
Add the following command to the PGM File. (If you use the Program
Wizard (see Control
The Program Wizard), the command is added
automatically).
0.000
Inject
When Chromeleon executes the program, it runs the commands that occur
before the Inject command and then waits for the signal from the remote
input device. A message in the audit trail is displayed: "Wait for inject
response on remote start." When the inject signal occurs, program
execution resumes.
Example PGM File: AS40 and DX-120
Data_Collection_Rate
Pump =
SRS =
EluentPressure =
Column =
ECD_TTL_1.State =
5.00
On
On
On
A
5v
Note 1
Wait
RinseComplete
Note 2
-0.100
ECD_TTL_1.State =
0v
Note 3
0.000
ECD.Autozero
Note 4
Inject
Note 5
ECD_1.AcqOn
10.000
ECD_1.AcqOff
End
632
Device Control
Notes:
1. TTL Out 1 turned off.
2. The TTL_1 5v and TTL_1 0 v commands must be separated by one or
more commands or they must occur at different times in the program.
3. TTL Out 1 turned on. AS40 Load Cycle starts.
4. Program execution waits until inject signal is received.
5. AS40 Ready Out signals the DX-120 Inject TTL Input. Injection occurs.
Program execution resumes.
Priming the Syringe (ASI-100 Series)
The PrimeSyringe command allows removing air from the syringe without
dismantling the syringe from the instrument. First, the syringe is filled 5
times with washing liquid. Then, the procedure is repeated using eluent.
Before executing the PrimeSyringe command:
•
Make sure that the pump flow is off. Else, the pump would deliver
eluent to the wash vial when the injection valve switches into the Inject
position (see step 3).
•
Set the WashSpeed and the DispSpeed to values that correspond to
the installed syringe. (Note: When a wash vial is used, the WashSpeed
command is required instead of the DrawSpeed command.)
The recommended settings and the required time are listed in the table
below:
Syringe
100 µL
250 µL
1000 µL*
2500 µL*
WashSpeed [µL/s]
5
10
25
25
DispSpeed [µL/s]
5
10
50
50
SyringeDelay [s