Surveyor Plus Getting Started with Xcalibur 2.0 - SA-LE

Surveyor Plus Getting Started with Xcalibur 2.0 - SA-LE
Surveyor Plus™
Getting Started with Xcalibur 2.0
60053-97103 Revision D
January 2007
© 2007 Thermo Fisher Scientific Inc. All rights reserved.
Surveyor™ and Xcalibur™ are trademarks of Thermo Fisher Scientific Inc. Windows® and Excel® are
registered trademarks of Microsoft Corporation. PEEK™ is a trademark of Victrex PLC.
Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use
in the product operation. This document is copyright protected and any reproduction of the whole or
any part of this document is strictly prohibited, except with the written authorization of Thermo
Fisher Scientific Inc.
The contents of this document are subject to change without notice. All technical information in this
document is for reference purposes only. System configurations and specifications in this document
supersede all previous information received by the purchaser.
Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or
error-free and assumes no responsibility and will not be liable for any errors, omissions, damage or
loss that might result from any use of this document, even if the information in the document is
followed properly.
This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a
purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which
Terms and Conditions of Sale shall govern all conflicting information between the two documents.
Software Revision: Xcalibur 2.0 and higher
Regulatory Compliance
Thermo Electron San Jose performs complete testing and evaluation of its products to ensure full compliance
with applicable domestic and international regulations.
Changes that you make to your instrument may void compliance with one or more of these EMC and safety
standards. Changes to your system include replacing a part or adding components, options, or peripherals
not specifically authorized and qualified by Thermo Electron. To ensure continued compliance with EMC
and safety standards, replacement parts and additional components, options, and peripherals must be ordered
from Thermo Electron or one of its authorized representatives.
This section contains regulatory compliance information for the following devices of the Surveyor Plus family
of LC instruments:
•
•
•
Surveyor LC Pump Plus
Surveyor MS Pump Plus
Surveyor Autosampler Plus
•
•
Surveyor PDA Plus Detector
Surveyor UV/Vis Plus Detector
iii
Surveyor LC Pump Plus
When the Surveyor LC Pump Plus is delivered to you, it meets all pertinent electromagnetic compatibility
(EMC) and safety standards as described below
EMC Directive 89/336/EEC amended by 92/31/EEC and 93/68/EEC
EMC compliance has been evaluated by Underwriters Laboratories Inc.
EN 55011
1998
EN 61000-4-3
2002
EN 61000-3-2
1995, A1; 1998, A2; 1998, A14; 2000
EN 61000-4-4
1995, A1; 2001, A2; 2001
IEC 61000-3-2
2000
EN 61000-4-5
1995, A1; 2001
EN 61000-3-3
1995
EN 61000-4-6
1996, A1; 2001
IEC 61000-3-3
1994
EN 61000-4-11
1994, A1; 2001
EN 61326-1
1997
EN 61000-4-2
1995 A1; 1998 A2; 2001
CISPR 11
1999, A1; 1999, A2; 2002
FCC Class A, CFR 47 Part 15 Subpart B: 2004
Low Voltage Safety Compliance
Low voltage safety compliance has been evaluated by TUV Rheinland of North America, Inc. This device
complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
Surveyor MS Pump Plus
When the Surveyor MS Pump Plus is delivered to you, it meets all pertinent electromagnetic compatibility
(EMC) and safety standards as described below
EMC Directive 89/336/EEC amended by 92/31/EEC and 93/68/EEC
EMC compliance has been evaluated by Underwriters Laboratories Inc.
EN 55011
1998
EN 61000-4-3
2002
EN 61000-3-2
1995, A1; 1998, A2; 1998, A14; 2000
EN 61000-4-4
1995, A1; 2001, A2; 2001
IEC 61000-3-2
2000
EN 61000-4-5
1995, A1; 2001
EN 61000-3-3
1995
EN 61000-4-6
1996, A1; 2001
IEC 61000-3-3
1994
EN 61000-4-11
1994, A1; 2001
EN 61326-1
1997
EN 61000-4-2
1995 A1; 1998 A2; 2001
CISPR 11
1999, A1; 1999, A2; 2002
FCC Class A, CFR 47 Part 15 Subpart B: 2004
Low Voltage Safety Compliance
Low voltage safety compliance has been evaluated by TUV Rheinland of North America, Inc.This device
complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
iv
Surveyor Autosampler Plus
When the Surveyor Autosampler Plus is delivered to you, it meets all pertinent electromagnetic compatibility
(EMC) and safety standards as described below
EMC Directive 89/336/EEC amended by 92/31/EEC and 93/68/EEC
EMC compliance has been evaluated by Underwriters Laboratories Inc.
EN 55011
1998
EN 61000-4-3
2002
EN 61000-3-2
1995, A1; 1998, A2; 1998, A14; 2000
EN 61000-4-4
1995, A1; 2001, A2; 2001
IEC 61000-3-2
2000
EN 61000-4-5
1995, A1; 2001
EN 61000-3-3
1995
EN 61000-4-6
1996, A1; 2001
IEC 61000-3-3
1994
EN 61000-4-11
1994, A1; 2001
EN 61326-1
1997
EN 61000-4-2
1995 A1; 1998 A2; 2001
CISPR 11
1999, A1; 1999, A2; 2002
FCC Class A, CFR 47 Part 15 Subpart B: 2004
Low Voltage Safety Compliance
Low voltage safety compliance has been evaluated by TUV Rheinland of North America, Inc. This device
complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
Surveyor PDA Plus Detector
When the Surveyor PDA Plus Detector is delivered to you, it meets all pertinent electromagnetic
compatibility (EMC) and safety standards as described below
EMC Directive 89/336/EEC amended by 92/31/EEC and 93/68/EEC
EMC compliance has been evaluated by Underwriters Laboratories Inc.
EN 55011
1998
EN 61000-4-3
2002
EN 61000-3-2
1995, A1; 1998, A2; 1998, A14; 2000
EN 61000-4-4
1995, A1; 2001, A2; 2001
IEC 61000-3-2
2000
EN 61000-4-5
1995, A1; 2001
EN 61000-3-3
1995
EN 61000-4-6
1996, A1; 2001
IEC 61000-3-3
1994
EN 61000-4-11
1994, A1; 2001
EN 61326-1
1997
EN 61000-4-2
1995 A1; 1998 A2; 2001
CISPR 11
1999, A1; 1999, A2; 2002
FCC Class A, CFR 47 Part 15 Subpart B: 2003
Low Voltage Safety Compliance
Low voltage safety compliance has been evaluated by TUV Rheinland of North America, Inc.This device
complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
v
Surveyor UV/Vis Plus Detector
When the Surveyor UV/Vis Detector is delivered to you, it meets all pertinent electromagnetic compatibility
(EMC) and safety standards as described below
EMC Directive 89/336/EEC amended by 92/31/EEC and 93/68/EEC
EMC compliance has been evaluated by Underwriters Laboratories Inc.
EN 55011
1998
EN 61000-4-3
2002
EN 61000-3-2
1995, A1; 1998, A2; 1998, A14; 2000
EN 61000-4-4
1995, A1; 2001, A2; 2001
IEC 61000-3-2
2000
EN 61000-4-5
1995, A1; 2001
EN 61000-3-3
1995
EN 61000-4-6
1996, A1; 2001
IEC 61000-3-3
1994
EN 61000-4-11
1994, A1; 2001
EN 61326-1
1997
EN 61000-4-2
1995 A1; 1998 A2; 2001
CISPR 11
1999, A1; 1999, A2; 2002
FCC Class A, CFR 47 Part 15 Subpart B: 2004
Low Voltage Safety Compliance
Low voltage safety compliance has been evaluated by TUV Rheinland of North America, Inc.This device
complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
vi
FCC Compliance Statement
The following statements apply to all the devices of the Surveyor Plus family of LC instruments.
THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS
SUBJECT TO THE FOLLOWING TWO CONDITIONS: (1) THIS DEVICE MAY NOT
CAUSE HARMFUL INTERFERENCE, AND (2) THIS DEVICE MUST ACCEPT ANY
INTERFERENCE RECEIVED, INCLUDING INTERFERENCE THAT MAY CAUSE
UNDESIRED OPERATION.
CAUTION: Read and understand the various precautionary notes, signs, and symbols
contained inside this manual pertaining to the safe use and operation of this product before
using the device.
Notice on Lifting and Handling of
Thermo Electron San Jose Instruments
For your safety, and in compliance with international regulations, the physical handling of this
Thermo Electron San Jose instrument requires a team effort for lifting and/or moving the instrument. This
instrument is too heavy and/or bulky for one person alone to handle safely.
Notice on the Proper Use of
Thermo Electron San Jose Instruments
In compliance with international regulations: If this instrument is used in a manner not specified by
Thermo Electron San Jose, the protection provided by the instrument could be impaired.
Notice on the Susceptibility
to Electromagnetic Transmissions
Your instrument is designed to work in a controlled electromagnetic environment. Do not use radio
frequency transmitters, such as mobile phones, in close proximity to the instrument.
vii
WEEE Compliance
This product is required to comply with the European Union’s Waste Electrical & Electronic
Equipment (WEEE) Directive 2002/96/EC. It is marked with the following symbol:
Thermo Electron has contracted with one or more recycling/disposal companies in each EU
Member State, and this product should be disposed of or recycled through them. Further
information on Thermo Electron’s compliance with these Directives, the recyclers in your
country, and information on Thermo Electron products which may assist the detection of
substances subject to the RoHS Directive are available at www.thermo.com/WEEERoHS.
WEEE Konformität
Dieses Produkt muss die EU Waste Electrical & Electronic Equipment (WEEE) Richtlinie
2002/96/EC erfüllen. Das Produkt ist durch folgendes Symbol gekennzeichnet:
Thermo Electron hat Vereinbarungen mit Verwertungs-/Entsorgungsfirmen in allen EUMitgliedsstaaten getroffen, damit dieses Produkt durch diese Firmen wiederverwertet oder
entsorgt werden kann. Mehr Information über die Einhaltung dieser Anweisungen durch
Thermo Electron, über die Verwerter, und weitere Hinweise, die nützlich sind, um die Produkte
zu identifizieren, die unter diese RoHS Anweisung fallen, finden sie unter www.thermo.com/
WEEERoHS.
Conformité DEEE
Ce produit doit être conforme à la directive européenne (2002/96/EC) des Déchets
d'Equipements Electriques et Electroniques (DEEE). Il est marqué par le symbole suivant:
Thermo Electron s'est associé avec une ou plusieurs compagnies de recyclage dans chaque état
membre de l’union européenne et ce produit devrait être collecté ou recyclé par celles-ci.
Davantage d'informations sur la conformité de Thermo Electron à ces directives, les recycleurs
dans votre pays et les informations sur les produits Thermo Electron qui peuvent aider la
détection des substances sujettes à la directive RoHS sont disponibles sur www.thermo.com/
WEEERoHS.
Contents
Preface ............................................................................................ xv
About This Guide ..................................................................... xv
Related Documentation ............................................................ xv
Safety and Special Notices......................................................... xv
Contacting Us.......................................................................... xvi
Assistance .............................................................................. xvi
Changes to the Manual and Online Help.............................. xvi
Thermo Electron Corporation
Chapter 1
Introduction to the Surveyor Plus Integrated LC/MS System .....1
Analytical Pump..........................................................................3
Surveyor MS Pump Plus ..........................................................3
Surveyor LC Pump Plus...........................................................4
Surveyor Autosampler Plus..........................................................7
Tray Compartment ..................................................................7
Injection System.......................................................................8
Injection Modes .....................................................................14
Temperature Control .............................................................16
Surveyor PDA Plus Detector.....................................................17
Surveyor UV/Vis Plus Detector.................................................18
Communication with Xcalibur..................................................19
Synchronizing the Surveyor LC Devices....................................20
Status LEDs ..............................................................................21
Chapter 2
Configuring Your Surveyor Plus LC Instrument ...........................23
Checking the Communication Hardware..................................24
Configuring the Surveyor Plus LC Devices ...............................25
Opening the Instrument Configuration Dialog Box...............25
Adding Devices to the Instrument Configuration ..................26
Configuring the Devices.........................................................27
Configuring the Surveyor UV/Vis Plus Detector....................37
Exiting the Instrument Configuration Dialog Box .................38
Chapter 3
Preparing for Operation ...................................................................39
Turning On the Power to Each LC Device ...............................40
Launching Xcalibur...................................................................41
Surveyor Plus Getting Started with Xcalibur
xi
Contents
Checking the Status of the LC Devices .....................................42
Displaying the Information View - Status Page ......................42
Viewing the Status of Each Device .........................................44
Turning On the LC Devices from Info View ............................50
Opening the Instrument Setup Window ...................................51
Removing Air from the Solvent Lines........................................52
Purging the LC Pump............................................................52
Purging the MS Pump ...........................................................54
Flushing the Autosampler Syringe..........................................56
Checking the Firmware Version of the Surveyor LC Pump.......58
Priming the Pulse Dampener of the MS Pump .........................59
Calibrating the PDA Detector...................................................61
Dark Current Calibration ......................................................61
Wavelength Calibration .........................................................61
xii
Chapter 4
Creating Instrument Methods ........................................................ 63
Opening the Instrument Setup Window ...................................64
Entering the Method Parameters for the MS Pump ..................65
Parameters for the Surveyor MS Pump Plus ..........................65
Parameters for the Pump........................................................65
Programming the Surveyor MS Pump Plus ............................67
Entering the Method Parameters for the LC Pump ...................72
Entering the Method Parameters for the Autosampler...............76
Parameters for the Surveyor Autosampler Plus .......................76
Programming the Surveyor Autosampler Plus ........................83
Entering the Method Parameters for the PDA Detector ............86
Parameters for the Surveyor PDA Plus Detector.....................86
Programming the Surveyor PDA Plus Detector......................93
Entering the Method Parameters for the UV/Vis Detector........96
Adding a Sample Preparation Routine to the Method ...............98
Opening the Sample Preparation Page ...................................98
Building the Sample Preparation List .....................................99
Sample Preparation Rules.....................................................101
An Example of a Sample Preparation Routine......................103
Saving the Method..................................................................109
Chapter 5
Creating and Running Sequences............................................... 113
Creating a Single Sample Sequence .........................................114
Opening the Sequence Setup Window.................................114
Creating the Sequence..........................................................116
Saving the Sequence .............................................................119
Equilibrating Your Column and Warming Up the D2 Lamp..121
Warming Up the Deuterium Lamp......................................121
Equilibrating the Chromatographic Column........................122
Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
Contents
Loading the Surveyor Autosampler .........................................125
Starting Data Acquisition........................................................126
Viewing the Data As It Is Acquired .........................................130
Reviewing Real-Time Data ..................................................131
Adding Cells to the Display .................................................132
Chapter 6
Using Qual Browser .......................................................................135
Opening a Raw Data File in Qual Browser .............................136
Working with the Cell Grid ....................................................140
Cell States ............................................................................140
Cursor Actions .....................................................................142
Changing the Font Size of the Display ....................................146
Viewing a Report of the Instrument Method ..........................148
Creating a Layout for PDA Data.............................................149
Specifying the Chromatogram Range ...................................149
Setting the Display Options for the Chromatogram Cell .....154
Specifying the Spectrum Range ............................................156
Setting the Display Options for the Spectrum View.............157
Inserting Cells ......................................................................159
Saving a New Layout ...........................................................163
Viewing the Spectrum for a Specific Time Point.....................164
Viewing the Chromatogram for a Specific Wavelength ...........165
Determining Peak Areas..........................................................166
Calculating the Purity of the Chromatographic Peaks .............169
Appendix A Calibration Procedures..................................................................173
Verifying the Performance of the PDA Detector .....................174
Turning On the Lamps ........................................................174
Adjusting the Light Output .................................................176
Performing a Wavelength Calibration ..................................181
Performing a Dark Current Calibration ...............................191
Calibrating the Autosampler ...................................................197
Column Oven Calibration ...................................................197
Vial Tray Metal Sensor Calibration......................................201
Well Bottom Distance Calibration.......................................206
Calibrating the LC Pump........................................................211
Accessing the Calibration Options for the LC Pump............211
Calibration Options .............................................................212
Calibration Procedures.........................................................216
Index..................................................................................................223
Thermo Electron Corporation
Surveyor Plus Getting Started with Xcalibur
xiii
Preface
About This Guide
Welcome to the Surveyor Plus Integrated LC/MS system and the Xcalibur™
data system. The Surveyor Plus™ is part of the Thermo Electron family of
LC instruments.
This Surveyor Plus Getting Started with Xcalibur manual contains an
introduction to the devices of the Surveyor Plus LC System and guides you
through the process of preparing your LC devices for a run, creating an
instrument method, making your first injection, and reviewing the results in
the Qual Browser window of the Xcalibur data system.
Related
Documentation
Safety and Special
Notices
In addition to this guide, Thermo Electron provides the following
documents for the Surveyor Plus system:
•
Surveyor Plus Preinstallation Requirements Guide
•
Surveyor Plus Getting Connected
•
Surveyor Autosampler Plus Hardware Manual
•
Surveyor LC Pump Plus Hardware Manual
•
Surveyor MS Pump Plus Hardware Manual
•
Surveyor UV/Vis Plus Detector Hardware Manual
•
Surveyor PDA Plus Detector Hardware Manual
•
Surveyor UV/Vis Plus Detector Hardware Manual
Make sure you follow the precautionary statements presented in this guide.
The safety and other special notices appear in boxes.
Safety and special notices include the following:
CAUTION Highlights hazards to humans, property, or the environment.
Each CAUTION notice is accompanied by an appropriate CAUTION
symbol.
Thermo Electron Corporation
Surveyor Plus Getting Started with Xcalibur
xv
Preface
Contacting Us
IMPORTANT Highlights information necessary to avoid damage to
software, loss of data, invalid test results, or information critical for
optimal performance of the system.
Note Highlights information of general interest.
Tip Helpful information that can make a task easier.
Contacting Us
Assistance
There are several ways to contact Thermo Electron Corporation.
For new product updates, technical support, and ordering information,
contact us in one of the following ways:
Visit Us on the Web
www.thermo.com/finnigan
Contact Technical Support
Phone:
Fax:
E-mail:
1-800-685-9535
1-561-688-8736
[email protected]
Find software updates and utilities to download at
http://mssupport.thermo.com
Contact Customer Service
In the US and Canada for ordering information:
Phone:
1-800-532-4752
Fax:
1-561-688-8731
Web site: www.thermo.com/finnigan
Changes to the Manual
and Online Help
xvi
Surveyor Plus Getting Started with Xcalibur
To suggest changes to this guide or to the online Help, use either of the
following methods:
•
Fill out a reader survey online at www.thermo.com/lcms-techpubs
•
Send an e-mail message to the Technical Publications Editor at
[email protected]
Thermo Electron Corporation
Chapter 1
Introduction to the Surveyor
Plus Integrated LC/MS
System
The Surveyor Plus LC system, which integrates with the Thermo Electron
family of mass spectrometers, consists of an analytical pump, an
autosampler, an optional photodiode array detector or dual wavelength
UV/Vis detector, and a solvent platform. See Figure 1.
Solvent Platform
Detector
Autosampler
Analytical Pump
Figure 1.
Thermo Electron Corporation
Surveyor Plus LC instrument
Surveyor Plus Getting Started with Xcalibur
1
1
Introduction to the Surveyor Plus Integrated LC/MS System
Two analytical pumps are available: the Surveyor LC Pump Plus and the
Surveyor MS Pump Plus. Each pump is a dual-piston, quaternary,
low-pressure mixing solvent delivery system with a built-in vacuum degasser
and pulse dampener. The LC pump operates in the flow rate range from 1
to 9999 μL/min. It provides precise gradients over a flow rate range of 200
to 2000 μL/min. The MS pump operates in the flow rate range from 1 to
2000 μL/min. It provides precise gradients in the flow rate range from 25 to
800 μL/min.
Two autosamplers are available: the Surveyor Autosampler Plus and the
Surveyor Autosampler Plus Lite. Both autosamplers automate sample
injections and sample preparation. Their tray compartments accept various
vials, 96-well plates, and 384-well plates. In addition to these features, the
full-featured Surveyor Autosampler Plus includes a built-in column oven
(5 to 95 °C) and sample compartment temperature control (0 to 60 °C).
Two detectors are available: the Surveyor PDA Plus Detector and the
Surveyor UV/Vis Plus Detector. The Surveyor PDA Plus Detector, in
combination with the 5 cm LightPipe flowcell, provides the highest level of
sensitivity available in photodiode array detection for HPLC. The Surveyor
UV/Vis Plus Detector is a dual-wavelength UV/Vis detector. Both detectors
have dual-lamp optical bench that covers the UV-visible spectrum from 190
to 800 nm.
The solvent platform, which is located on the top of the Surveyor Plus LC
stack, holds four 1-L solvent reservoir bottles and one 1-L wash bottle. Four
1/8-in. OD, 1/16-in. ID, FEP solvent lines carry solvent from the reservoir
bottles down to the vacuum membrane degasser, which is built into the
analytical pump. One 1/8-in. OD, 1/16-in. ID, FEP solvent line carries
solvent from the wash bottle to the 2-position syringe valve of the
autosampler.
This chapter provides a brief description of the Surveyor Plus LC system
and contains the following sections:
• Analytical Pump
• Surveyor Autosampler Plus
• Surveyor PDA Plus Detector
• Surveyor UV/Vis Plus Detector
• Communication with Xcalibur
• Synchronizing the Surveyor LC Devices
• Status LEDs
2
Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Analytical Pump
Introduction to the Surveyor Plus Integrated LC/MS System
Analytical Pump
Thermo Electron offers two analytical pumps in the Surveyor Plus series:
the Surveyor MS Pump Plus, which provides optimum performance in the
lower flow rate ranges needed for mass spectrometry and the Surveyor LC
Pump Plus, which provides optimum performance in the flow rate ranges
needed for chromatography.
Both analytical pumps contain a built-in solvent degassing system that
consists of four independent chambers maintained at a constant vacuum of
approximately 27-in. Hg. Each chamber contains an 18-in. length of
0.045-in. ID Teflon® AF tubing. This translates to a volume of less than
500 μL per channel, adding very little to the quantity of solvent that is
required to purge the lines when you replace an eluent.
The distinguishing features of these analytical pumps are described in the
following topics contained in this section:
• Surveyor MS Pump Plus
• Surveyor LC Pump Plus
Surveyor MS Pump Plus
The Surveyor MS Pump Plus is a dual-piston, quaternary, low-pressure
mixing pump with a built-in vacuum degasser and pulse dampener. The
pumping system provides flow rates from 0.1 to 2000 μL/min, the range
needed to perform LC/MS applications. You can run precise gradients from
25 to 800 μL/min.
The built-in pulse dampening assembly consists of a low volume
T-connector through which the mobile phase passes. Attached to the side
leg of the T-connector is a permeable isolation membrane made of sintered
Teflon followed by a 2 mL coil of stainless steel tubing. The coil is
terminated with a priming valve. When the priming valve is open, the
dampening coil can be flushed or filled with an appropriate solvent such as
methanol or isopropanol. When the priming valve is closed, the diffusion
barrier cartridge is shut off from the flow path and the dampening coil
absorbs pump pulsations. Even at elevated pressures, the pump will show
only minimal pulsation when the pulse dampener is used. Because the 2 mL
coil is shut off from the flow path, the low volume T-connector of the pulse
dampener adds only 3 μL of delay volume to your system.
The Surveyor MS Pump Plus is remotely controlled by way of an RS232
serial communication link from a PC using Xcalibur software (version 1.3
or higher). The only manual control is the power switch located on the
front of the pump in the lower left corner below the door. See Figure 2.
Thermo Electron Corporation
Surveyor Plus Getting Started with Xcalibur
3
1
Introduction to the Surveyor Plus Integrated LC/MS System
Analytical Pump
Power Switch
Figure 2.
Vacuum
Degasser
Liquid
Displacement
Assembly
Pulse
Dampener
Front view of Surveyor MS Pump Plus
Surveyor LC Pump Plus
The Surveyor LC Pump Plus is a dual-piston, quaternary, low-pressure
mixing pump with a built-in vacuum degasser and pulse dampener. The
pumping system provides flow rates from 0.001 to 9.999 mL/min, the
range that is needed to perform high performance liquid chromatography.
You can run precise gradients from 0.200 to 2.000 mL/min.
The Surveyor LC Pump Plus is a bench-top unit for inclusion in the
Surveyor Plus high performance liquid chromatograph (HPLC). It is
remotely controlled by way of an Ethernet communication link from a PC
using Xcalibur (version 2.0 or higher). The only manual control is the
power switch located on the front of the pump in the lower left corner
below the door. Direct control of the pump is achieved through the
Xcalibur data system.
4
Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Introduction to the Surveyor Plus Integrated LC/MS System
Analytical Pump
The Surveyor LC Pump Plus, shown in Figure 3, has five major
components: the vacuum degassing assembly; the solvent proportioning
assembly; the pump head assemblies; the purge manifold assembly; and the
pulse dampening assembly, which adds 400 μL of gradient delay volume to
the system. In addition, the LC pump has status LEDs on the front door,
and a low voltage power supply.
Note Do not overtighten the fingertight fitting that connects the LC
pump to the autosampler. See Figure 3. Overtightening this fitting will
cause leaking.
Solvent Lines Entering and Exiting
the Degassing Assembly
IN
Pump Head
Assemblies
Line Body
Containing
Filter Frit
OUT
A
N
PE
DRAIN
O
SE
Right
LO
Left
C
B
C
Purge
Manifold
Assembly
D
On/Off Switch
Figure 3.
Solvent
Proportioning
Assembly
Pulse Dampening Assembly
(Behind the Front Panel)
Front view of Surveyor LC Pump Plus
Thermo Electron Corporation
Surveyor Plus Getting Started with Xcalibur
5
1
Introduction to the Surveyor Plus Integrated LC/MS System
Analytical Pump
You can purge the LC pump from the Xcalibur data system after you open
the purge valve. You open the purge valve by turning the knob on the front
of the purge manifold assembly counter-clockwise. See Figure 4.
Open Position
LO
SE
PE
N
C
Figure 4.
DRAIN
O
PE
N
DRAIN
O
SE
LO
C
Closed Position
Drain valve positions
Figure 5 shows the line body fitting, located at the top of the purge
manifold assembly. The line body fitting contains an in-line filter frit that
captures particulate matter shed by the pump seals. This prevents particulate
matter from contaminating the tubing that connects the pump to the
autosampler. As this frit becomes clogged, your system backpressure rises.
Replace this frit when your system backpressure rises above its typical
operating limits.
Figure 5.
6
Surveyor Plus Getting Started with Xcalibur
Line body and filter frit
Thermo Electron Corporation
1
Surveyor Autosampler
Plus
Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
The Surveyor Autosampler Plus allows you to inject samples and perform
sample dilutions automatically. Thermo Electron offers two Surveyor Plus
autosampler models: the full-featured Surveyor Autosampler Plus and the
Surveyor Autosampler Plus Lite. The full-featured autosampler includes a
built-in column oven (5 to 95 °C) and tray/sample temperature control
(0 to 60 °C). The autosampler tray compartment, the injection system, the
injection modes, and the temperature control features are described in the
following topics:
• Tray Compartment
• Injection System
• Injection Modes
• Temperature Control
Tray Compartment
The tray compartment can hold up to five conventional sample trays or one
microwell carrier. The tray compartment also holds up to four 12 mL
capacity reservoir vials that can be used to hold solvent, reagent, or diluent.
The reservoir vials are located behind the wash station and are designated
RV1, RV2, RV3, and RV4. See Figure 6.
XYZ Arm
Mechanism
RV4
RV3
RV2
RV1
Figure 6.
Thermo Electron Corporation
Wash
Station
Injection
Port
Tray compartment, showing brackets for needle tubing assembly
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Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
The five conventional sample trays, from the left side to the right side of the
tray compartment, are designated A, B, C, D, and E. Each sample tray holds
up to 40 standard 1.8 mL vials for a total capacity of 200 samples. Overlays
allow the sample trays to accommodate different vial sizes. The microwell
carrier can hold up to three low-density 96-well microplates or up to three
high-density 384-well microplates. The microplates are designated A, B,
and C.
The tray compartment door contains a magnetic switch. The magnet is
located in the door and the switch is attached to the chassis. When you open
the door, the switch signals the autosampler that the door is open.
If you select the Verify Door Is Closed check box when you configure the
autosampler, the XYZ arm will automatically move to the back of the tray
compartment when you open the door so that you can remove trays or
replace vials. Opening the door while the autosampler is making an
injection does not interrupt the current run. The XYZ arm moves to the
back of the compartment after the injection is complete, whereupon the
sequence is halted and the tray can be removed. After you close the
autosampler door, the sequence resumes.
Injection System
The main components of the injection system include the following:
• Syringe Drive Assembly and Syringe Valve
• Wash Bottle Reservoir and Tubing
• Interchangeable Syringe
• XYZ Arm Mechanism
• Needle Assembly and Needle Tubing Assembly
• Injection Port and Transfer Tubing Assembly
• Injection Valve and Sample Loop
8
Syringe Drive Assembly and
Syringe Valve
The syringe drive assembly consists of a stepper motor drive mechanism, a
syringe valve, and fittings that hold the interchangeable syringe. The syringe
drive assembly is mounted to the front of the autosampler with three
mounting pins. On the back of the assembly, holes with grommets mate
with these pins. These rubber grommets minimize the effects of vibration
and cause the connection between the syringe drive assembly and the body
of the autosampler to feel slack. See Figure 7.
Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
Wash Bottle Tubing
Two-Way
Syringe Valve
10-Pin Connector
Needle
Tubing
Syringe
Fitting
Front
Figure 7.
Mating Hole
to Vertical
Mounting Stud
Mating Holes
to Horizontal
Mounting Studs
Syringe drive assembly, showing front and back of assembly
The syringe valve is a 2-position rotary valve. In the wash bottle position,
solvent is drawn into the syringe barrel as the syringe plunger descends. In
the needle position, sample is drawn into the needle tubing as the syringe
plunger descends and is pushed out of the needle tubing assembly as the
syringe plunger ascends. Sample is never drawn into the barrel of the
syringe.
Note The needle tubing holds approximately 560 μL of solvent. If you
have installed the 2500 μL standard syringe (which must be special
ordered) and you plan to make large volume injections, ensure that you
have also installed the 1-mL needle tubing extension that is shipped with
this syringe.
Wash Bottle Reservoir and
Tubing
Thermo Electron Corporation
The wash bottle rests in the solvent platform on the top of the Surveyor Plus
tack. It is connected to the syringe valve by way of the wash bottle tube.
Both of the direct commands, Flush (from bottle) and Wash Needle (from
bottle), draw solvent from the wash bottle. In addition, both the partial loop
injection and the no waste injection modes draw transfer solution from the
wash bottle. If the wash bottle runs dry, the wash bottle tubing and the
syringe barrel will fill with air. If the syringe runs dry, the autosampler
cannot draw sample into the needle tubing.
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Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
Interchangeable Syringe
The standard configuration for the Surveyor Autosampler Plus consists of a
250 μL concentric syringe. The concentric syringe consists of a small, inner
plunger, and a larger, outer plunger. See Figure 8.
Inner Plunger
Outer Plunger
Figure 8.
Concentric Syringe
The inner plunger is used to draw and deliver sample amounts equal to or
less than the maximum capacity of the syringe, which is 265 μL for the
250 μL concentric syringe. The outer plunger is used to draw and expel
large volumes of solvent.
Concentric syringes are available in 100, 250, and 500 μL sizes. In addition,
a 2500 μL standard syringe is available. The volume of the outer plunger
region, which is 565 μL, is the same for all three concentric syringes. The
size of the inner plunger and the injection mode determine the available
injection volume range.
Because injections are always performed by using the inner plunger of the
syringe, the size of the syringe does not affect the precision of an injection.
However, for sample preparation routines, the outer plunger of the syringe
is used for solvent transfers larger than the nominal size of the syringe.
Solvent transfers performed with the outer plunger of the syringe are less
precise than those performed with the inner plunger. Therefore, you might
want to order the 500 μL concentric syringe or the 2500 μL standard
syringe if you plan to use the autosampler to routinely perform sample
dilutions.
XYZ Arm Mechanism
10
Surveyor Plus Getting Started with Xcalibur
The XYZ arm mechanism moves the needle along the x-y plane to the
requested vial or well location. After the needle is positioned above the vial
or well, the XYZ arm mechanism lowers the needle along the z-axis to the
Thermo Electron Corporation
1
Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
requested needle height. After the needle withdraws sample, the XYZ arm
mechanism moves along the x-y plane, back to the home position, which is
above the injection port of autosampler. The needle is then lowered into the
injection port where it expels the sample.
IMPORTANT Because the XYZ arm moves to the sample position to
withdraw sample, it is important that you do not place objects taller
than 1.8 inches into the tray compartment. Tall objects will stall the
XYZ arm.
If you select the Verify Door Is Closed check box when you configure the
autosampler, the XYZ arm will automatically move to the back of the tray
compartment when you open the door so that you can remove trays or
replace vials.
When you submit a Wash Needle direct command from the data system,
the XYZ arm mechanism moves the needle to the wash station position.
When you submit a Remove Needle direct command from the data system,
the XYZ arm mechanism moves to the middle front of the tray
compartment to allow easy access to the needle tubing assembly.
Needle Assembly and Needle
Tubing Assembly
The needle assembly consists of a blunt-tip needle, a latch nut, a flag, a
compression spring, and a 10-32 fitting that connects to the needle tube
assembly. See Figure 9. The needle is inserted into the needle mount on the
XYZ arm.
Fitting to Syringe Valve
Needle Assembly
10-32 Fitting
PEEK Fitting
to Needle Assembly
Figure 9.
Needle Guide
Needle tubing assembly and needle assembly
Thermo Electron Corporation
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Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
The needle tube assembly consists of a piece of low-pressure tubing, a
PEEKTM fitting that connects to the needle assembly, a fitting and
flangeless ferrule that connects to the right side of the syringe valve, and a
needle tube guide that is inserted into the X-axis positioning frame. To
prevent the needle tubing from interfering with the movement of the XYZ
arm, the needle tubing is secured with a clip on the Z-axis of the XYZ arm
and a clip on the X-axis positioning frame. In addition to routing the needle
tubing through these clips, make sure that you route the needle tubing
behind the syringe drive assembly as you mount it.
Injection Port and Transfer
Tubing Assembly
The injection port of the autosampler is located behind the syringe drive
assembly. A 0.012-in. ID transfer tube connects the injection valve of the
autosampler to port 2 of the Valco injection valve. See Figure 10.
Note The label attached to the transfer tube assembly specifies its
internal volume. You must enter this value in the Surveyor Autosampler
Plus Configuration dialog box. See “Configuring the Surveyor
Autosampler Plus” on page 28.
Wash
Station
Transfer Tube Assembly Label
(specifying the Dead Volume)
Injection
Port
22µL
Transfer
Tube
Figure 10. Injection port and transfer tube
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Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Injection Valve and Sample Loop
Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
The injection valve is a six-port Valco valve that introduces sample onto the
column by way of the sample loop. The sample loop is a section of stainless
steel tubing with end fittings. It is an interchangeable part that is attached to
the Valco valve at ports 1 and 4. The Surveyor Autosampler Plus ships with
a 25 μL sample loop. The available sizes are listed in Table 1.
Table 1.
Available sample loop sizes
Sample Loop Size (μL)
Part Number
5
00109-99-00007
10
00109-99-00008
20
00109-99-00009
25
00109-99-000010
50
00109-99-000011
100
00109-99-000012
500
00109-99-000013
1000
00109-99-000014
There are two positions for the six-port injection valve:
• Fill Position
• Inject Position
Fill Position
In the fill position, the sample loop is isolated from the mobile phase
stream. As the mobile phase bypasses the sample loop, sample is pushed into
the front of the loop that is connected to port 1 of the Valco injection valve.
After the loop is filled, excess solution is pushed out the back of the loop to
waste. See Figure 11.
Transfer Tubing
1
2
To
Column
6
3
To
Waste
4
Mobile Phase
Wash Solvent
5
From
Pump
Figure 11. Injection valve in the fill position
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Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
Inject Position
In the inject position, mobile phase enters the sample loop from the back,
backflushing the contents of the loop onto the column. Excess sample left in
the transfer tube is expelled directly to waste. To allow ample rinsing of the
sample loop with mobile phase, the injection valve remains in the inject
position during the entire run. See Figure 12.
Transfer Tubing
1
2
To
Column
6
3
To
Waste
4
Mobile Phase
Wash Solvent
5
From
Pump
Figure 12. Injection valve in the inject position
Injection Modes
The Surveyor Autosampler Plus can operate in any of the following
three modes:
• No Waste Injection Mode
• Partial Loop Injection Mode
• Full Loop Injection Mode
The optimum injection mode depends upon the amount of sample that you
have and the degree of precision that your application requires.
No Waste Injection Mode
The no waste injection mode is a technique that withdraws only the exact
amount of sample requested from the sample vial. Of the three injection
modes, the no waste injection mode uses the least amount of sample, but it
is also the least precise. Use this injection mode to conserve sample. The
minimum allowable injection volume is 0.1 μL.
Note To perform accurate no waste injections, use a sample loop that is
at least 5 μL larger than the injection volume.
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Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Partial Loop Injection Mode
Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
The partial loop injection mode is a technique that withdraws 22 μL of
excess sample from the vial in addition to the requested injection volume.
Approximately one-half of the excess volume is expelled to waste before the
center of the sample bolus is metered into the front of the sample loop. The
second portion of excess sample is expelled to waste after the sample bolus is
backflushed onto the column.
Partial loop injections are useful when you have a limited volume of sample.
Using the partial loop injection mode, you can inject variable amounts of
sample, ranging from a minimum of 0.1 μL to a working maximum of
one-half the volume of your sample loop. This maximum volume limitation
is caused by the laminar flow of fluid within the stainless steel sample loop.
Note To make precise partial loop injections, use a sample loop that is at
least twice the size of the injection volume.
Full Loop Injection Mode
The full loop injection mode is a technique that withdraws a sample volume
from the vial that is sufficient to overfill the sample loop by a minimum
factor of two. Because the actual injection volume is determined by the
physical size of the sample loop, not the metering action of the stepper
motor, a full loop injection is very reproducible. However, because the
intent of the full loop injection mode is to completely fill the sample loop,
you cannot inject variable amounts of sample.
Full loop injection is useful when you want maximum precision and have
unlimited sample. If you want to change the injection volume, you must
change the sample loop size. For the available loop sizes, see Table 1 on
page 13.
Note In Xcalibur, full loop injections are limited to the size of the
configured sample loop.
In the full loop injection mode, the autosampler withdraws a large excess of
solution from the sample vial according to the following equation:
Amount Withdrawn = 3 × Injection Volume + Dead Volume + 7.5 μL
Where:
Dead Volume = Volume of Transfer Tube + Volume of Injection Port
and Rotor Slot
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Surveyor Plus Getting Started with Xcalibur
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1
Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor Autosampler Plus
This equation is valid until the maximum capacity of the syringe is reached,
at which point only the maximum capacity of the syringe is withdrawn. The
maximum capacity of the 250 μL concentric syringe is 265 μL.
Temperature Control
The full-featured Surveyor Autosampler Plus has two built-in temperature
control features:
• Tray Temperature Control
• Column Oven Control
Tray Temperature Control
Column Oven Control
The tray temperature control feature provides temperature control of the
samples in the range from 0 to 60 °C. A Peltier device maintains the tray
temperature.
The built-in column oven controls the temperature of the air surrounding
the chromatographic column. Isothermal temperature control is achieved
using a Peltier device. The Peltier device is a solid-state, heat-transferring
assembly used to heat or cool the column oven. The range of temperature
control is 5 to 95 °C.
Between the analytical pump and the autosampler injection valve, the
mobile phase is diverted through approximately 120 cm of 0.020-in. ID
stainless steel high-pressure tubing that is located behind the column oven.
As it passes through this tubing, the mobile phase equilibrates to the
temperature of the column oven before it reaches the Valco injection valve.
Although it is useful for temperature sensitive applications, this additional
tubing also adds 250 μL of gradient delay volume to the Surveyor Plus LC
system. If you are performing low flow gradient applications, you might
want to bypass this tubing by connecting the outlet of the pump directly to
port 5 of the Valco injection valve.
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Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Surveyor PDA Plus
Detector
Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor PDA Plus Detector
The Surveyor PDA Plus Detector is a full-featured, time-programmable,
photodiode array detector. Detection can be carried out in the full
ultraviolet-visible range from 190 to 800 nm. Data can be taken in this
range at a rate of up to 20 Hz with 20-bit digital conversion.
The optimal location for the PDA detector is above the autosampler and
below the solvent platform. The PDA detector is controlled remotely over
an Ethernet communication link from a PC using the Xcalibur software.
The PDA detector consists of a dual-light source, an optical bench, a
photodiode array, a low voltage power supply, several printed circuit boards
(PCBs), and four status LEDs.
The dual-light source includes a deuterium lamp for detection in the
ultraviolet wavelength range (190 to 360 nm) and a tungsten-halogen lamp
for detection in the visible wavelength range (360 to 800 nm). There is
some overlap between the two lamps in the 300 to 500 nm range.
Attenuators are used to adjust the intensity of light reaching the photodiode
array.
The optical bench contains a beam combiner, focusing lens, filter wheel,
flow cell, beam shaper, folding mirror and grating. The beam combiner
reflects the light coming from the tungsten-halogen lamp so that it is
parallel to and coincident with the light from the deuterium lamp. The
combined beam is then focused on the inlet window of the flow cell through
the filter wheel. The standard filter wheel has two positions. Position 1
(Open) is used for normal operation. Position 2 contains a sealed, quartz
cuvette filled with a holmium oxide / perchloric acid solution (NIST
Traceable) and is used for wavelength accuracy verification and calibration.
See Figure 13.
Beam
Combiner
Focusing
Lens
Filter
Wheel
Beam
Shaper
Flow Cell
Deuterium
Lamp
Attenuators
Folding
Mirror
1 x 512
Photo Diode
Array
Fixed
Grating
Tungsten-Halogen
Lamp
Figure 13. Optical Bench of the Surveyor PDA Plus detector
Thermo Electron Corporation
Surveyor Plus Getting Started with Xcalibur
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Introduction to the Surveyor Plus Integrated LC/MS System
Surveyor UV/Vis Plus Detector
Surveyor UV/Vis Plus
Detector
The Surveyor UV/Vis Plus Detector is a full-featured, time-programmable,
variable-wavelength UV/Vis (ultraviolet / visible) absorbance detector. It
operates in either the single wavelength mode or the dual wavelength mode.
The wavelength range in the single wavelength mode is 190 nm to 800 nm.
In the dual wavelength UV mode, the range is 190 nm to 450 nm. In the
dual wavelength Visible mode, the range is 366 nm to 700 nm.
The wavelength time table is available in all three modes. The time table can
contain up to 10 lines. If the Zero On Wavelength Change feature is
enabled, the absorbance of the baseline is re-zeroed between each line in the
time table, even if the wavelengths remain the same. The absorbance of the
baseline is not zeroed between the last two lines in the table.
To provide a complete spectrum of ultraviolet and visible light, the detector
uses a deuterium lamp for the UV range (190-365 nm) and a tungsten lamp
for the visible range (366-800 nm). The lamps are protected by a cover with
a special safety interlock to reduce the possibility of human exposure to
harmful UV light.
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Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Communication with
Xcalibur
Introduction to the Surveyor Plus Integrated LC/MS System
Communication with Xcalibur
Communication with the Xcalibur data system is established with an
Ethernet connection for the Surveyor LC Pump Plus, the Surveyor
Autosampler Plus, the Surveyor PDA Plus Detector, and the Surveyor
UV/Vis Plus Detector. To establish communications with each of these
devices, you must connect one end of a Ethernet cable (shielded, category 5,
RJ-45, 7-ft. length cable with ferrite) to the Ethernet port on the back panel
of the device and the other end of the cable to an Ethernet switch. The
Ethernet switch is connected by way of a second Ethernet cable to the PC
on which the Xcalibur data system resides.
The Xcalibur data system is connected to the Surveyor MS Pump Plus with
a serial communication link. The MS pump accessory kit contains a serial
cable with nine pin adaptors on each end.
In addition to connecting the communication cables, you must also enter
the appropriate stack addresses when you configure the devices of your
instrument. The stack address that you enter when you add a device to the
configuration for your instrument must match the Unit ID setting on the
back panel of the device. For more information on configuring the Surveyor
Plus LC devices, see Chapter 2, “Configuring Your Surveyor Plus LC
Instrument.”
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Surveyor Plus Getting Started with Xcalibur
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1
Introduction to the Surveyor Plus Integrated LC/MS System
Synchronizing the Surveyor LC Devices
Synchronizing the
Surveyor LC Devices
A system interconnect cable is used to coordinate the run control signals
between the Surveyor Plus devices.
Thermo Electron provides two versions of this cable: the older version has
5-combicon connectors and the newer version has 7-combicon connectors.
See the Surveyor Plus Getting Connecting manual for details on connecting
one of these cables to the devices in your Surveyor Plus stack.
During a run the system issues the following sequence of run control signals:
1. A request to perform an injection is issued from the data system PC.
2. When the autosampler becomes ready, it issues the A/S Ready signal.
The autosampler goes into the Ready state when all of the configuration
and Instrument Method conditions are met. These conditions can
include the following: the tray door is closed, and the sample tray and
column oven temperature zone readings are within tolerance of their
setpoints.
3. When the pump pressure stabilizes, the pump issues the Pump Ready
signal to the autosampler.
Note The Surveyor LC Pump Plus issues a Pump Ready signal. The
Surveyor LC Pump does not issue this signal.
The autosampler switches the injection valve to the load position, and
then loads sample into the sample loop.
4. The autosampler issues the Gradient Start signal to the pump. This
signal commands the pump to start its gradient program.
5. When its piston cam reaches the home position, the pump issues the
Inject Hold release signal to the autosampler.
Note The Surveyor LC Pump Plus issues an Inject Hold Release
signal. The Surveyor LC Pump does not issue this signal.
6. The autosampler injects the sample and issues a momentary Inject Out
signal to the detector.
The injection valve of the autosampler switches to the inject position,
allowing the mobile phase to backflush the contents of the sample loop
onto the column. The detector starts to collect data.
20
Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
1
Status LEDs
Table 2.
Introduction to the Surveyor Plus Integrated LC/MS System
Status LEDs
Each of the Surveyor Plus LC devices has a panel of four status LEDs
(light-emitting diodes) located on its front-right door. All of the devices
have these three LEDs: Power, Comm, and Run. The fourth LED on the
detector is the Lamps LED. The fourth LED on the autosampler in the
Temp LED. The fourth LED on the analytical pump is the Degas LED.
The states of these status LEDs and their meanings are described in Table 2.
States of the status LEDs on the front panels of the Surveyor devices
Location
All Devices
LED
State
Meaning
Power
Steady Green
The Power switch is switched on and the detector is receiving power.
Amber
The detector is not receiving power.
Steady Green
Communication to the data system has been established.
Amber
There is no communication with the data system.
Flashing Amber
Firmware is being downloaded to the CPU of the detector. The rotary switches on the
back panel of the detector are set to 00.
Steady Green
The power is on and no run is in progress.
Flashing Green
A run is in progress
Flashing Amber
An error condition has occurred.
Steady Green
One or both lamps are On.
Amber
The lamp specified in the method is not On.
Steady Green
The autosampler is in the steady state.
Flashing Green
An injection or a timed event is in progress.
Flashing Amber
An error condition has occurred.
Steady Green
The column oven and tray temperature zones are in equilibrium at the set
temperature. You must select the Wait for temperature ready check box when you
configure the autosampler for this feature to be active. If the Wait for temperature
ready feature is not enabled, the Temp LED remains green.
Steady Amber
A temperature change is in progress. The column oven, or the tray temperature zones,
or both are reaching the set temperature.
Steady Green
The pump is On and no run is in progress.
Flashing Green
An run is in progress.
Flashing Amber
An error condition has occurred.
Steady Amber*
There is no communication with the data system PC, the motor is stopped, the pistons
are homing, the system pressure is stabilizing, or the pump is being purged.
Steady Green
The Degas LED changes to green when sufficient vacuum has developed for
chromatography to be performed.
Steady Amber
The degas unit is building vacuum.
Flashing Amber
The degasser has lost vacuum.
Comm
Run
Detectors
Lamps
Run
Autosampler
Temp
Run
Analytical Pump
Degas
* This LED state pertains to the Surveyor LC Pump Plus only.
Thermo Electron Corporation
Surveyor Plus Getting Started with Xcalibur
21
Chapter 2
Configuring Your Surveyor
Plus LC Instrument
After you set up and connect your Surveyor Plus LC instrument, you are
ready to configure the devices of your instrument. This chapter contains the
information for configuring the Surveyor Plus LC devices of your integrated
Surveyor Plus LC/MS instrument. For information on configuring your
MS detector, see its online Help.
Before you configure your instrument, check the unit ID setting on the rear
panel of each device. The unit ID consists of two rotary switches, which are
set to 01 at the factory. See Figure 14.
2 3
0 1
9
5 6
5 6
9
4
4
0 1
2 3
7 8
7 8
Unit ID
(set to 1)
Figure 14. Unit ID with a stack address of 1
This chapter contains the following sections:
• Checking the Communication Hardware
• Configuring the Surveyor Plus LC Devices
Thermo Electron Corporation
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2
Configuring Your Surveyor Plus LC Instrument
Checking the Communication Hardware
Checking the
Communication
Hardware
The Surveyor LC Pump Plus, Surveyor Autosampler Plus, and Surveyor
PDA Plus Detector communicate with the Xcalibur data system by way of
an Ethernet connection. Each device has an Ethernet port on its back panel.
A shielded, CAT 5 Ethernet cable with ferrite is used to connect each device
to an Ethernet switch, which in turn is connected by way of an Ethernet
cable to the data system computer. The Unit ID setting on the back panel of
the LC pump, autosampler, and detector must match the Stack Number in
the configuration.
Unlike the other Surveyor Plus devices, the Surveyor MS Pump Plus
communicates with the Xcalibur data system through an RS232 serial
connection.
Before you configure your instrument from the data system, do the
following:
1. Check the setting of the rotary switches on the back panels of your
devices.
2. If you are using the MS pump, verify that the serial communication
cable connecting the MS pump to your PC is attached to COM port 1.
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Thermo Electron Corporation
2
Configuring the
Surveyor Plus LC
Devices
Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
To configure your Surveyor Plus LC Instrument, perform the following
procedures in this section as listed:
1. Opening the Instrument Configuration Dialog Box
2. Adding Devices to the Instrument Configuration
3. Configuring the Devices
4. Exiting the Instrument Configuration Dialog Box
Opening the Instrument
Configuration Dialog Box
To open the Instrument Configuration dialog box
• From the Windows taskbar, choose Start > All Programs > Xcalibur >
Instrument Configuration.
• Alternatively, from the Windows desktop, double-click the Instrument
Configuration icon.
The Instrument Configuration dialog box is displayed. The Instrument
Configuration dialog box shown in Figure 15 contains a list of all the
available devices that were installed with the Xcalibur data system when it
was loaded onto your computer.
Go to the next topic: Adding Devices to the Instrument Configuration.
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2
Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
Figure 15. Instrument Configuration dialog box
Adding Devices to the
Instrument Configuration
To add the devices of your Surveyor Plus instrument to its configuration,
double-click each of your Surveyor Plus devices in the Available Devices
list box.
As you double-click a device in the Available Devices list, it is added to the
list of configured devices. See Figure 16.
After you add the devices that make up your Surveyor Plus LC system to the
Configured Devices list, go to the next topic: Configuring the Devices.
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2
Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
Figure 16. Instrument Configuration dialog box, showing added devices
Configuring the Devices
Now that you have added your LC devices to the Configured Devices list,
you are ready to specify configuration options for each device.
Perform the procedures provided in this topic that apply to the devices of
your instrument:
• Configuring the Surveyor Autosampler Plus
• Configuring the Surveyor MS Pump Plus
• Configuring the Surveyor LC Pump Plus
• Configuring the Surveyor PDA Plus Detector
• Configuring the Surveyor UV/Vis Plus Detector
You can configure the specific devices in any order. After you finish
configuring the LC devices, go to “Exiting the Instrument Configuration
Dialog Box” on page 38.
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
Configuring the Surveyor
Autosampler Plus
To configure the Surveyor Autosampler Plus
1. Double-click the Surveyor AS button in the Configured Devices
list box.
Xcalibur opens the Surveyor Autosampler Configuration dialog box
with the Tray page displayed. See Figure 17.
Figure 17. Surveyor Autosampler Configuration dialog box - Tray page
2. Select the tray options:
a. From the Tray Type list, select the tray type.
Table 3 lists the 11 tray type selections.
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Table 3.
Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
Tray type selections
Tray Type
Requires Well Bottom
Distance Calibration
1.8 mL Vial, 5 trays, 40 vials each
No
Custom Vial Setting
Yes
96 Well Microplate + Tall Microwell Carrier
No
96 Well Microplate + Short Microwell Carrier +Riser Plate
No
1 mL or 2 mL Deep Well Plate + Short Microwell Carrier
No
96 Well Microplate + Short Microwell Carrie
No
96 Well PCR Plate + Cooling Adapter + Short Microwell Carrier
No
Custom 96 Well Setting
Yes
384 Well Microplate + Tall Microwell Carrier
No
384 Well Microplate + Short Microwell Carrier + Riser Plate
No
Custom 384 Well Setting
Yes
b. If you chose the 1.8 mL Vial option or the Custom Vial Setting,
proceed to step 3. Otherwise continue at step 2c.
c. In the A1 Well Position area, select either the Top Left or the
Bottom Right option button.
d. In the Well Ordering area, click the button that represents the
order in which you want the sequence injections to occur.
3. Click the Communication tab to display the Communication page
shown in Figure 18.
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
Enter the value specified on
the label attached to the
transfer tube assembly.
Figure 18. Surveyor Autosampler Configuration dialog box – Communication
page
4. In the Communication page, make the following entries and selections:
a. In the Stack Address box, type the appropriate stack address or use
the up down arrows to select the appropriate stack address.
The stack address must match the Unit ID setting located on the
back panel of the Surveyor Autosampler Plus. The Surveyor
Autosampler Plus is shipped with the Unit ID set to 01. The value
of 00 is reserved for service functions.
b. From the Syringe Type list, select the size of the syringe that is
attached to the autosampler.
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
c. (optional) To activate the optional Wait For Temperature Ready
control, select the Wait For Temperature Ready check box.
Activating the Wait For Temperature Ready control prevents the
Surveyor Autosampler Plus from triggering a run until the column
oven temperature and/or the sample tray temperature have reached
their setpoint values.
d. (optional) To activate the Verify Door Is Closed control, select the
Verify Door Is Closed check box.
Activating the Verify Door Is Closed control prevents the Surveyor
Autosampler Plus from starting a run until the tray door is closed. If
the tray door is opened during a run, then the XYZ arm moves to its
home position.
e. (optional) To activate the maintenance log, select the Enable
Maintenance Log check box.
Activating the maintenance log allows the Surveyor Autosampler
Plus to keep an internal count of the total injections, total valve
cycles, total needle usage, and total syringe cycles. If any of the
counters exceed the user set Scheduled Maintenance Time (SMT),
the autosampler will not start a run until you perform the scheduled
maintenance or you clear the check box.
f. In the Vial Bottom Sensing area, select the type of vial bottom
sensing that is appropriate for your application:
− Select the On option to activate vial bottom sensing for every
injection in a sequence.
− Select the Auto option to activate vial bottom sensing for only
the first injection in a sequence.
− Select the Off option to deactivate vial bottom sensing.
Each tray type has a stored value for the distance that the needle
must travel to reach the bottom of the vial or well. If you activate
vial bottom sensing, the autosampler performs a search routine to
determine the actual location of the vial or well bottom. If the search
routine determines a new value for the bottom distance, it is stored
until the tray type is modified. Deactivate vial bottom sensing if you
do not want the needle to touch the bottom of a vial or well.
g. In the Dead Volume box, enter the value specified on the label
attached to the transfer tube assembly. See Figure 10 on page 12.
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
h. In the Sample Loop Volume box, type the nominal size of the
sample loop attached to the Valco injection valve.
The Surveyor Autosampler Plus ships with a 25 μL sample loop.
5. Click the Signal Polarity tab to display the Signal Polarity page.
6. Use the following criteria to select the appropriate check boxes for the
Input signal polarities:
• For the Surveyor LC Pump Plus with firmware versions 2.0 and
higher or the Surveyor MS Pump Plus, make the selections shown in
Figure 19.
• For the Surveyor LC pump with firmware versions below 2.0, make
the selections shown in Figure 20.
Note If you select the Input check boxes in the Signal Polarity
page and your system has a Surveyor LC Pump with a firmware
version below 2.0, the Waiting for Contact Closure status
message will be displayed indefinitely for the Surveyor LC Pump
after you make an injection. See “Checking the Firmware
Version of the Surveyor LC Pump” on page 58.
7. In the Output area, leave the check boxes deselected if your instrument
consists entirely of Surveyor or Surveyor Plus devices.
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
Figure 19. Surveyor Autosampler Configuration dialog box - Signal Polarity
page, showing selections for the Surveyor LC Pump Plus or the
Surveyor MS Pump Plus
Figure 20. Surveyor Autosampler Configuration dialog box - Signal Polarity
page, showing selections for the Surveyor LC Pump
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
8. Click the Firmware tab to display the Firmware page. See Figure 21.
9. If you have upgraded your Surveyor Autosampler Plus firmware, upload
the firmware version:
a. Click Upload Firmware Version.
After you click the Upload Firmware Version button, the firmware
version of your Surveyor Autosampler Plus appears next to Current
Version. If you have a full-featured Surveyor Autosampler Plus, the
temperature controlled features will be listed as installed.
b. Click OK to save the settings and close the Surveyor Autosampler
Configuration dialog box.
If you have finished configuring all of your LC devices, go to “Exiting the
Instrument Configuration Dialog Box” on page 38.
Figure 21. Surveyor Autosampler Configuration dialog box - Firmware page
Configuring the Surveyor MS
Pump Plus
The Surveyor MS Pump Plus communicates with the Xcalibur data system
by way of an RS232 serial connection. Determine which COM port the
serial communication cable for the MS pump is attached to.
To configure the Surveyor MS Pump Plus
1. In the Configured Devices list, double-click the Surveyor MS Pump
Plus button.
The Surveyor MS Pump Plus dialog box appears. See Figure 22.
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
Figure 22. Surveyor MS Pump Plus dialog box
The value in the Serial number box is read from the firmware for the
pump. It is not a user-editable parameter.
2. Click OK to exit the Surveyor MS Pump Configuration dialog box.
If you have finished configuring all of your LC devices, go to “Exiting the
Instrument Configuration Dialog Box” on page 38.
Configuring the Surveyor LC
Pump Plus
The Surveyor LC Pump communicates with the Xcalibur data system by
way of an Ethernet connection. To make this connection, the Ethernet
cable on the back of the detector must be connected to the Ethernet switch
and the Unit ID setting on the back panel of the pump must match the
Stack address in the configuration.
To configure the Surveyor LC Pump Plus
1. Double-click the Surveyor LC Pump button to open the Surveyor LC
Pump dialog box. See Figure 23.
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Configuring the Surveyor Plus LC Devices
2. Make the following entries and selections:
• From the Pressure Units list, select the pressure units that you want
use to display the backpressure of your system.
Note 1 MPa = 10 bar = 145 psi
• In the Stack Number box, type an appropriate value.
The value must match the Unit ID setting on the back panel of the
Surveyor LC Pump.
• If you want your Sequences to pause following a degasser error,
check the Pause Sequence Following Degasser Error check box.
3. Click OK to exit the Surveyor LC Pump dialog box.
Note If you are adding a Surveyor LC Pump (prior to the Plus
version) with a firmware version below 2.0 to your instrument,
confirm that the Input signal polarities for the autosampler are
deselected as shown in Figure 20 on page 33.
Figure 23. Surveyor LC Pump dialog box
Configuring the Surveyor PDA
Plus Detector
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Surveyor Plus Getting Started with Xcalibur
The Surveyor PDA Plus Detector communicates with the Xcalibur data
system through an Ethernet connection. To make this connection, the
Ethernet cable on the back of the detector must be connected to the
Ethernet switch and the unit ID setting on the back panel of the detector
must match the stack number in the configuration.
Thermo Electron Corporation
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Configuring Your Surveyor Plus LC Instrument
Configuring the Surveyor Plus LC Devices
To configure the Surveyor PDA Plus Detector
1. Double-click the Surveyor PDA Plus button.
The Surveyor PDA Plus Configuration dialog box shown in Figure 24
appears.
2. In the Stack Number box, type the appropriate number (unit ID).
Figure 24. Surveyor PDA Plus Configuration dialog box
3. Click OK to close the Surveyor PDA Plus Configuration dialog box.
When you have finished configuring all of your LC devices, go to
“Exiting the Instrument Configuration Dialog Box” on page 38.
Configuring the Surveyor
UV/Vis Plus Detector
LC Devices 2.0.2 supports the Surveyor UV/Vis Plus Detector.
To configure the Surveyor UV/Vis Plus Detector
1. Double-click the Surveyor UV/Vis button.
The Surveyor UV/Vis Configuration dialog box shown in Figure 25
appears.
2. In the Stack Number box, type the appropriate number (unit ID).
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Configuring the Surveyor Plus LC Devices
Figure 25. Surveyor UV/Vis Configuration dialog box (for the
Surveyor UV/Vis Plus Detector)
3. Click OK to close the Surveyor UV Vis Configuration dialog box.
When you have finished configuring all of your LC devices, go to the
next topic: Exiting the Instrument Configuration Dialog Box.
Exiting the Instrument
Configuration Dialog Box
Before you can open the Xcalibur application program, you must close the
Instrument Configuration dialog box.
To save your instrument configuration and exit the Instrument
Configuration dialog box, click Done at the bottom of the Instrument
Configuration dialog box.
You return to the Windows desktop.
Note You must exit the Instrument Configuration dialog box before you
launch Xcalibur. The two applications cannot be open simultaneously.
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Chapter 3
Preparing for Operation
Now that you have installed and configured your Surveyor Plus LC system,
you are ready to prepare the system for operation. Not all of the procedures
contained in this chapter will apply to your system. For example, if your
system contains a Surveyor LC Pump Plus rather than a Surveyor MS Pump
Plus, you will not need to prime the pulse dampener. And if your system
does not contain an optional Surveyor PDA Plus Detector, you do not need
to calibrate it.
Perform the procedures that are provided in this chapter and that apply to
your system in the order listed:
1. Turning On the Power to Each LC Device
2. Launching Xcalibur
3. Checking the Status of the LC Devices
4. Turning On the LC Devices from Info View
5. Opening the Instrument Setup Window
6. Removing Air from the Solvent Lines
7. Checking the Firmware Version of the Surveyor LC Pump
8. Priming the Pulse Dampener of the MS Pump
9. Calibrating the PDA Detector
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Preparing for Operation
Turning On the Power to Each LC Device
Turning On the Power
to Each LC Device
Turn on the power to your LC devices, and then observe their status LEDs.
The ON/OFF power switch for each device is located below the left door of
the module. Figure 26 shows the location of the status LEDs and the power
switches.
Shortly after you turn on the power, all the LEDs except the Comm LEDs
turn green. In addition, the autosampler syringe goes through its
initialization process.
If any of the Power LEDs remain amber, make sure that the power line to
the affected device(s) is connected. If the Degas LED on the pump is
flashing amber, the degas unit has failed to produce a vacuum, and you need
to call your local Thermo Electron representative for repairs. If the Lamp
LED remains amber, both of the lamps are turned off.
For information on turning on the lamps, see “Warming Up the Deuterium
Lamp” on page 121.
LEDs
Power
Switches
Figure 26. Surveyor Plus LC Stack
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Launching Xcalibur
Preparing for Operation
Launching Xcalibur
You control the Surveyor devices from the Xcalibur data system. The only
manual control for each Surveyor LC device is its ON/OFF power switch.
To launch Xcalibur
1. Double-click the Xcalibur icon on your desktop to display the Home
Page shown in Figure 27.
Shortly after you open Xcalibur, the Comm LEDs on the front panels
of your LC devices turn green.
2. If the Comm LEDs remain amber, do the following:
• Check that the communication cable to each module is connected
to the data system computer.
• Check that the stack addresses in the configuration for the
autosampler, LC pump, and detector match the setting of the
unit IDs on their back panels.
• Check that the serial communication cable for the MS pump is
attached to the Com port selected in the configuration of the pump.
For information on configuring your instrument devices, see
Chapter 2, “Configuring Your Surveyor Plus LC Instrument.”
Figure 27. Xcalibur Home Page, showing the Roadmap view and the Info view
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Preparing for Operation
Checking the Status of the LC Devices
Checking the Status
of the LC Devices
After you turn on the power to the LC devices and open the Xcalibur data
system, check the status of each device.
To check the status of the LC devices, perform the procedures that are
provided in this section in the order listed:
1. Displaying the Information View - Status Page
2. Viewing the Status of Each Device
Displaying the Information
View - Status Page
The status of the Surveyor devices can be monitored from the Information
view. This view is normally displayed on the left side of the Home Page
window. If this view is not displayed, the view has been turned off. From
the Home Page window, choose View > Info View to toggle the
Information view On and Off.
To display the Information View - Status page
1. In the Home Page window, ensure that the Information View is
displayed by choosing View > Info View.
2. Click the Status tab to display the Information View – Status page. See
Figure 28.
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Checking the Status of the LC Devices
Figure 28. Information View - Status Page
If you have just recycled the power and have not yet downloaded a
method, you see the following status readouts in the Status page:
• Initializing appears while Xcalibur is attempting to connect to an
instrument module.
• Lamp Warm-up appears for the PDA detector while the deuterium
lamp is igniting.
• Ready to download appears after communication has been
established with an instrument module and after each run has
ended.
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Preparing for Operation
Checking the Status of the LC Devices
Viewing the Status of
Each Device
The Surveyor Plus Integrated LC system contains an autosampler and an
analytical pump. In addition, your system might contain an optional PDA
or UV/Vis detector.
Check the status of each device by clicking its name in the Device Tree list.
The status information for a specific device is displayed in the lower portion
of the Status page.
The Status view for the full-featured Surveyor Autosampler Plus contains
four pages: AS Status, Oven, Tray, and Error Log. See Figure 29. The Status
view for the Surveyor Autosampler Plus Lite contains two pages.
The status page for the Surveyor Autosampler Plus displays one of the
following eight states:
1. Initializing appears while Xcalibur is attempting to connect to the
autosampler.
2. Ready to download appears after communication has been established
with the autosampler and after each run has ended.
3. Ready to run appears for a very brief period after a method is
downloaded to the autosampler and before the injection process
has begun.
4. Running appears during the injection process. If the method contains a
time function for the autosampler, the running status message is
displayed until the time function has expired.
5. Direct control appears when the direct control or calibration windows
are open.
6. Busy appears while the autosampler is performing a direct control
operation.
7. Error appears when an error condition other than the loss of
communication occurs.
8. Off appears after Xcalibur has attempted to connect to the autosampler
and failed five times. The most common reasons for Xcalibur failing to
connect with the Surveyor Autosampler Plus are that the 10-pin
connector of the syringe drive assembly is unplugged from the
autosampler or that the XYZ arm is stalled.
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Checking the Status of the LC Devices
Figure 29. Status view - AS Status page for the Surveyor Autosampler Plus,
showing the autosampler in the Ready to Download state
See Figure 30, Figure 31, Figure 32 and Figure 33, which respectively show
the Status views for the Surveyor MS Pump Plus, the Surveyor LC Pump
Plus, the Surveyor PDA Plus Detector, and the Surveyor UV/Vis Detector.
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Preparing for Operation
Checking the Status of the LC Devices
Figure 30. Status view for Surveyor MS Pump Plus
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Checking the Status of the LC Devices
Figure 31. Status view for the Surveyor LC Pump Plus
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Preparing for Operation
Checking the Status of the LC Devices
Figure 32. Status view for the Surveyor PDA Plus Detector
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3 Preparing for Operation
Checking the Status of the LC Devices
Figure 33. Status view for the Surveyor UV/Vis Detector
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Preparing for Operation
Turning On the LC Devices from Info View
Turning On the LC
Devices from Info
View
You can turn your LC devices On or Off from their respective device
listings in Info View.
To turn on a device, right-click its device listing in the Status window.
Then, choose Turn Device On.
The analytical pump begins pumping solvents from the last set of
downloaded parameters. The detector turns on the lamps. The autosampler
adjusts its controlled temperature zones to the last set of downloaded
parameters. See Figure 34.
Figure 34. Info view – Status page, showing shortcut menu
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Opening the Instrument Setup Window
Opening the
Instrument Setup
Window
The Direct Control dialog boxes are accessed through the Instrument Setup
window.
To open the Instrument Setup window
1. If it is not already open, open the Roadmap view by clicking the
Roadmap View button in the toolbar.
2. Click the Instrument Setup button on the toolbar or the larger
Instrument Setup icon in the Roadmap View.
The Instrument Setup window opens to the first module that is
displayed in the View Bar. The View bar is a vertical bar on the left of
the Instrument Setup window. It contains buttons for each of your
configured devices. See Figure 35.
Tip To return to Home Page, click the Home Page button in the
toolbar.
Figure 35. Instrument Setup window – Surveyor AS (full-featured) view
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Preparing for Operation
Removing Air from the Solvent Lines
Removing Air from the
Solvent Lines
If you have just connected the solvent lines to the built-in degasser of the
analytical pump and attached the wash bottle tubing to the left-side of the
autosampler syringe valve, you will notice that you have air in these lines.
To remove air from these low-pressure solvent lines, perform the procedures
provided in this section that apply to your system.
• Purging the LC Pump
• Purging the MS Pump
• Flushing the Autosampler Syringe
Purging the LC Pump
To purge air from the solvent lines or the pump head assemblies
Tip Cyclical baseline noise can be an indication of air in the pump heads.
Note If the solvent line connecting the solvent reservoir bottle to the
degassing assembly is completely dry, attach a syringe to the drain line
and open the drain valve. Make sure that the pump power is on, and
then draw the syringe barrel back, pulling solvent into the line.
1. Open the drain valve by turning it counter-clockwise 180° to the purge
position. The word DRAIN on the knob will appear upside down as
shown in Figure 36.
Open Position
LO
SE
PE
N
C
DRAIN
O
PE
N
DRAIN
O
SE
LO
C
Closed Position
Figure 36. Drain valve knob in purge position
2. In the Instrument Setup viewbar, click the Surveyor LC Pump button
to open its Instrument Setup view.
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Removing Air from the Solvent Lines
3. From the menu bar, choose Surveyor LC Pump > Direct
Control > Operation to open the Operation dialog box for the
LC pump. See Figure 37.
Figure 37. Operation – Surveyor LC Pump Direct Control page
4. In the Purge area, do the following:
a. In the Purge Time box, type an appropriate purge time.
The allowable range is 0.1 to 9.9 min. Each 1/16-in. ID tube
connecting a solvent reservoir bottle to the inlet port of the degasser
holds approximately 3 mL of solvent.
b. From the Solvent Valve list, select the solvent valve for the solvent
line that you want to purge.
Note Clicking the Pump On button while the drain valve is in
the purge position can generate a pump error. You must clear this
error before you can run the pump. To clear a pump error,
remedy the source of the error, and then click Clear in the Error
Condition area.
c. Click Purge. The pistons of the pump begin to move rapidly,
increasing the flow rate to 9.99 mL/min.
5. After you finish purging the solvent lines, close the drain valve.
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Preparing for Operation
Removing Air from the Solvent Lines
Purging the MS Pump
After you have filled the solvent bottles, connected the solvent lines to
the degasser, and configured the MS pump to communicate with the
Xcalibur data system, prepare the MS pump for operation by removing the
air from the solvent lines.
To draw solvent through the solvent lines quickly
1. Insert the tip of the 10 mL syringe into the tubing that is connected to
the wingnut located on the left-side of the LDA. See Figure 38.
2. Open the wingnut by turning it counter-clockwise.
3. Turn on the power to the pump.
Tip When the power to the pump is Off, the proportioning valves are
closed. Before you take the LDA apart to perform maintenance, turn
off the power to the pump to prevent solvent from leaking out of the
proportioning assembly.
Figure 38. Liquid Displacement Assembly (LDA) of MS pump
4. From the Instrument Setup View, open the Direct Control dialog box
for the MS pump:
a. Click the Surveyor MS Pump Plus button in the viewbar.
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Removing Air from the Solvent Lines
b. Choose Surveyor MS Pump > Direct Control to open the Direct
Control dialog box for the MS pump. See Figure 39.
Figure 39. Direct Control dialog box for MS pump
5. In the Direct Control dialog box, do the following:
a. Select the Take pump under control check box.
b. Type percentages in the Inlet boxes for the solvent lines through
which you want to draw solvent.
c. In the Flow box, type 2000.
d. Click the Start Run button
to start the pump flow.
Because each 1.5 m (5-ft.) section of tubing holds approximately
3 mL of solvent, you need to stop the pump flow and empty the
syringe periodically.
e. After you finish purging the solvent lines, click the Stop button
to stop the pump flow.
6. Verify that the solvent lines are free of air.
7. Turn the wingnut clockwise to close the vent valve.
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Removing Air from the Solvent Lines
Flushing the Autosampler
Syringe
To ensure the proper performance of the autosampler, remove air from the
wash bottle tubing and the autosampler syringe before you make your first
injection. Once you have your system running, periodically check the level
of solvent in the Wash bottle and remove air from the syringe as needed.
To flush air out of the wash bottle tubing and the autosampler syringe
1. From the Instrument Setup window, click the Surveyor AS button to
display the Instrument Setup view for the Surveyor Autosampler.
2. Choose Surveyor AS > Direct Control to display the Direct Control
dialog box for the Surveyor Autosampler. See Figure 40.
Figure 40. Flush Syringe direct control command
3. Initiate the Flush Syringe direct control command:
a. Click the down-arrow to display the list of commands.
b. Select Flush Syringe from the list of commands. The parameters for
the command appear below the list.
c. Select Flush Bottle from the list of Reservoir choices.
d. Type an appropriate flush volume in the Volume box. The
maximum allowable flush volume is 6000 μL.
e. Click Apply to download the command to the autosampler.
4. Verify that the wash bottle tubing and syringe are free of air. See
Figure 41.
5. Close the Direct Control dialog box.
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Removing Air from the Solvent Lines
Note To flush the sample loop as well as the syringe, select the Flush
With Syringe In Fill Position command from the Command list.
2-Way Syringe Valve
Needle Tubing Assembly
Outer plunger moves upward,
pushing solvent out of the needle tubing,
into the injection port of the autosampler,
through the transfer tube, and into the
Rheodyne 6-port injection valve
Wash Tube
Wash Bottle
Concentric
Syringe
Solvent Path for
Flush with Syringe in Fill Position Direct Command
Solvent Path for
Flush Syringe Direct Command
Injection Port
of Autosampler
Injection Port
of Autosampler
Transfer Tube
To
Column
1
2
Waste Tube
Transfer Tube
To
Column
1
2
6
6
3
3
4
5
4
From
Pump
Valco Injection Valve
in the Inject Position
Waste
Container
5
From
Pump
Valco Injection Valve
in the Inject Position
Waste
Container
Mobile Phase
Wash Solvent
Figure 41. Schematic of solvent path for flushing the syringe
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Checking the Firmware Version of the Surveyor LC Pump
Checking the
Firmware Version of
the Surveyor LC Pump
If your Surveyor LC Pump has a firmware version below 2.0, it does not
send a Pump Ready signal to the Surveyor Autosampler. If you selected
either of the Input check boxes in the Signal Polarity page when you
configured the Surveyor Autosampler, the status of the Surveyor LC Pump
will indefinitely remain at Waiting for Contact Closure after you make an
injection. To prevent this mismatch, verify the firmware version of your
Surveyor LC Pump. If necessary, modify the configuration for the
autosampler signal polarities accordingly.
To check the version of your Surveyor LC Pump Plus
1. Open the Instrument Setup window as described on page 51.
2. In the Instrument Setup viewbar, click the Surveyor LC Pump button
to open its Instrument Setup view.
3. From the menu bar, choose Surveyor LC Pump > Direct
Control > Configuration to open the Configuration – Surveyor LC
Pump Direct Control dialog box. See Figure 42.
4. Note the firmware version of the Surveyor LC Pump. If the version is
lower than 2.0, check the configuration of the Surveyor Autosampler.
See “Configuring the Surveyor Autosampler Plus” on page 28.
Figure 42. Configuration – Surveyor LC Pump Direct Control dialog box
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Priming the Pulse Dampener of the MS Pump
Priming the Pulse
Dampener of the MS
Pump
Before you can operate your MS pump, you must prime its built-in pulse
dampener. Priming the pulse dampener involves filling the dampening coil
of the pulse dampener with a solvent such as methanol or isopropanol. After
the coil is filled with solvent, it can effectively dampen pressure pulsations
from the rest of your system.
Closing the manual bleeder valve after you fill the coil shuts the dampening
coil off from the mobile phase stream. See Figure 43. However, only a
permeable membrane separates the mobile phase stream from the
dampening coil. Therefore, if you use buffered mobile phases, there will be a
gradual buildup of electrolytes in the dampening coil. Due to the pressure
gradient, the reverse does not occur. Therefore, you do not need to match
the priming solvent to the mobile phase because the solvent in the coil
cannot diffuse into the mobile phase stream.
If you are not pumping buffered mobile phases, you need to prime the pulse
dampener only before you initially operate the MS pump. In contrast, you
will occasionally need to reprime the pulse dampener if you are pumping
buffered mobile phases. Flushing the pulse dampener following the use of
buffered eluents helps to prevent salt build-up and its associated corrosion.
Note Do not fill the pulse dampener with an aggressive acid or a
buffered solution. The recommended filling solvents are methanol,
acetonitrile, or isopropyl alcohol. If you accidentally fill the pulse
dampener with an acidic or buffered solvent, flush the loop with a
miscible solvent, and then refill it with methanol, acetonitrile, or
isopropyl alcohol.
Dual Valve
Cartridge
Diffusion Barrier
Cartridge
Manual Bleeder
Valve - for Priming
2 mL Dampening Loop
Figure 43. Pulse dampening assembly for MS pump
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Preparing for Operation
Priming the Pulse Dampener of the MS Pump
To prime the pulse dampener
1. Fill a solvent reservoir with particulate-free, HPLC grade methanol.
2. Connect an HPLC column or a flow restrictor to the pulse dampener
outlet.
3. Insert the tip of the 10 mL syringe into the tubing that is connected to
the manual bleeder valve of the pulse dampener. Then, open the valve
by turning it counter-clockwise.
4. Set the flow rate to 1 mL/min (1000 μL/min).
5. Fill the loop completely, to expel any air that might be trapped in the
dampener loop.
6. Set the flow rate to one that is appropriate for your system.
7. Close the manual bleeder valve of the pulse dampener (see Figure 44) by
turning the valve clockwise.
Unless you need to refill the dampener loop of the pulse dampener with
fresh solvent, keep the manual bleeder valve closed.
Outlet of Pulse Dampener
Manual Bleeder Valve
with Attached Tubing
Figure 44. Manual bleeder valve and attached tubing
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Preparing for Operation
Calibrating the PDA Detector
Calibrating the PDA
Detector
There are two types of calibration for the PDA detector:
• Dark Current Calibration
• Wavelength Calibration
You must perform both calibrations after the initial installation of your
detector. Thereafter, Thermo Electron recommends that you perform a
dark current calibration monthly and a wavelength calibration whenever
you move your detector or it is physically jolted. The instructions for
performing these calibrations are included in “Verifying the Performance of
the PDA Detector” on page 174.
Dark Current Calibration
The function of the dark current calibration is to measure and correct for
the background signal that is produced by the photodiode array. Even when
both lamps are turned Off, the array still produces a small amount of
background signal. Typical dark current values range from 1500 to
3000 counts.
Before intensity values can be converted to absorbance units, the dark
current must be subtracted from the intensity measurements. Following
dark current calibration, light intensity values are automatically corrected
for dark current. Remember that because the dark current signal is a
function of temperature both lamps must be On during calibration. You
typically warm-up the lamps for one hour before making injections.
Therefore, it is important to warm-up the lamps for one hour before
performing a dark current calibration to ensure that the temperature within
the detector during calibration matches the temperature within the detector
during normal operation.
Perform a dark current calibration monthly.
Wavelength Calibration
The alignment of the spectrum on the diode array depends upon the
physical alignment of various components of the optical bench. The
alignment can become offset if the detector is sharply jolted, for example, in
shipping. Such bumps and jars can slightly change the wavelength of light
reaching the photodiode array.
The automated wavelength calibration allows you to determine wavelength
accuracy. It also allows the PDA detector to mathematically compensate for
minor changes in the alignment of the components of the optical bench.
Perform a wavelength calibration if you move the detector or if it receives a
physical jolt.
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Chapter 4
Creating Instrument Methods
To automate the control of your LC devices, you must create an Instrument
Method from the Instrument Setup window of the Xcalibur data system.
This chapter describes the instrument control parameters for your Surveyor
Plus LC devices. It does not describe the instrument control parameters for
your MS detector. For information on the instrument control parameters
for your MS detector, see its Getting Started manual.
To create an instrument method containing the appropriate
chromatographic conditions, perform the following procedures that are
provided in this chapter and that pertain to your LC devices in the order
listed:
1. Opening the Instrument Setup Window
2. Entering the Method Parameters for the MS Pump
3. Entering the Method Parameters for the LC Pump
4. Entering the Method Parameters for the Autosampler
5. Entering the Method Parameters for the PDA Detector
6. Entering the Method Parameters for the UV/Vis Detector
7. (Optional) Adding a Sample Preparation Routine to the Method
8. Saving the Method
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Creating Instrument Methods
Opening the Instrument Setup Window
Opening the
Instrument Setup
Window
To open the Instrument Setup window
1. Double click the Xcalibur icon on your desktop to display the Xcalibur
Home Page.
2. On the Xcalibur Roadmap - Home Page, click the Instrument Setup
button in the Road Map or choose GoTo > Instrument Setup to open
the Instrument Setup window. See Figure 45.
The Viewbar on the left-side of the window contains an icon for each
configured device of your instrument. Clicking an icon for a device
opens the Instrument Setup view for that device.
Figure 45. Instrument Setup window
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Entering the Method Parameters for the MS Pump
Entering the Method
Parameters for the
MS Pump
The instrument control parameters for the Surveyor MS Pump Plus that
control the composition and flow rate of the mobile phase are entered in the
Surveyor MS Pump Instrument Setup view.
To open the Surveyor MS Pump view, click the Surveyor MS Pump icon in
the Viewbar of the Instrument Setup window.
If you are unfamiliar with the Surveyor MS Pump Plus, review the
description of its instrument control parameters before you create the pump
program.
This section contains the following topics:
• Parameters for the Surveyor MS Pump Plus
• Programming the Surveyor MS Pump Plus
Parameters for the
Surveyor MS Pump Plus
The following topics describe the instrument control parameters for the
Surveyor MS Pump Plus:
• Surveyor MS Pump View - General Page
• Surveyor MS Pump View - Gradient Page
Surveyor MS Pump
View - General Page
The Surveyor MS Pump View – General page has the following areas:
Parameters for the Pump
The General page contains the parameters listed below. The values that you
enter in these boxes will be reported in the Instrument Method report
attached to the raw file. This report can be accessed in the Qual Browser
view by choosing View > Reports > Instrument Method.
Name
Use this box to select the pump if you are running a dual pump system.
Comments
Use this box to enter more information about the method, for example,
the type of LC column being used.
Solvent
Select the check box to the left of these boxes to make them available,
and then type a description of the solvent.
Operating mode
Use this list to select the operating mode: 0 to 7000 PSI (standard) or 0
to 16000 PSI (ultra-high performance).
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Entering the Method Parameters for the MS Pump
Start settings
Use this list to select how runs are triggered. The available selections are
Autosampler logic and manual. Select Surveyor AS injection logic if
you want to use the autosampler as the start instrument that triggers the
run.
Method finalizing
Use this list to select the mobile phase composition at the end of the run.
the available selections are First line conditions, Last line conditions, and
Stop after the end. Select Stop after the end if you want to use the
method to turn off the pump flow.
Min pressure
Use this box to enter a minimum pressure below which the pump will
stop operating. Entering an appropriate minimum pressure prevents the
pump from operating when the solvent reservoirs have run dry or the
system plumbing has developed a leak. Running the pump without
solvent will quickly ruin the piston seals. The range is 0 to 400 bar
(5800 psi) with a default value of 0 bar. Type a value that is well below
the typical operating pressure for your application in this box. In the
event that the pressure falls below this limit, the pump automatically
stops and sends an error message to the computer.
Max pressure
Use this box to enter a maximum operating pressure for the pump.
Entering an appropriate maximum pressure prevents the pump from
operating with a restriction on the outlet side of the pump. Excess
pressure can damage the HPLC column and any other component
between the restriction and the pump. The range is 0 to 400 bar
(5800 psi) with a default value of 0 bar. In this box, type a value that is
well above the typical operating pressure for your application, but also
below the pressure that can damage your system. In the event that the
pressure rises above this limit, the pump automatically stops and sends
an error message to the computer.
Pressure stability
Use this box to adjust the stability condition required for the pump to
go into a Ready mode. A lower value requires greater pressure stability
before the pump becomes Ready. A higher value is more forgiving of
pressure pulsations.
Home before run
This check box is available when you select Manual from the Start
settings list. Selecting this check box increases the reproducibility of
chromatographic runs when you are performing manual injections.
Pressure units
Use this list to select the units for the backpressure readout. The
available selections are bar, PSI, and MPa.
Surveyor MS Pump
View - Gradient Page
The Surveyor MS Pump View – Gradient page has the following areas:
• Gradient Table Area
• Gradient Profile Area
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Gradient Table Area
Each row in the Gradient Table defines the solvent composition and flow
rate for a specific time point. Between time points, the solvent composition
changes linearly, whereas, the flow rate changes as a discontinuous
step-function. The Gradient Table can contain up to 80 time lines.
The time parameter (min box) specifies at what point in the run the
associated solvent composition and flow rate become effective. The first
time line remains set to 0 min.
The solvent composition parameters (%A, %B, %C, and %D boxes) work
interactively with each other and specify the mobile phase composition at
the time point specified in the associated Min box. The mobile phase
composition changes linearly between two consecutive time points. The
default composition setting is 100% A.
The flow rate parameter (μL/min) box specifies the flow rate of the mobile
phase at the time specified in the associated Min box. The default setting
flow rate setting is 1000 μL/min.
Gradient Profile Area
The Gradient Profile area graphically displays the values entered in the
Gradient Table. The Y-axis represents percent composition and the X-axis
represents time in minutes. Each solvent is color-coded for better
visualization of the programmed gradient.
Programming the Surveyor
MS Pump Plus
The Surveyor MS Pump Plus contains a built-in solvent proportioning
assembly that is capable of proportioning up to four solvents to create
binary, tertiary, and quaternary mobile phases. This capability reduces the
need to make premixed mobile phases. You can run the MS pump in either
the isocratic mode or the gradient mode. In the isocratic mode, the same
proportions of solvent are maintained throughout the run.
To program the pump
1. In the General page (see Figure 46), type or select the following:
a. Type the names of the solvents that make up the mobile phase
b. Select the operating mode: 0-7000 PSI.
c. From the Start settings list, select how the system is triggered to
start a run.
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The available selections are Surveyor AS injection logic and Manual.
To trigger a run automatically from the autosampler, select
Surveyor AS injection logic.
d. In the Method finalizing list, select the ending conditions for
the run.
The available selections are First line conditions, Last line
conditions, and Stop after the end. If you are creating a shutdown
method that turns off the pump flow, select Stop after the end.
e. Type or select the minimum allowable operating pressure.
f. Type or select the maximum allowable operating pressure.
g. Type or select an appropriate backpressure stability value.
The pump will not return a Ready signal until the system pressure
reaches this limit.
h. Select the pressure units for the backpressure display
Note 1 bar = 14.5 psi
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Figure 46. General page for the Surveyor MS Pump Plus
2. Click the Gradient Program tab.
3. Type the flow rate and solvent composition in the first time line of the
Time Program table, and then press ENTER.
The allowable flow rate range for the Surveyor MS Pump Plus is 0 to
2000 μL/min. The first time line in the Time Program table is set to
0.00 min. This time value of 0.00 min cannot be changed. The second
time line in the program is a placeholder line and has no effect on the
pumping conditions. For example, the pump program shown in
Figure 47 will produce a proportioned mobile phase consisting of 80%
solvent A and 20% solvent B (v/v). The proportions of the solvents will
remain constant throughout the run. The flow rate of the mobile phase
will remain at 1 mL/min throughout the run.
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Entering the Method Parameters for the MS Pump
4. If you are creating an isocratic pump program, skip the next step, which
describes how to create a gradient program, and go to “Entering the
Method Parameters for the Autosampler” on page 76.
Placeholder Line
Figure 47. An example of an isocratic pump program for the MS pump
5. To create a gradient pump program, enter at least two steps in the
Gradient Table. For each line in the program, type the solvent
composition and the flow rate. Then, press ENTER.
The first time line in the Time Program table is set to 0.00 min. This
time value of 0.00 min cannot be changed. The last time line in the
program is a placeholder line and has no effect on the pumping
conditions. The allowable flow rates are 0 to 2000 μL/min.
The gradient table and gradient profile shown in Figure 48 produce the
gradient program described in Table 4.
Table 4.
Gradient program
Time (min)
Solvent Composition
0.00 to 5.00
Held constant at 90% solvent B / 10% solvent A
5.00 to 25.00
Linear ramp from 10% solvent A to 90% solvent A
25.00 to 25.01
Stepped down to initial solvent composition of
90% solvent B / 10% solvent A
25.01 to 40.00
Column is equilibrated at the initial solvent composition of
90% solvent B / 10% solvent A
At 25.01 minutes, the program returns to its initial solvent
composition. To equilibrate the column prior to the next run, the
solvent composition is held at the initial solvent composition for a
period of 15 minutes. The last line in the Gradient Table is a
placeholder and has no affect on the pump conditions.
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Entering the Method Parameters for the MS Pump
Note When you are performing a sequence of gradient runs, add an
equilibration step at the end of the Gradient Table.
After you enter the pump program, go to “Entering the Method Parameters
for the Autosampler” on page 76.
Placeholder Line
Figure 48. An example of a gradient program for the MS pump
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Creating Instrument Methods
Entering the Method Parameters for the LC Pump
Entering the Method
Parameters for the LC
Pump
The instrument control parameters for the Surveyor LC Pump Plus that
control the composition and flow rate of the mobile phase are entered in the
Surveyor LC Pump Instrument Setup view. To open the Surveyor LC
Pump Plus view, click the Surveyor LC Pump icon in the Viewbar of the
Instrument Setup window.
To create a pump program for the Surveyor LC Pump Plus
1. In the General page (see Figure 49), enter the following:
• The names of the solvents that make up the mobile phase
• A description of the LC column
• The minimum allowable operating pressure
Note Pressure drops can indicate the following problems:
• Mobile phase in the solvent reservoir bottles has run out.
• A leak has occurred in the flow line(s).
• An excessive amount of air has entered the pump head
assemblies.
• The maximum allowable operating pressure
Note The maximum pressure setting is 43 MPa, 431 bar, or
6258 psi. Reduce the maximum pressure setting for PEEK
fittings to 28 MPa, 276 bar, or 4000 psi.
• The pressure units for the backpressure display
Note 1 bar = 14.5 psi
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Figure 49. General page for the Surveyor LC Pump Plus
2. Click the Gradient Program tab to open the Gradient Program page.
3. Type the initial flow rate and the initial solvent composition for your
pump program in the first time line of the time program table, and then
press ENTER.
To create an isocratic pump program, you need to enter only one time
line. If you are not creating a gradient program, skip the next step and
go to “Entering the Method Parameters for the Autosampler” on
page 76.
Note If you set the limit for the maximum pressure to less than
21 MPa, the allowable flow rate range is 0.000 to 9.999 mL/min. If
you set the maximum pressure limit to 21 MPa (215 bar, 3129 psi)
or higher, the maximum allowable flow rate is 5.000 mL/min.
The first time line in the time program table is set to 0.00 min. This
time value of 0.00 min cannot be changed. The second time line in the
program is a placeholder line and has no affect on the pumping
conditions.
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Entering the Method Parameters for the LC Pump
For example, the pump program shown in Figure 50 will produce a
proportioned mobile phase consisting of 80% methanol and 20% water
(v/v). The proportions of the solvents will remain constant throughout
the run. The flow rate of the mobile phase will remain at 1 mL/min
throughout the run.
Placeholder Line
Figure 50. An example of an isocratic pump program for the Surveyor LC Pump Plus
4. To create a gradient pump method, you must enter at least two steps in
the Gradient Program table. For each additional line in the program,
type a time value in the Time column, a flow rate in the Flow column,
and the solvent percentages for the mobile phase in the solvent columns.
Then press ENTER.
The Gradient Program table shown in Figure 51 creates the gradient
profile shown in Table 5.
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Table 5.
Gradient program
Time (min)
Solvent Composition
0.00 to 5.00
Held constant at 90% water / 10% methanol
5.00 to 25.00
Linear ramp from 10% methanol to 90% methanol
25.00 to 25.01
Stepped down to initial solvent composition of 90%
water / 10% methanol
25.01 to 40.00
Column is equilibrated at the initial solvent composition of 90%
water / 10% methanol
At 25.01 minutes, the program returns to its initial solvent
composition. To equilibrate the column prior to the next run, the
solvent composition is held at the initial solvent composition for a
period of 15 minutes. The last line in the gradient table is a placeholder
and has no effect on the pump conditions.
Note When you are performing a sequence of gradient runs, add an
equilibration step at the end of the Gradient Program.
5. After you enter your pump program, go to the next section: “Entering
the Method Parameters for the Autosampler” on page 76.
Placeholder
Line
Figure 51. An example of a gradient program for the LC pump
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Entering the Method Parameters for the Autosampler
Entering the Method
Parameters for the
Autosampler
The parameters for the autosampler are entered in the Surveyor
Autosampler Instrument Setup view. To open this view, click the Surveyor
Autosampler icon in the Viewbar of the Instrument Setup window. If you
are unfamiliar the Surveyor Autosampler Plus, review the description of the
instrument control parameters for the Surveyor Autosampler before you
enter your injection parameters.
This section contains the following topics:
• Parameters for the Surveyor Autosampler Plus
• Programming the Surveyor Autosampler Plus
Parameters for the
Surveyor Autosampler
Plus
The Surveyor AS Instrument Setup view contains four pages. The following
three pages are described in this section. The Sample Preparation page is
described in the next section “Adding a Sample Preparation Routine to the
Method” on page 98.
• Surveyor AS Method Page
• Reservoir Content Page
• Timed Events Page
Surveyor AS Method Page
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You set the injection and the temperature control parameters for the
autosampler in the Surveyor AS Method page. See Figure 52.
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4 Creating Instrument Methods
Entering the Method Parameters for the Autosampler
Figure 52. Surveyor AS Instrument Setup view – Surveyor AS Method page
The available parameters are:
• Injection Volume
• Needle Height From Bottom
• Syringe Sample Speed
• Flush Volume
• Flush/Wash Source
• Wash Volume
• Flush Speed
• Post Injection Valve Switch Time
• Loop Loading Speed
• Injection Mode
• Tray Temperature Control
• Column Oven Control
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Injection Volume
The Injection Volume box displays the injection volume in microliters of
sample that will be placed in the sample loop prior to opening the loop up
to the mobile phase stream. The available injection volumes are determined
by the size of the syringe. For the No Waste and the Partial Loop injection
modes, the sample loop size limits the practical injection volume range.
For optimal performance, refer to the following suggested injection
volumes:
• For no waste injections, limit the injection volume range to a minimum
of 0.5 μL and a maximum equal to 5 μL less than the nominal sample
loop size.
• For partial loop injections, limit the injection volume range to a
minimum of 0.1 μL and a maximum equal to one-half the nominal
sample loop size.
• For full loop injections, the Injection Volume box is grayed out,
limiting the injection volume to the configured sample loop size. The
Surveyor Autosampler is shipped with a 25 μL sample loop.
Needle Height From Bottom
The Needle Height From Bottom box displays the height in millimeters
from the bottom of the vial or well at which the needle withdraws liquid.
The range of values is 0.1 to 18.0 mm in increments of 0.1 mm. The
default value is 2.0 mm. Figure 53 shows the needle descending to a depth
of 2 mm from the bottom of a standard 1.8 mL vial.
2mm
Figure 53. View of needle descending to 2 mm from the bottom of a vial
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Syringe Sample Speed
The Syringe Sample Speed box displays the rate in microliters per second at
which the syringe withdraws liquid from a tray vial or a plate well. The
syringe speed range is determined by the size of the syringe. You select the
size of the syringe in the Syringe Type list in the Communication page of
the Surveyor Autosampler Configuration dialog box. Use a syringe speed
lower than the default for viscous samples. Also use a syringe speed lower
than the default for samples of very low viscosity or surface tension to
prevent the sample bolus from breaking apart during the transport process.
Flush Volume
The Flush Volume box displays the volume in microliters of liquid that is
used to flush the syringe. During an internal flush, the syringe needle is
moved to the injection port to dispense a specified volume of solvent from
the bottle or reservoir vial. The injector is switched to the Inject position to
prevent solvent from entering the loop. The internal flush is used to remove
residual sample. The range of values is 0.0 to 6000.0 μL. The default
volume is 400.0 μL.
Note A flush is accomplished by pushing the amount of solvent that you
specify into the injection port of the autosampler, through the transfer
tubing to the Valco injection valve, and out port 3 of the injection valve
to waste. If the specified flush volume exceeds the syringe volume, then
the autosampler takes multiple draws from the reservoir or bottle to
equal the specified flush volume.
Flush/Wash Source
The Flush/Wash Source list displays the flush/wash source. The flush/wash
source can be either the wash bottle or a reservoir vial (RV1 to RV4). To
change the flush/wash source, click the arrow to display the list of source
options.
Wash Volume
The Wash Volume box displays the volume in microliters of liquid that is
used to wash the needle. During an external wash, the syringe needle is
moved to the wash station to dispense a specified volume of solvent from
the bottle or reservoir vial. The range of volumes is 0.0 to 6000.0 μL. The
default value is 0.0 μL.
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Note A wash is accomplished by washing the interior and exterior of the
syringe needle with solvent. If the specified wash volume exceeds the
syringe volume, then the autosampler takes multiple draws from the
reservoir or bottle to equal the specified wash volume.
Flush Speed
The Flush Speed box displays the rate in microliters at which the syringe
draws flush solvent into its chamber and expels flush solvent through the
needle during a flush cycle. The range of values for the concentric syringes is
0.83 to 661.38 μL/s. The default value is 100.0 μL/s. The range of values
for the 2500 standard syringe is 8.27 to 330.85 μL/s. The default value is
82.71 μL/s.
Note The default flush speed of 100 μL/s for the 250 μL concentric
syringe is the maximum flush speed for a solvent of high viscosity such as
water. At high flush speeds, viscous solvents cause the autosampler to
make a grinding sound.
Post Injection Valve Switch Time
The default time for the Post Injection Valve Switch Time box is 0. If you
leave this value at 0, the valve remains in the inject position throughout the
entire run, which allows ample rinsing of the sample loop with mobile
phase.
Any nonzero value equal to or greater than 0.1 min is the time at which the
injection valve will switch from the inject position to the load position
following an injection. To reduce the gradient delay volume for low flow
gradient applications, consider switching the injection valve from the inject
position to the load position during the run by entering a nonzero value in
this box. In the load position, flow from the pump bypasses the sample loop
and goes directly to the column.
Loop Loading Speed
The Loop Loading Speed box displays the speed at which the sample is
loaded onto a guard column or a trap. This parameter is only available in
the no waste injection mode. The syringe pushes the sample bolus to the
injection port at syringe speed. The injection valve then switches to the load
position and the syringe meters the sample bolus onto the guard column or
trap at loop loading speed.
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Injection Mode
The option buttons in the Injection Mode area allow you to select the
injection mode. The injection modes are No Waste, Partial Loop, and Full
Loop and are described in detail in Chapter 1, “Introduction to the
Surveyor Plus Integrated LC/MS System.” See “Injection Volume” on
page 78 for information on the appropriate injection volumes for the three
injection modes.
Tray Temperature Control
You to set the temperature for the sample tray in the Tray Temperature
Control area. The temperature control feature performs heating or cooling
of the sample tray to establish an isothermal condition.
You select the Enable Tray Temperature Control check box to activate the
Temperature box. The Temperature box displays the temperature of the
sample tray in degrees Celsius. The range of values is 0 to 60 °C. The default
value is 30 °C. The precision is 1 °C. As a safety feature, a thermostat
disables power to the sample tray if the temperature reaches 65 °C.
Column Oven Control
You to set the temperature for the column oven in the Column Oven
Control area. The temperature control feature performs isothermal heating
or cooling of the column oven to establish an isothermal condition.
You select the Enable Column Oven Control check box to activate the
Temperature box.
The Temperature box displays the temperature in degrees Celsius of the
column oven. The range of values is 5 to 95 °C. The default value is 30 °C.
The precision is 1 °C. As a safety feature, a thermostat disables power to the
column oven if the temperature reaches 110 °C.
Reservoir Content Page
You enter descriptions for the contents of the four reservoir vials and the
wash solvent in the Reservoir Content Page shown in Figure 54. You can
type up to 80 characters in each box.
To open the Reservoir Content page, click the Reservoir Content tab in the
Surveyor AS Instrument Setup View.
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Figure 54. Surveyor AS Instrument Setup view – Reservoir Content page
Timed Events Page
You set timed events for the time function terminals (TF1 to TF4) located
on the rear panel of the Surveyor AS (See Figure 56) in the Timed Events
page shown in Figure 55. You can use the TF terminals to control
peripheral devices that are not controlled by Xcalibur.
Timed Event output signals are issued after the Inject Out signal in the
signal sequence. See “Synchronizing the Surveyor LC Devices” on page 20.
The TF terminal output signal is LO (Closed) by default. You can change
the polarity of the TF output signal to HI (Open) by selecting the Timed
Events Active High check box in the Signal Polarity page of the Surveyor
Autosampler Configuration dialog box.
To display the Timed Events page, click the Timed Events tab in the
Surveyor AS Instrument Setup view. You program timed events by adding
entries to the Timed Events table.
The Timed Events table contains Time boxes and event (TF1, TF2, TF3,
and TF4) lists. In the Time box, you specify the time in minutes when the
Surveyor Autosampler TF terminal (TF1 to TF4) signals an event. Time 0.0
is defined as the time when the Surveyor Autosampler issues an Inject Out
signal. The range of values is 0.0 to 9999.9 min.
In the event (TF1, TF2, TF3, and TF4) lists, you select whether the TF1,
TF2, TF3, and TF4 output terminal is On or Off at the time specified in
the Time box.
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Figure 55. Surveyor AS Instrument Setup view – Timed Events page
Figure 56. Time function event terminals on the back panel of the Surveyor
Autosampler Plus
Programming the Surveyor
Autosampler Plus
To program the injection parameters for your Instrument Method
1. Click the Surveyor Autosampler icon in the Viewbar of the Instrument
Setup window to open the Surveyor Autosampler view.
2. In the Surveyor AS Method page (see Figure 52 on page 77) make the
following entries and selections:
a. Select an injection mode: Partial Loop, Full Loop, or No Waste.
Then, do one of the following:
• If you selected the Full Loop injection mode, continue at
step 2d. The injection volume is automatically set to the
configured value for the sample loop size.
• If you selected the No Waste injection mode, go to step 2b.
• If you selected the Partial Loop injection mode, go to step 2c.
b. For the No Waste injection mode, select a loop loading speed from
the Loop Loading Speed box. For most applications, leave the loop
loading speed at its default value.
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c. For the Partial Loop or No Waste variable volume injection modes,
type an injection volume in the Injection Volume box. See
“Injection Volume” on page 78 for information on the appropriate
injection volumes for these injection modes.
d. From the Needle Height From Bottom box, select the depth to
which the needle of the autosampler will descend before it
withdraws sample from the vial / well location. The allowable values
are 0.1 to 18 mm. Entering a lower value causes the needle to
descend closer to the bottom of the vial or well. The standard
1.8 mL vials supplied with the Surveyor Autosampler have a depth
of approximately 20 mm. See Figure 53 on page 78.
e. For most applications, leave the syringe speed at its default value
(8 μL/s for the 250 μL concentric syringe). If you notice a break up
of the sample in the needle tubing as the autosampler withdraws
sample from a vial / well location, adjust the syringe speed.
f. Type a volume from 0 to 6000 μL in the Flush volume box or leave
the value at its default of 400 μL.
g. Select a flush / wash solution from the Flush / Wash list. For most
applications, select the Wash bottle. If you select one of the reservoir
vial locations as your flush / wash source, remember to load the
autosampler with a reservoir vial. See “Tray Compartment” on
page 7.
h. For most applications, leave the wash volume at its default value of
0 μL. If you want to wash the exterior of the needle after each
injection, type a value between 0 to 6000 μL in the Wash Volume
box.
i. Leave the flush speed at its default (250 μL/s for the 250 μL
concentric syringe) if you are using a flush solvent of low viscosity
such as methanol. Reduce the flush speed if you are using water or a
methanol / water mixture as the flush solvent.
Note If the flush speed is too high, the autosampler makes a grinding
noise as it performs a flush.
j.
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For most applications, leave the post injection valve switch time at
its default of 0 min. This leaves the sample loop in the inject
position during the entire run, which generally allows ample rinsing
of the sample loop between injections.
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Entering the Method Parameters for the Autosampler
k. For the full-featured Surveyor Autosampler Plus, do the following:
• If you want to control the temperature of the tray compartment,
select the Enable Tray Temperature Control check box. Then,
type an appropriate temperature from 0 to 60 °C in the
Temperature box.
• If you want to control the temperature of the LC column, select
the Enable Column Oven Control check box. Then, type an
appropriate temperature from 5 to 95 °C in the Temperature
box. Controlling the temperature of the LC column increases
the reproducibility of the chromatographic retention times.
3. In the Reservoir Content page (see Figure 54 on page 82), identify the
solvents contained in the 15 mL solvent reservoir vials and the 1 L wash
bottle by typing their names in their respective boxes.
4. If you want to use the Surveyor Autosampler Plus to control external
devices make the appropriate entries in the Timed Events page (see
Figure 55 on page 83).
5. Complete the instrument method:
• If your LC system contains a Surveyor PDA Plus Detector, go to the
next section: Entering the Method Parameters for the PDA
Detector.
• If your LC system contains a Surveyor UV/Vis Plus Detector, go to
• If your system does not contain a detector, but you want to add
sample preparation steps to your method, go to “Adding a Sample
Preparation Routine to the Method” on page 98.
• If your system does not contain a detector and you do not want to
add sample preparation steps to your method, go to “Saving the
Method” on page 109.
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Entering the Method Parameters for the PDA Detector
Entering the Method
Parameters for the
PDA Detector
The instrument control parameters for the Surveyor PDA Plus Detector are
entered in the Surveyor PDA Instrument Setup view.
To open the Surveyor PDA Plus view
Click the Surveyor PDA Plus icon in the Viewbar of the Instrument Setup
window.
This section contains the topics listed below. If you are unfamiliar with the
Surveyor PDA Plus Detector, review the description of its instrument
control parameters before you program it.
• Parameters for the Surveyor PDA Plus Detector
• Programming the Surveyor PDA Plus Detector
Parameters for the
Surveyor PDA Plus
Detector
The Surveyor PDA Method page contains parameters that define the data
that is collected during an analysis. See Figure 57. The Surveyor PDA
Method page contains the following areas:
• Run Area
• Units Area
• Spectra Area
• Channels Area
• Timed Events Area
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Figure 57. Surveyor PDA Plus Instrument Setup View – Surveyor PDA Plus
Method page
Run Area
The run area box contains the following boxes:
• Run Length Box
• Filter Rise Time List
Run Length Box
The Run Length box specifies the time during which the detector will
collect spectral and discrete wavelength data. The maximum run length is
600 minutes.
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Filter Rise Time List
In the Rise Time list, you select the response time of the detector, in
seconds, to the signal. Rise time is inversely proportional to the amount of
baseline noise: the longer the rise time, the less noise detected. Setting the
rise time to a value greater than one-tenth the width of the chromatographic
peak at half-height results in peak broadening as shown in Figure 58. The
one-second default value is appropriate for most LC applications.
Note The baseline noise inevitably increases as the deuterium lamp
reaches the limit of its expected lifetime. Track the expected signal to
noise levels for your chromatographic method to determine an
appropriate replacement schedule for the deuterium lamp.
0.1 s Rise Time
2 s Rise Time
mAU
5 s Rise Time
10 s Rise Time
Minutes
Figure 58. Effect of rise time on band broadening
Units Area
In the Units area, you select the units that are displayed in the spectrum
plots. You can select to display either absorbance (default) or intensity.
Absorbance displays the absorbance signal from the detector. In this case,
the X-axis corresponds to wavelength in nanometers. Intensity displays the
light intensity detected by the photo diodes of the diode array. The X-axis,
therefore in this case, corresponds to photo diode number.
The controls in this area are the Wavelength/Absorbance option button and
the Diode/Intensity option button.
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There are 512 diodes in the Surveyor PDA diode array. Diodes 0 and 1 are
not used. Diode 2 corresponds to wavelength 190 nm and diode 511
corresponds to 800 nm. This gives a spacing of 610 /510 diodes =
1.2 nm/diode. The data is interpolated to give integer values.
Spectra Area
To collect spectral scans, select the Collect Spectral Data check box, and
then fill in the following scan parameters:
• Start Wavelength
• End Wavelength
• Wavelength Step
• Sample Rate
• Filter Bandwidth
Start Wavelength
In the Start Wavelength box, you enter a starting wavelength for the scan.
The range of acceptable values is from 190 to 800 nm. The default starting
wavelength is 200 nm.
End Wavelength
In the End Wavelength box, you enter an ending wavelength for the scan
data. The range of acceptable values is from 191 to 800 nm. The default
ending wavelength is 600 nm. The end wavelength must be greater than the
start wavelength and the difference between the end wavelength and the
start wavelength must be an integral multiple of the wavelength step.
Wavelength Step
In the Wavelength Step box, you enter an appropriate scan data increment.
The default is 1 nm. (At a value of 1 nm, the detector will collect data at
every nanometer within the scan range.). Depending on the wavelength
range, the allowable values are 1 to 610 nm.
Note If you plan to create Spectrum files from the scan data acquired
with this method, use a 1 nm wavelength step.
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Sample Rate
In the Sample Rate list, you select an appropriate sample rate for your
application. The acceptable values are 0.5, 1, 2, 4, 5, 10, and 20 Hz with
1 Hz being the default. This sampling rate is the rate at which data points
are collected for each wavelength in the scan range. The size of your data
files increases as the sampling rate and number of wavelengths scanned
increases.
Filter Bandwidth
In the Filter Bandwidth box, you enter an appropriate bandwidth for the
Savitsky-Golay filter. Increasing the value of the bandwidth filter can
improve the signal-to-noise ratio for your chromatograms, but compromises
spectral resolution. The default setting is 1.00 nm for the highest spectral
resolution. See Figure 59, which shows the loss of spectral resolution for a
spectrum of toluene as the bandwidth is raised from 1 nm to 11 nm.
1 nm Bandwidth
11 nm
Bandwidth
Figure 59. Spectra of toluene, effect of bandwidth on spectral resolution
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Bandwidth values are limited to the following subset of odd integers: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 43, 45, 47,
and 49. In addition, the allowable scan range for a particular bandwidth is
limited as follows:
• Start Wavelength (minimum) = 190 nm + (bandwidth / 2)
• End Wavelength (maximum) = 800 nm – (bandwidth / 2)
According to these equations, the maximum scan range for a bandwidth of
49 nm is 214 nm to 776 nm.
Note Wider bandwidths decrease both spectral noise and the resolution
of the spectra. In general, the filter bandwidth should not exceed 10% of
the bandwidth at half-height of the narrowest spectral feature of interest.
Channels Area
The Channels area contains option buttons to select None, One, Two or
Three single-wavelength channels. There is also a sample rate box, which is
applied to all chromatogram-channels (A, B, and, C).
Sample Rate
In the Sample Rate list, you select a data-sampling rate for the discrete
channels. The available values are: 0.5, 1, 2, 4, 5, 10, and 20 Hz (1 Hz = 1
point per second), with the default of 10 Hz. A sampling rate that is too low
can affect the integration of narrow peaks.
None/A/B/C Select
You must select the appropriate channel option button to collect data for
that channel. You can select any combination of channels or none. Default
is Three Discrete channels.
Wavelength
In the Wavelength box, you enter an appropriate wavelength for your
application. The acceptable range of values is from 190 nm to 800 nm, in
increments of 1 nm. The default wavelengths are 214, 254, and 280 nm.
Note The combination of the bandwidth and wavelength settings
cannot extend beyond the range of the detector. For example, a
bandwidth of 49 nm is not allowed for a wavelength of 200 nm because
it would require an analysis range from 176 nm to 224 nm. The
maximum allowable bandwidth for 200 nm is 21 nm, which requires an
analysis range of 190 nm to 210 nm.
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Filter Bandwidth
In the Filter Bandwidth box, you enter an appropriate bandwidth for the
Savitsky-Golay filtering. The acceptable range is from 1 to 49 nm in
odd-number increments, with 1 nm meaning no filtering. The default value
is 9 nm.
Note Enter a wider bandwidth to improve the signal-to-noise ratio.
Timed Events Area
In the Timed Events area, you program actuation of a rear panel contact
closure as a function of either time or the absorbance level from one of the
discrete wavelength channels. This feature can be used to trigger an external
device, such as a fraction collector. See your Surveyor PDA Plus Detector
Hardware manual for information on connecting an external device to the
PDA detector.
The Time Events table lists all of the external events in chronological order.
The type, time, channel, level, and delay for each event is included for each
external event. You can add, modify, and remove events to the external
events table by double-clicking the event setting you want to modify, or by
using New, Delete, or Delete All buttons.
In the Timed Event dialog box, which is accessed by clicking the
New button or an event in the Time Events table, you select the
following parameters:
• Type
• Time
• Channel
• Level
• Delay
The Type list describes how the event is actuated. The selections are Event
Off, Event On, Zero Data, and Level Trigger. The Event Off option turns the
external Event contact Off. The Event On option turns the Event On. The
Level Trigger option turns the Event On when a specified absorbance level is
reached. The Zero Data option performs a zeroing of the detector signal.
The Event Off option is the default selection.
The Time box specifies at what point in the analysis the external Event is
triggered. The range is 0.00 to 600 min. The maximum allowable time is
the run time. The default value is 1.00 min.
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The Channel list designates which discrete wavelength channel is monitored
for the absorbance level. Either A, B, or C can be selected. The A wavelength
channel is the default selection.
The Level box specifies the absorbance level at which the external Event is
triggered if the Type is set to Level. The range is from -2000 to
4000.00 mAU. The default value is 0.00 mAU.
The Delay box is used in conjunction with the Level box to compensate for
the volume between the detector flow cell and the end of the outlet line
going to a fraction collector or other instrument. The range is 0.00 to
65535.00 s. The default value is 0 s.
Programming the Surveyor
PDA Plus Detector
To program the Surveyor PDA Plus Detector
1. Type a run time from 0 to 600 min in the Run Time box.
2. Select a rise time from 0 to 10.0 s in the Filter Rise Time list.
3. To collect spectral data, select the Spectra check box. Then, make the
following entries and selections:
a. Type a starting wavelength from 190 to 799 nm in the Start
Wavelength box.
b. Type an ending wavelength from 191 to 800 nm in the End
Wavelength box. The ending wavelength must be greater than the
starting wavelength.
c. Type a value for the wavelength interval in the Wavelength Step
box. Depending on the wavelength range defined by the starting
and ending wavelengths, the allowable values are 1 to 610 nm. The
wavelength range must be an integral number of wavelength steps. If
you are collecting spectral data for a spectral library, use a
wavelength step of 1 nm.
d. Select a sample rate from the Sample Rate box. As you increase the
sampling rate you also increase the data file size.
e. Type a bandwidth in the Filter Bandwidth box. Depending on the
wavelength range defined by the starting and ending wavelengths,
the allowable values are the subset of odd integers from 1 to 49 nm.
See “Filter Bandwidth” on page 90. Increasing the bandwidth
decreases the spectral resolution.
f. For normal data collection, select the Wavelength / Absorbance
option in the Units area.
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4. To collect discrete channel data, select one of the option buttons in the
Channels area. Then, make the following entries:
a. Select a sampling rate (in data points per second) from the Sample
Rate list. Higher data rates for discrete channels do not add
significantly to the data file size.
b. For each discrete channel, type a wavelength from 190 to 800 nm in
the Wavelength box and a filter bandwidth in the Filter Bandwidth
box.
Depending on the discrete wavelength, the acceptable bandwidth
range is from 1 to 49 nm in odd-number increments with 1 nm
meaning no filtering. Bandwidth values outside the range of the
detector are not allowed. For example, for the discrete wavelength of
200 nm, the maximum allowable bandwidth is 21 nm. At a
bandwidth setting of 21 nm, the reported absorbance value for
200 nm will be a weighted average from 190 nm to 210 nm. A value
greater than 21 nm would be outside the lower range limit of the
detector, which is 190 nm.
5. If you want to add a timed event to your Instrument Method, such as
the triggering of a fraction collector as a peak begins to elute, do the
following:
a. In the Timed Events area (see Figure 57 on page 87), click New.
Xcalibur displays the Timed Event dialog box shown in Figure 60.
b. Select the appropriate parameters from the Timed Event dialog box.
See “Timed Events Area” on page 92.
c. Click OK to exit the Timed Event dialog box.
Figure 60. Timed Event dialog box
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6. Complete the Instrument Method:
• If you want to add sample preparation steps to your method, go to
“Adding a Sample Preparation Routine to the Method” on page 98.
• If you do not want to add sample preparation steps to your method,
go to “Saving the Method” on page 109.
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Entering the Method Parameters for the UV/Vis Detector
Entering the Method
Parameters for the
UV/Vis Detector
The instrument control parameters for the Surveyor UV/Vis Plus Detector
are entered in the Surveyor UV/Vis Instrument Setup view shown in
Figure 61.
To open the Surveyor UV/Vis Plus view
Click the Surveyor UV/Vis icon in the Viewbar of the Instrument Setup
window.
Figure 61. Surveyor UV/Vis Instrument Setup view
To program the Surveyor UV/Vis Plus Detector
1. In the Rise Time list, select a rise time from 0 to 10.0 seconds
Rise time is the response time of the detector, in seconds, to the signal.
Increasing the rise time decreases the baseline noise; however, setting the
rise time to a value greater than one-tenth the width of the
chromatographic peak at half-height results in peak broadening as
shown in Figure 58 on page 88. The one-second default value is
appropriate for most LC applications
2. In the Data Rate list, select a data rate from 4 to 20 points per second.
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3. To zero the baseline at a specific time point, select the Enable autozero
check box and type a time in the Autozero time (min) box.
The detector resets its output voltage to zero at this time point.
4. In the Program type area, select one of the following options:
• Select the Single wavelength 190 to 800 nm option to collect one
chromatogram during a run. You can program time wavelength
changes in the program table.
• Select the Dual wavelength UV 190 to 450 nm option to collect two
chromatograms in the UV range during a run. You can program
time wavelength changes in the program table.
• Select the Dual wavelength Vis 366 to 700 nm option to collect two
chromatograms in the visible range during a run. You can program
time wavelength changes in the program table.
5. To re-zero the baseline when a programmed wavelength change occurs,
select the Zero on wavelength change check box.
The detector does not zero the baseline on the first or last rows of a
wavelength program. If the wavelength program contains three or more
rows, the detector zeroes the baseline on the second row and all
successive rows until it reaches the last row. The detector zeroes its
output signal even if the same wavelength is listed in the second through
second to last rows of the table.
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Adding a Sample Preparation Routine to the Method
Adding a Sample
Preparation Routine
to the Method
In Xcalibur, sample preparation routines are part of the Instrument
Method. In the Sample Preparation page, you create a multi-task routine,
which can contain up to 512 tasks. A task consists of a sample preparation
operation and its associated parameters.
The ability to add 512 tasks to a sample preparation routine gives you
considerable flexibility, however, the arrangement of the tasks in the task list
must follow a logical order. For example, you cannot add a task that
deposits liquid before you add a task that draws liquid. In addition, if you
are using the 250 μL concentric syringe that ships with the Surveyor
Autosampler, you must follow an additional set of rules that allow for the
proper positioning of the inner and outer plungers of the syringe.
This section contains the following topics:
• Opening the Sample Preparation Page
• Building the Sample Preparation List
• Sample Preparation Rules
• An Example of a Sample Preparation Routine
Opening the Sample
Preparation Page
To open the Sample Preparation page
1. Double-click the Xcalibur icon on your desktop to display the Xcalibur
Home Page.
2. On the Xcalibur Home Page, click the Instrument Setup button in the
Road Map or choose Goto > Instrument Setup.
3. Click the Surveyor AS button to display the Instrument Setup view for
the Surveyor Autosampler.
4. Click the Sample Preparation tab to display the Sample Preparation
page. See Figure 62.
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Figure 62. Surveyor AS Instrument Setup view – Sample Preparation page
Building the Sample
Preparation List
To build a sample preparation routine
1. If you have a stored instrument method that contains all or part of the
sample preparation routine that you want to use, go to step 2.
Otherwise, go to step 3.
2. Import the sample preparation routine from a stored instrument
method as follows:
a. Click Import to display the Open files dialog box.
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b. Browse through your Methods folder and select the appropriate
Instrument Method. Then, click Open.
Only the sample preparation task list contained in the stored
Instrument Method will be imported into the current Instrument
Method.
3. For each task that you want to add to the sample preparation routine,
do the following:
a. Click the individual task in the Prep Operations list.
Table 6 lists the thirteen tasks that you can use to create a sample
preparation routine. The parameters available for the task are
displayed below the Prep Operations list.
b. Enter the appropriate values for each parameter.
c. Click Add To Task List.
The task is copied to the Task List list in the Method area.
Note If the previous task used the small bore of the syringe, you
must add a Flush to Waste or a Wash Needle task before you can
add a task that requires the use of the large bore of the syringe. In
addition if the previous task used the small bore of the syringe,
the transfer volume of the current task will be limited to the
nominal syringe size or less. See “Sample Preparation Rules” on
page 101 for more information.
4. Edit the task list as needed:
• To remove the last task in the list, click Remove Task.
• To clear the entire task list, click Clear All Tasks.
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Table 6.
Sample Preparation Rules
Pretreatment Tasks
Task
Description
Deposit Liquid in Sample
Deposits liquid into a designated sample vial or well.
Deposit Liquid in Reservoir
Deposits liquid held in the needle tubing into a
reservoir vial.
Draw from Sample
Withdraws liquid from a sample vial or a well.
Draw from Reservoir
Withdraws liquid from a reservoir vial.
Flush to Waste
Moves the needle to the injection port and dispense
a specified volume of solvent drawn from the wash
bottle or a reservoir vial.
Mix at Sample
Mixes the contents at a sample location by
aspirating the sample into the needle tubing and
expunging it back into the sample location.
Mix at Reservoir
Mixes the contents of a reservoir vial. Mixing is
accomplished by aspirating and expunging the
contents of the reservoir vial.
Transfer from Sample to Reservoir
Moves liquid from a sample vial or well to a reservoir
vial.
Transfer from Sample to Sample
Moves liquid from one sample vial or well to a
second sample vial or well.
Transfer from Reservoir to Reservoir
Moves liquid from one reservoir vial to another
reservoir vial.
Transfer from Reservoir to Sample
Moves liquid from a reservoir vial to a sample vial or
well.
Wait Time
Pauses the sample preparation activity for a
specified period of time in min
Wash Needle
Moves the needle to the waste station and dispense
a specified volume of solvent drawn from the wash
bottle or a reservoir vial.
The maximum volume that can be deposited, drawn, or transferred during a
sample preparation task depends on the syringe type, the task requested, and
the previous step in the sample preparation method.
For the 250 μL concentric syringe, the manner in which the various sample
preparation tasks can be linked together is affected by whether the task takes
place in only the large bore of the syringe, in only the small bore of the
syringe, or in either bore of the syringe.
The sample preparation tasks can be divided into three groups depending
their bore usage as shown in Table 7.
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Table 7.
Sample preparation tasks arranged in groups according to bore usage
Small Bore Only
Large Bore Only
Small or Large Bore
Draw from Sample
Mix at Sample
Draw from Reservoir
Transfer from Sample to Sample
Mix at Reservoir
Deposit Liquid in Sample
Transfer from Sample to
Reservoir
Wash Needle
Deposit Sample in Reservoir
Flush to Waste
Transfer from Reservoir to Sample
Transfer from Reservoir to
Reservoir
For the 250 μL concentric syringe, the arrangement of the tasks in the
sample preparation list is restricted by the following rules:
Note To save a method, it must conform to these rules.
• For tasks performed with the small bore of the concentric syringe, the
maximum volume (sample + air bubble) that you can draw, deposit, or
transfer is limited to the nominal size of the syringe, 250 μL.
If a task can be performed from either the small or the large bore, the
concentric syringe uses the small bore if the requested volume (liquid +
air bubble) plus any volume left in the needle tubing from a previous
step is less than the nominal syringe size.
• For tasks performed with the large bore of the concentric syringe, the
maximum volume (sample + air bubble) that you can draw, deposit, or
transfer is limited to 500 μL, except for the Wash Needle and Flush to
Waste tasks.
If a task can be performed from either the small or the large bore, the
syringe uses the large bore if the requested volume (liquid + air bubble)
is greater than the nominal syringe size.
• Crossover between bores is not allowed. Therefore, you cannot add a
task that uses the large syringe bore immediately following a task that
uses the small syringe bore. To switch from the small bore to the large
bore of the syringe, you must insert a Flush to Waste step or a Wash
Needle step. These tasks home the position of the syringe plungers.
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An Example of a Sample
Preparation Routine
This topic contains an example that describes how to create a sample
preparation routine to dilute samples ten-fold.
To add a sample preparation routine that dilutes samples ten-fold
1. From the Instrument Setup window - Surveyor AS view, click the
Sample Preparation tab to display the Sample Preparation page.
2. Open the [.meth] file that you want to modify.
3. Create the sample preparation routine:
a. Add a Transfer From Sample To Sample prep operation to the
routine:
i. In the Prep Operations area, select the Transfer From Sample To
Sample preparation operation from the list.
ii. The parameters for the Transfer From Sample To Sample
preparation operation are listed below the Add To Task List
button.
iii. Keep all the parameters for the Transfer From Sample To
Sample preparation operation set to the default settings except
those that are shown below and are reflected in Figure 63.
Parameter
Setting
Result
Source Sample
Location
Location
Relative Location
Current
Specifies that sample will be
withdrawn from the current
vial location listed in the
sequence table.
Destination Sample
Location
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Relative Location
Location
Current + 1
Specifies that sample will be
deposited in the current + 1
vial location listed in the
sequence table.
Volume
50
Specifies that 50 μL of sample
will be transferred
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Figure 63. Parameters for Transfer From Sample To Sample
preparation operation
iv. Click Add To Task List to add the task to the sample
preparation routine.
When this task is performed, 50 μL of sample is withdrawn from
the current vial location in the sequence table and deposited in the
current + 1 vial location.
b. Add two Transfer from Reservoir To Sample prep operation tasks:
i. In the Prep Operations area, select the Transfer From Reservoir
To Sample preparation operation from the list.
The parameters for the Transfer From Reservoir To Sample
preparation operation are listed below the Add To Task List button.
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ii. Keep all the parameters for the Transfer From Reservoir To
Sample preparation operation set to the default settings except
those that are shown below and are reflected in Figure 64.
Figure 64. Parameters for the Transfer From Reservoir To Sample
preparation operation
iii. Click Add To Task List twice to add this task to the sample
preparation routine twice.
When this task is performed, 225 μL of diluent is withdrawn from
the reservoir vial 1 and deposited in the current + 1 vial location.
This task is performed twice to transfer a total of 450 μL of diluent
to the current + 1 vial location. Two aliquots of 225 μL each are
transferred rather than one aliquot of 450 μL because fluid transfers
performed by the inner plunger of the concentric syringe are more
precise than those performed with the outer plunger.
c. Add a Wash Needle prep operation task:
i. In the Prep Operations area, select the Wash Needle preparation
operation from the list.
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The parameters for the Wash Needle preparation operation are
listed below the Add To Task List button.
ii. Keep all the parameters for the Wash Needle prep operation set
to the default settings as reflected in Figure 65.
Figure 65. Parameters for the Wash Needle preparation operation
iii. Click Add To Task List to add this task to the sample
preparation routine.
The Wash Needle operation is performed at this point in the
routine to home the concentric syringe.
d. Add a Mix At Sample prep operation task:
i. In the Prep Operations area, select the Mix At Sample prep
operation from the list.
The parameters for the Mix At Sample preparation operation are
listed below the Add To Task List button.
ii. Keep all the parameters for the Mix At Sample prep operation
set to the default settings except those that are shown on
page 107 and are reflected in Figure 66.
iii. Click Add To Task List to add this task to the sample
preparation routine.
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When this operation is performed, the solution in the current + 1
vial location will be aspirated and expunged 10 times.
Parameter
Setting
Result
Sample Location
Location
Relative
Location
Specifies that sample will be aspirated and
expunged from the current + 1 vial location
listed in the sequence table.
Location
Current + 1
Volume
250
Specifies that 250 μL of sample will be
aspirated and expunged
Cycles
10
Specifies that the sample will be aspirated
and expunged 10 times
Figure 66. Parameters for the Mix At Sample preparation operation
4. Review the task list by expanding the tasks in the Method list. See
Figure 67.
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5. Save your method by choosing File > Save.
To perform this sample routine, you need to insert empty vials into your
sample tray. For example, to dilute five samples, place the samples in
vial locations A1, A3, A5, A7, and A9. Place empty vials in vial locations
A2, A4, A6, A8, and A10. Fill reservoir vial 1 with an appropriate
diluent that matches the sample matrix.
If you do not want to inject the diluted samples, create a five line
sequence that lists the vial locations of the original samples (A1, A3, A5,
A7, and A9). The original samples will be injected.
If you want to inject the diluted samples as well, create a ten line
sequence that lists all the vial locations (A1 to A10). Ensure that you use
the method that contains the sample preparation routine on only the
odd rows.
Figure 67. Method area, showing expanded task list
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Creating Instrument Methods
Saving the Method
To save the instrument method as a file of type [.meth]
1. Choose File > Save As to open the Save As dialog box. See Figure 68.
Figure 68. Save As dialog box
2. Browse through the directory tree to find the Drive:\Xcalibur\methods
directory.
3. Type a file name in the File Name box.
4. Click Save to open the File Summary Information dialog box. See
Figure 69.
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Saving the Method
Figure 69. File Summary Information dialog box
5. Type a description of the Method file in the Description box.
6. Click OK.
If the Authorization Manager - Comment check box is enabled, the File
Save - Audit Trail dialog box appears. See Figure 70.
Figure 70. File Save – Audit Trail dialog box
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Saving the Method
7. Type a comment concerning the changes you made to the instrument
method in the Comment box.
8. Click Continue to close the File Save – Audit Trail dialog box and save
the Instrument Method.
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Chapter 5
Creating and Running
Sequences
This chapter describes how to acquire and view chromatographic and PDA
spectral data for a single sample and contains the following sections:
• Creating a Single Sample Sequence
• Equilibrating Your Column and Warming Up the D2 Lamp
• Loading the Surveyor Autosampler
• Starting Data Acquisition
• Viewing the Data As It Is Acquired
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Creating a Single Sample Sequence
Creating a Single
Sample Sequence
To set up a Sequence to inject a single sample, perform the following
procedures contained in this section in the order listed:
1. Opening the Sequence Setup Window
2. Creating the Sequence
3. Saving the Sequence
Opening the Sequence
Setup Window
To open the New Sequence Template Dialog box
1. Click the Sequence Setup button on the Home Page to open the
Sequence Setup view.
A view similar to that in Figure 71 is displayed.
2. From the Sequence Setup window, choose File > New.
Xcalibur displays the New Sequence Template dialog box. See
Figure 72.
Figure 71. Sequence Setup window
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Figure 72. New Sequence Template dialog box
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Creating a Single Sample Sequence
Creating the Sequence
To create a new Sequence
1. In the General area, make the following entries and selections:
a. Type a name for the raw data file in the Base File Name box.
b. Browse to the data file directory where you want to store your raw
data files.
Xcalibur adds the [.raw] file extension to the data files that contain
the chromatographic and spectral data.
c. Browse to the instrument method that you want to use to acquire
your raw data files.
Instrument Setup methods have a [.meth] file extension. The
Instrument Setup view for the Surveyor devices is described in
Chapter 4, “Creating Instrument Methods.” .
d. If you have not yet created a processing method that contains the
information needed to quantitate your unknowns, leave the
Processing Method box blank.
You can create a processing method and reprocess your stored data
files at a later date. Processing methods have a .pmd file extension.
For information on performing tests to determine the suitability of
your chromatographic method and on creating calibration curves to
quantitate your unknowns, see the Xcalibur Getting Productive:
Quantitative Analysis manual.
2. In the Samples area, type the following:
a. Type 1 in the Number Of Samples box.
b. Type 1 in the Number Of Injections box.
c. Type the vial position in the Initial Vial Location box.
The vial positions for the conventional trays are shown in Figure 73.
d. Type an identifying name for the sample in the Sample ID box.
This box entry is optional. If you do not enter a Sample ID,
Xcalibur automatically uses the vial position as the Sample ID. If
you enter a Sample ID, Xcalibur automatically appends the vial
position to your entry.
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Slot
for Tray
Vial
Position
A:20
Creating and Running Sequences
Creating a Single Sample Sequence
Tray
Runner
Vial
Position
E:40
RV4
RV3
RV2
RV1
Standard
1.8 mL vial
Vial
Position
A:1
A
B
C
D
E
Vial
Position
E:21
Tray
Handle
Figure 73. Vial positions for conventional sample trays
3. Click OK to display your sequence spreadsheet. See Figure 74.
The injection volume displayed in the Inj Vol column matches the
injection volume contained in your instrument setup method. You can
override this injection volume value.
4. If you want to change the injection volume, double-click the
spreadsheet cell containing the injection volume value that you want
to change, highlight the current value, and then type a new value in
the cell.
For full details of all the parameters in the New Sequence Template
dialog box, see the Online Help or your Xcalibur Getting Productive:
Qualitative Analysis or Xcalibur Getting Productive: Quantitative Analysis
manuals.
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Creating a Single Sample Sequence
Figure 74. Sequence Setup view, showing newly created one line sequence
5. To alter the current column arrangement, click the Column
Arrangement toolbar button to display the Column Arrangement
dialog box. See Figure 75. Then do one of the following:
• To add a column to the sequence, select the column from the
Available Columns list, and click Add.
• To remove a column from the sequence, select the column from the
Displayed Columns list, and click Remove.
• To alter the position of the columns in the sequence, select the
column from the Displayed Columns list, and click either Move
Up or Move Down as appropriate.
Figure 75. Column Arrangement dialog box
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Creating and Running Sequences
Creating a Single Sample Sequence
To save the sequence
1. Choose File > Save As.
Xcalibur displays the File Summary Information dialog box. See
Figure 76.
2. Type an appropriate description in the Description box.
Then click OK.
Xcalibur displays the Save As dialog box. See Figure 77.
3. Browse to the appropriate folder in which you want to save the
sequence.
4. Type a file name in the File Name box.
5. Click Save.
Figure 76. File Summary Information dialog box
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Figure 77. Save As dialog box
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5 Creating and Running Sequences
Equilibrating Your Column and Warming Up the D2 Lamp
Equilibrating Your
Column and Warming
Up the D2 Lamp
Before you inject your sample, equilibrate your chromatographic column
and warm up the deuterium lamp, if necessary. For the best results, warm up
the deuterium lamp for a minimum of 1 hour and equilibrate your column
for a minimum period of time equivalent to 15 column volumes.
Note Warming up the deuterium lamp and equilibrating your LC
column helps to reduce baseline drift.
This section contains the following procedures:
• Warming Up the Deuterium Lamp
• Equilibrating the Chromatographic Column
Warming Up the
Deuterium Lamp
To warm up the deuterium lamp of the PDA detector
1. Open the Surveyor PDA Plus Instrument Setup view.
2. Choose Surveyor PDA Plus > Direct Control.
3. Click the Configuration tab.
The Configuration page appears as shown in Figure 125 on page 175.
4. In the Deuterium Lamp area, click Turn On.
To warm up the deuterium lamp of the UV/Vis detector
1. Open the Surveyor UV/Vis Instrument Setup view.
2. Choose Surveyor UV/Vis > Direct Control.
The Direct Control dialog box for the Surveyor UV/Vis Detector
appears as shown in Figure 78.
3. In the Deuterium Lamp area, click Lamp On.
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Figure 78. Direct Control dialog box for the Surveyor UV/Vis Detector
Equilibrating the
Chromatographic Column
To equilibrate your LC column
1. Choose GoTo > Instrument Setup to display the Instrument Setup
window.
2. Download the same solvent percentages and flow rate as those contained
in your method to the pump:
• For the LC pump, see Starting the LC Pump Solvent Flow.
• For the MS pump, see Starting the MS Pump Solvent Flow.
Starting the LC Pump Solvent
Flow
To start the solvent flow from the LC pump
1. Click the Surveyor LC Pump button in the viewbar to display the
Surveyor LC Pump view.
2. Choose Surveyor LC Pump > Direct Control > Operation to open
the Operation dialog box. See Figure 79.
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Figure 79. Operation - Surveyor LC Pump Direct Control dialog box
3. In the Method area, enter the appropriate values for the flow rate and
the solvent percentages.
4. Click Download.
5. Click Pump On to start the pump motor.
6. Click Close to exit the dialog box.
Starting the MS Pump Solvent
Flow
To start the solvent flow from the MS pump
1. Click the Surveyor MS Pump Plus button in the viewbar to display the
Surveyor MS Pump view.
2. Choose Surveyor MS Pump Plus > Direct Control to open the Direct
Control dialog box. See Figure 80.
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Figure 80. Direct Control dialog box for the MS pump
3. In the Direct Control dialog box, do the following:
a. Select the Take pump under control check box.
b. Type percentages in the Inlet boxes to create the same mobile phase
as the one in the instrument method you plan to use.
c. In the Flow box, type an appropriate flow rate.
d. Click the Start Run button
to start the pump flow.
e. After you finish equilibrating the LC column, click the Stop button
to stop the pump flow.
4. Close the Direct Control dialog box.
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Autosampler
Creating and Running Sequences
Loading the Surveyor Autosampler
Before you load your samples into the autosampler, ensure that your
samples are completely soluble in the mobile phase and that you have
filtered your samples through a 0.5-micron filter (if necessary). These
techniques minimize sample precipitation in the lines and remove
particulate matter that could obstruct the flow through the autosampler
injector or the column. In addition, make sure that the vial caps are securely
fastened onto the vials.
Before you start a sequence run, ensure that you have samples in the
locations specified in your sequence.
Note To trigger the vial sensor, custom vials must be positioned in the
tray such that the top of the vial reaches the minimum height of 1.55-in.
If you place vials that fall below this minimum height in the tray, the vial
sensor will not detect them, your sequence will be halted, and the
message Vial Not Found will appear.
To load a conventional tray into the Surveyor Autosampler Plus
1. Open the left door of the autosampler.
If you selected the Verify Door Is Closed configuration option for the
autosampler, the XYZ arm moves to the back of the tray compartment.
2. If the XYZ arm does not move to the back of the tray compartment, use
the Position Arm to Access Tray command to move it as follows:
a. From the Instrument Setup window, click the Surveyor
Autosampler button in the Viewbar to display the Surveyor
Autosampler view.
b. Choose Surveyor AS > Direct Control to display the Direct
Control dialog box.
c. Click the down-arrow to display the list of commands. Then select
Position Arm To Access Tray from the list.
d. Click Apply to download and execute the command.
e. Close the Direct Control dialog box.
3. Hold the tray handle, tilting the back end of the tray down. Insert the
tray runner into the slot at the rear of the tray compartment. Lower the
front of the tray into place. Then press down firmly to seat the tray.
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Starting Data Acquisition
Starting Data
Acquisition
To inject a sample and start data acquisition
1. Open the sequence file containing the information for the sample that
you want to inject:
a. From the Home Page – Sequence Setup view, choose File > Open
to display the Open dialog box. See Figure 81.
Figure 81. Open dialog box, showing the selection of a Sequence file
b. Browse to the appropriate folder.
c. Select the sequence that contains the sample you want to run.
Sequence files are identified by their [.sld] file extension.
d. Click Open.
Xcalibur opens the selected sequence.
2. Highlight the sequence row that you want to run. Do this even if the
sequence contains just one row. See Figure 82.
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Figure 82. Sequence Setup view, showing sequence with first row selected
3. Confirm that you have a vial in the position specified in the
sequence row.
4. From the toolbar, click the Run Sample button to open the Run
Sequence dialog box. See Figure 83.
In the Run Sequence dialog box, notice that the User box contains your
login name and that the Run Rows box contains the number of the row
that you selected in the sequence spreadsheet.
5. In the Acquisition Options area, confirm the following:
a. Confirm that the MS detector and LC components are configured
for operation as Xcalibur devices in the Instrument list.
b. Confirm that the autosampler is configured as the start instrument.
After the injection valve of the autosampler switches to the inject
position, the autosampler sends a signal to the detector to begin data
collection.
Note If you want to change the components of your LC system, click
Change Instruments to open the Change Instruments In Use dialog box.
A list of components that have been configured for operation as Xcalibur
devices is displayed. Select the appropriate LC component(s) you want to
add to your LC system and deselect the component(s) that you want to
remove from your LC system. For more information about the Change
Instruments in Use dialog box, see the online Help.
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Figure 83. Run Sequence dialog box
c. To start the run automatically, select the Start When Ready
check box.
Xcalibur will start the run after you click OK in the Run Sequence
dialog box. The run begins after the pump sends a pump ready
signal to the autosampler. The MS pump does not indicate the
Ready state until it monitors a stable backpressure as defined in your
instrument method.
Note To manually start an acquisition, clear the Start When Ready
check box and choose Actions > Start Analysis from the Sequence Setup
menu. To subsequently control the acquisition, choose Actions > Pause
Analysis or Actions > Stop Analysis.
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d. In the After Sequence Set System area, select one of the three
options:
• Select the On option if you want Xcalibur to leave the devices in
the On state.
• Select the Off option if you want Xcalibur to turn off the pump
flow and the lamps of the PDA detector after the data file is
acquired.
• Select the Standby option if you want to turn off the pump flow
but leave the lamps on after the data file is acquired.
6. Leave the parameters in the other areas at their defaults.
The Instrument Method and Programs areas in the Run Sequence
dialog box are used to specify particular acquisition or processing
requirements. For full details of these facilities, see the Xcalibur
online Help.
7. Click OK to start the run.
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Viewing the Data As It Is Acquired
Viewing the Data As It
Is Acquired
To view the data as it is acquired
1. Choose View > Real Time Plot View or click the Real Time Plot View
toolbar button on the Home Page.
2. If the display is not already locked, click the Lock Display button to
lock the display.
Locking the display allows you to monitor the real-time progress of
your run.
Locked
Unlocked
In the unlocked position, you cannot monitor the real-time progress of
your run, but you can review your data. For example, you can display
the spectrum for a particular peak that has already eluted. Data
collection continues off screen as you review your data.
If you are collecting PDA scan data, a view similar to that shown in
Figure 84 is displayed. The view contains three cells: a Chromatogram
cell, a Spectrum cell, and a Total Scan cell.
The sample shown in Figure 84 contains a mixture of uracil, benzene,
toluene, anthracene, and pyrene. The peak for pyrene is eluting at 7.5 min.
The PDA detector parameters for the data file being acquired are as follows:
• Run Time: 10.0 min
• Rise Time: 1.0 s
• Scan Range: 220 nm - 400 nm
• Wavelength Step: 1 nm
• Bandwidth: 1 nm
• Three Channels: 239 nm, Bandwidth: 9 nm; 254 nm, Bandwidth:
9 nm; 357 nm, Bandwidth: 9 nm
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Lock Display Button
Figure 84. Real Time Plot view, showing the acquisition of PDA scan data and one discrete channel
Reviewing Real-Time Data
You can review the data as it is being collected.
To view the spectrum for a particular peak in the chromatogram
1. Unlock the display by clicking the Lock Display button.
After you unlock the display, data collection continues off screen.
2. Pin the Spectrum cell by clicking the pin in the upper-right corner of
the cell.
The pin in the upper-right corner of the Spectrum cell turns green.
Cursor actions in other cells such as the chromatogram cell will now
affect the view displayed in the Spectrum cell.
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3. Click the peak of interest in the Chromatogram cell.
In the Spectrum cell, a spectrum appears for the time-point that you
clicked.
4. Click the Lock Display button to resume monitoring real-time data
acquisition.
5. Pin the Chromatogram cell by clicking the pin in the upper-right corner
of the cell.
The pin in the upper-right corner of the Chromatogram cell turns
green. Cursor actions in other cells such as the Spectrum cell will now
affect the view displayed in the Chromatogram cell.
6. Click the wavelength of interest in the Spectrum cell.
In the Chromatogram cell that contained the Total scan chromatogram,
a scan chromatogram appears for the specific wavelength that you
clicked on.
7. Click the Lock Display button to resume monitoring real-time data
acquisition.
Adding Cells to the
Display
You can display multiple cells in the Real Time display view.
To display multiple chromatogram cells
1. Click the Chromatogram cell to make it the active cell with a gray
border.
2. Choose View > Ranges to open the Chromatogram Ranges dialog box.
See Figure 85.
3. For each cell that you want to add, do the following:
a. Select a Type check box.
b. Select a detector from the Detector list.
c. Select a Plot Type from the Plot Type list.
4. Click OK to close the Chromatogram Ranges dialog box.
5. Choose View > Lock Display to resume monitoring real-time data
acquisition.
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Figure 85. Chromatogram Ranges dialog box
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Chapter 6
Using Qual Browser
This chapter provides an introduction to the Xcalibur Qual Browser
facilities that you can use to review the PDA data contained in your [.raw]
data files.
This chapter contains the following sections:
• Opening a Raw Data File in Qual Browser
• Working with the Cell Grid
• Changing the Font Size of the Display
• Viewing a Report of the Instrument Method
• Creating a Layout for PDA Data
• Viewing the Spectrum for a Specific Time Point
• Viewing the Chromatogram for a Specific Wavelength
• Determining Peak Areas
• Calculating the Purity of the Chromatographic Peaks
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Opening a Raw Data File in Qual Browser
Opening a Raw Data
File in Qual Browser
The data files containing the raw chromatographic and spectral data have
the [.raw] file extension.
To open a Raw File in Qual Browser
1. Click the Qual Browser icon on the Home page - Roadmap view or
choose GoTo > Qual Browser to display the empty Qual Browser
window. See Figure 86.
Figure 86. The empty Qual Browser window
2. Choose File > Open to display the Open Raw Data File dialog box. See
Figure 87.
Figure 87. Open Raw File dialog box
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Opening a Raw Data File in Qual Browser
3. Select the .raw file that you want to review.
4. Select the window layout from the list at the bottom of the dialog box:
• Select Current Layout if you the current layout for the Qual
Browser window is different from the default layout and you want
to apply it to your data file.
• Otherwise, leave the window layout selection at its default of
Default Layout.
5. Click Open to open the raw file in the Qual Browser window.
If the default layout has not been modified and your raw file contains
MS data in addition to PDA data or UV data, the data file will open
with the MS TIC chromatogram in the upper cell and a mass range
spectrum in the lower cell. See “Working with the Cell Grid” on
page 140 for an explanation of cells.The Y-axis for these cells is set to
Relative Absorbance. See Figure 88.
Figure 88. Qual Browser view with a chromatogram cell displaying MS TIC data and a spectrum cell
displaying mass range data
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Opening a Raw Data File in Qual Browser
6. To apply a custom window layout to the Qual Browser view:
a. Choose File > Layout > Apply to open the Open Layout File
dialog box shown in Figure 89.
b. Select a layout file from the list.
c. Click Open to apply the layout to the [.raw] data file.
See Figure 90, which shows a custom layout file applied to a [.raw]
data file containing both MS data and PDA data. The custom
layout replaces the MS TIC chromatogram with a Total Scan
chromatogram for the PDA data. It also replaces the Mass Range
spectrum data from the MS detector with the spectral data from the
PDA detector.
See “Creating a Layout for PDA Data” on page 149 for instructions
on how to create a Layout file for your PDA data.
Figure 89. Open Layout File dialog box
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Figure 90. Qual Browser view, showing a chromatogram cell displaying a Total Scan from the PDA
detector and a spectrum cell displaying the spectral data from the PDA detector for time 0
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Working with the Cell Grid
Working with the Cell
Grid
To use the Qual Browser facility, you need to understand the concept of cell
states and the effect of cursor actions in a cell. This section contains the
following topics:
• Cell States
• Cursor Actions
Cell States
When you open a [.raw] data file in the Qual Browser window, the
information within the data file is displayed as a grid of cells.
There are three hierarchal states, which are described in this section, for a
cell within the grid:
• Inactive Cells
• Active but Unpinned Cells
• Active and Pinned Cells
The grid always contains either one active but unpinned cell or one
pinned cell. If the grid contains more than one cell, only one cell can be
active and the rest of the cells are inactive.
An unpinned cell has a gray pin icon in its upper right corner.
A pinned cell has a green pin icon in its upper right corner.
Inactive Cells
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Inactive cells are not highlighted with a gray border and the pin icon in their
upper right corners are gray. The cell in the lower portion of Figure 91 is
inactive as indicated by the absence of a gray border. Menu commands,
toolbar buttons, and cursor actions do not affect inactive cells. If you want
to zoom in on the contents of a cell or access its shortcut menu, you must
make it an active cell.
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Working with the Cell Grid
Gray Border
Shortcut Menu
Unpinned
Active Cell
Inactive Cell
Figure 91. Qual Browser window, displaying an active chromatogram cell and an inactive PDA
spectrum cell
Active but Unpinned Cells
An active but unpinned cell is highlighted with a gray border and the pin
icon in its upper right corner is gray. The cell in the upper portion of
Figure 91 is active but unpinned. Menu commands, toolbar buttons, and
cursor actions affect the active cell. Clicking an inactive cell in the grid will
make it the active cell.
Active and Pinned Cells
Clicking the pin in the upper right corner of a cell makes it the pinned cell
within the grid. A pinned cell is an active cell that cannot be made inactive
by clicking in another cell. Instead, actions performed in the inactive cells
affect the pinned cell as described in the next topic, “Cursor Actions” on
page 142. The lower cell in Figure 92 is a pinned cell.
If you want to automatically change the range of a cell by clicking in the
grid, you must pin the cell. For example, if you want to display the spectrum
for the 1 min time point without opening the Spectrum Ranges dialog box,
pin the Spectrum cell. Then, click the 1 min time point in the inactive
chromatogram cell.
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Working with the Cell Grid
Inactive Cell
Pinned Cell
Figure 92. Qual Browser window, displaying an inactive cell and a pinned cell
Cursor Actions
Within the cells of the grid, you can use the cursor in three ways:
• A click picks a point on the cell.
• A line dragged parallel to any axis picks a range.
• A line dragged in any diagonal direction selects an area.
The effect of these actions depends on the state of the cell. Within an active
cell, cursor actions rescale the plot. See Table 8.
Table 8.
Effect of cursor action in an active cell
Cursor Action
Effect
Drag parallel to X-axis
Rescale graph showing selected X range only, same Y range
Drag parallel to Y-axis
Rescale graph showing selected Y range only, same X range
Dragged area
Rescale graph showing both the selected X and Y ranges
If one of the cells is pinned, the cursor action in any of the inactive cells is
applied to the pinned cell. See Table 9.
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Table 9.
Effect of cursor action in an inactive cell on the pinned cell
Pinned cell
Cursor action
Effect
spectrum
Click in a chromatogram cell
The spectrum cell displays the
spectrum at that retention time.
chromatogram
Click in a spectrum cell
The chromatogram cell displays
chromatogram for the wavelength
selected in the spectrum cell.
In Figure 93, the Spectrum cell on the bottom of the view is pinned.
Clicking the 1.80 minute time point in the unpinned Chromatogram cell
causes the spectrum of benzene, which elutes at 1.8 minute, to be displayed
in the pinned Spectrum cell.
Pointer is set to 1.8 min time point
Figure 93. Qual Browser window, showing a pinned Spectrum cell and an unpinned Chromatogram cell,
with the cursor pointing to the 1.8 min time point
In Figure 94, the Chromatogram cell on the top of the view is pinned.
Clicking the wavelength in the unpinned Spectrum cell displays the
chromatogram of the scan wavelength in the pinned Chromatogram cell.
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Chromatogram of 251 nm
scan wavelength is displayed
Pointer is set to 251 nm
Figure 94. Qual Browser window, showing a pinned Chromatogram cell and an unpinned Spectrum cell,
with the cursor pointing to the 251 nm scan wavelength
Clicking the Full Size button in the toolbar sizes the active or pinned cell to
the full width and height of the window as shown in Figure 95.
Dragging across a region in the active or pinned cell zooms in on that region
as shown in Figure 96.
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Figure 95. Spectrum sized to full size of window
Figure 96. Full size view of Spectrum cell, zoomed in on the 320 nm to 380 nm region
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Changing the Font Size of the Display
Changing the Font
Size of the Display
Occasionally, you might want to change the font size of the data displayed
in the Xcalibur data system. For example, you might want to increase the
font size for screen captures that you plan to use for presentations.
To increase the font size
1. From Roadmap – Homepage, choose Tools > Configuration to open
the Xcalibur Configuration dialog box.
2. Click the Fonts tab to display the Fonts page. See Figure 97.
Figure 97. Xcalibur Configuration dialog box – Fonts page
3. To increase the font size of the chromatogram, spectrum, and map
axis labels:
a. In the Proportional Pitch Font area, select a larger font size from
the Max list.
b. Select a larger font size from the Min list.
4. To increase the font size for the Spectrum List, Scan Header, Scan
Filters, or Report, do the following:
a. In the Fixed Pitch Font area, select a larger font size from the Max
list.
b. Select a larger font size from the Min list.
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Changing the Font Size of the Display
See Figure 98, which shows the chromatogram axes labeled with a
proportional pitch font size of 8 and Figure 99, which shows the
chromatogram axes labeled with a proportional pitch font size of 16.
Figure 98. Proportional pitch font size = 8
Figure 99. Proportional pitch font size = 16
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Viewing a Report of the Instrument Method
Viewing a Report of
the Instrument
Method
After you open a data file, you might want to check the instrument
parameters that were used to acquire it.
To view a report that lists the instrument control parameters
1. Choose View > Report > Instrument Method.
The instrument method is displayed in the top cell of the window. The
instrument method is divided by device, with the parameters for each
device displayed on a separate page. See Figure 100.
2. Click the Show Previous and Show Next buttons to move through the
pages of your Instrument Method.
Figure 100. Qual Browser View, displaying the Surveyor PDA page of the
Instrument Method Report
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Creating a Layout for PDA Data
Creating a Layout for
PDA Data
This topic describes how to view chromatograms and spectral information
acquired by the Surveyor PDA detector. In addition, it describes how to save
your range and display settings for the PDA data in a Layout file.
Perform the following procedures provided in this section in the
order listed:
1. Specifying the Chromatogram Range
2. Setting the Display Options for the Chromatogram Cell
3. Specifying the Spectrum Range
4. Setting the Display Options for the Spectrum View
5. Inserting Cells
6. Saving a New Layout
Specifying the
Chromatogram Range
There are two types of chromatograms for the Surveyor PDA:
chromatograms acquired from any of the three discrete channels and
chromatograms interpolated from the scan data. This topic contains the
following procedures that describe how to display the chromatograms
acquired by the PDA detector in the Qual Browser window:
• Displaying Scan Chromatograms
• Displaying Discrete Chromatograms
Displaying Scan Chromatograms
To display a scan chromatogram for the PDA detector data
1. Pin the Chromatogram cell.
2. Right-click the Chromatogram cell to open a shortcut menu.
3. From the shortcut menu, choose Ranges to open the Chromatogram
Ranges dialog box. See Figure 101.
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Figure 101. Chromatogram Ranges dialog box, showing the selection of the Total Scan plot type
4. In the Range area, set the displayed time range of the chromatogram:
• For a generic layout file, leave an asterisk in the Time Range box.
When you open a .raw data file, the X-axis of the chromatogram is
scaled to the run time for your detector in the instrument setup
method used to acquire the data.
• To specify a specific time range, type a beginning time point and an
ending time point separated by a dash in the Time Range box.
5. In the Plot Properties area, select the following:
a. Select PDA from the Detector list.
b. Select Avalon from the Peak Algorithm list.
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c. Select a Plot Type:
• Select Wavelength from the Plot Type list to display the
chromatogram for a specific wavelength within your scan range
or to display the averaged results from a range of wavelengths in
your scan range.
• Select Total Scan from the Plot Type list to display the average
absorbance for each time point of all the wavelengths in your
scan range.
• Select Spectrum Maximum from the Plot Type list to display a
plot of the maximum absorbance values in your scan range for
each time point.
d. Select a wavelength range (If you selected the Total Scan plot type,
this box is unavailable.):
• To display the chromatogram for a specific scan wavelength,
type the wavelength number in the Range box.
• To display a plot of the average absorbance values for a range of
wavelengths, type the beginning wavelength number and the
ending wavelength number separated by a dash in the Range
box. For example, type 200-300 to display a plot of the average
absorbance values for the scanned wavelengths from 200 nm to
300 nm.
6. Click OK to exit the dialog box and view your scan chromatogram. See
Figure 102.
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220 nm Scan Wavelength
Figure 102. Chromatogram view, showing a chromatogram for a PDA scan wavelength
Displaying Discrete
Chromatograms
To display a discrete chromatogram for the Surveyor PDA Plus data
1. Pin the Chromatogram cell.
2. Right-click the Chromatogram cell to open a shortcut menu.
3. From the shortcut menu, choose Ranges to open the Chromatogram
Ranges dialog box. See Figure 103.
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Figure 103. Chromatogram Ranges dialog box, showing the selection of a discrete channel wavelength
4. In the Range area, set the displayed time range of the chromatogram:
• For a generic layout file, leave an asterisk in the Time Range box.
When you open a [.raw] data file, the X-axis of the chromatogram is
scaled to the run time for your detector in the instrument setup
method used to acquire the data.
• To specify a specific time range, type a beginning time point and an
ending time point separated by a dash in the Time Range box.
5. In the Plot Properties area, select the following:
a. Select UV from the Detector list.
b. Select Avalon from the Peak Algorithm list.
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c. Select Channel A, Channel B, or Channel C from the Plot Type list.
See Figure 103, which shows the selection of the discrete channel B
wavelength.
6. Click OK to exit the dialog box and view your discrete chromatogram.
See Figure 104, which shows the display of a discrete channel
chromatogram.
Discrete Channel B
Wavelength
Figure 104. Chromatogram view, showing a chromatogram for a PDA discrete channel
Setting the Display
Options for the
Chromatogram Cell
To set the display options for the Chromatogram cell
1. Right-click the pinned Chromatogram cell to open a shortcut menu.
2. From the shortcut menu, choose Display Options to open the Display
Options dialog box.
3. Specify the Normalization parameters:
a. Click the Normalization tab to open the Normalization page. See
Figure 105.
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b. In the Normalize Method area, select the Auto Range option
button.
This ensures that the entire dynamic range of the chromatogram is
displayed in the active view, normalized over the full range of the
Y-axis.
c. In the Normalize Each Plot To area, select the Largest Peak In
Selected Time Range option button.
Figure 105. Display Options dialog box – Normalization page
4. Specify the Axis parameters:
a. Click the Axis tab to open the Axis page. See Figure 106.
b. In the Units area, select the Absolute option button.
This sets the Y-axis to the absolute units of μAU.
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Figure 106. Display Options dialog box – Axis page
5. Specify the labels for the peaks in the chromatogram:
a. Click the Labels tab.
b. Select the check boxes associated with the labels that you want to
display, such as retention time, area, height, and name.
6. Click OK to exit the Display Options dialog box.
Specifying the Spectrum
Range
To display a spectrum for the Surveyor PDA Plus data
1. Pin the Spectrum cell.
2. Right-click the Spectrum cell to open a shortcut menu.
3. From the shortcut menu, choose Ranges to open the Spectrum Ranges
dialog box. See Figure 107.
4. In the Plot Properties area, select PDA from the Detector list.
5. For a generic layout file for PDA data, leave the other settings in the
Spectrum Ranges dialog box at their defaults as shown in Figure 107.
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Figure 107. Spectrum Ranges dialog box, showing the selection of the PDA detector
Setting the Display
Options for the Spectrum
View
To set the display options for the Spectrum cell
1. Right-click the pinned Spectrum cell to open a shortcut menu.
2. From the shortcut menu, choose Display Options to open the Display
Options dialog box.
3. Specify the Normalization parameters:
a. Click the Normalization tab to open the Normalization page. See
Figure 108.
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Figure 108. Display Options dialog box - Normalization page
b. In the Normalize Method area, select the Auto Range option
button.
This ensures that the entire dynamic range of the Spectrum is
displayed in the active view, normalized over the full range of
the Y-axis.
c. In the Normalize Each Plot To area, select the Largest Peak In Range
option button.
4. Specify the Axis parameters:
a. Click the Axis tab to open the Axis page. See Figure 109.
b. In the Units area, select the Absolute option button.
This sets the Y-axis to the absolute units of μAU.
5. Click OK to exit the Display Options dialog box.
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Figure 109. Display Options dialog box – Axis page
Inserting Cells
Occasionally, you might want to add more cells to the Qual Browser
window. For example, you might want to add a cell containing a Map plot
(contour or 3D) to the view screen or you might want to display several
discrete or scan wavelengths in separate cells.
To add a cell containing a Map plot to the window
1. Click a cell to make it the active cell.
2. Choose Grid > Insert Cells. Then, make a selection from the following
choices: Left, Right, Above, or Below.
The location of the new cell is relative to the active cell. Initially, the
new cell will contain the same information as the existing cell. See
Figure 110.
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Figure 110. Qual Browser window, showing three cells
3. Change the lower cell so that it displays the Map view (see Figure 111):
a. Pin the cell by clicking its pin button.
b. Click the View Map button in the toolbar.
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Figure 111. Qual Browser window, showing a Chromatogram view, a Spectrum view, and a Map view
4. Select the appropriate Range options for the Map view:
a. Right-click the Map cell to open its shortcut menu.
b. Choose Ranges to open the Map Ranges dialog box. See
Figure 112.
Figure 112. Map Ranges dialog box
c. Enter the wavelength range that you want to display in the
Wavelength box.
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d. Enter the time range that you want to display in the Time box.
5. Select the appropriate Display options for the Map view:
a. Right-click the Map cell to open its shortcut menu.
b. Choose Display Options to open the Display Options dialog box.
See Figure 113.
c. Click the Axis tab.
d. In the Units area, select the Absolute option button.
e. Click the Normalization tab.
f. In the Normalize Method area, select the Auto Range option
button.
g. Click the Style tab.
h. Select the appropriate style.
i. Click OK to exit the Display Options dialog box.
Figure 113. Display Options dialog box for the Map view
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Creating a Layout for PDA Data
Saving a New Layout
Now that you have created a layout for displaying your PDA data, save the
layout so that you can apply it to other data files containing PDA data.
To save the layout
1. Choose File > Layout > Save As to open the Save Layout File dialog
box. See Figure 114.
2. Type a file name in the File Name box.
3. Click Save.
Figure 114. Save Layout File dialog box
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Viewing the Spectrum for a Specific Time Point
Viewing the Spectrum
for a Specific Time
Point
To view a spectrum for a specific time point
1. Pin the Spectrum cell.
2. Click a time point in the Chromatogram cell.
The spectrum for the selected time point appears in the Spectrum cell as
shown in Figure 115.
Figure 115. Qual Browser window, displaying a spectrum for the 7.43 min time point
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Viewing the Chromatogram for a Specific Wavelength
Viewing the
Chromatogram for a
Specific Wavelength
To view the scan chromatogram for a specific wavelength
1. Pin the Chromatogram cell.
2. Click a wavelength in the Spectrum cell.
The scan chromatogram for the selected wavelength appears in the
Chromatogram cell as shown in Figure 116.
334 nm scan
wavelength
Figure 116. Qual Browser window, displaying a chromatogram cell and a spectrum cell
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Determining Peak Areas
Determining Peak
Areas
In a chromatogram, the area of an isolated peak is directly proportional to
the concentration of the analyte. Determining the area of a peak is therefore
an important feature of quantitation.
To make a peak area determination using Qual Browser
1. Access Qual Browser and open the [.raw] file that you are interested in.
See “Opening a Raw Data File in Qual Browser” on page 136.
2. Ensure that the Chromatogram view is the active cell. An active cell has
a gray border.
3. Select the chromatogram that you want to integrate:
a. Right-click the chromatogram and choose Ranges to open the
Chromatogram ranges dialog box.
b. Select the Detector type from the Detector list:
• To integrate a scan wavelength, select PDA.
• To integrate a discrete wavelength channel, select UV.
c. Ensure that Avalon is selected as the Peak Algorithm.
d. Select the appropriate wavelength:
• To display the chromatogram for a scan wavelength, select
Wavelength Range from the Plot Type list, and then type a value
for a wavelength within your scan range in the Range text box.
• To display the chromatogram for a discrete wavelength channel,
select Channel A, B, or C from the Plot Type list.
4. Turn on peak detection by right-clicking the Chromatogram view and
choosing Peak Detection > Toggle Detection in This Plot.
5. To display numerical values for areas of the chromatographic peaks:
a. Right-click the chromatogram and choose Display Options to open
the Display Options dialog box. See Figure 117.
b. Click the Labels tab to display the Labels page.
c. Select the Area check box.
d. Click OK to return to the Chromatogram view.
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Determining Peak Areas
Figure 117. Display Options dialog box – Labels page
6. To set the integration parameters:
a. Right-click the chromatogram and choose Peak Detection >
Settings to open the Avalon Peak Detection Settings page.
b. Click Auto Calc Initial Events to force the Avalon peak integration
algorithm to determine the “best” values for the following initial
events: Start Threshold, End Threshold, Peak Threshold, P-P
Threshold, Bunch Factor, Negative Peaks, and Tension.
See Figure 118, which shows a chromatogram for the 239 nm scan
wavelength that is integrated with the “best” initial integration
values as determined by the Avalon peak integration algorithm. The
peaks areas are shown above the apexes of the integrated peaks.
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Figure 118. Avalon Peak Detection Settings page and Chromatogram view
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Calculating the Purity of the Chromatographic Peaks
Calculating the Purity
of the
Chromatographic
Peaks
Xcalibur can calculate the spectral purity of your chromatographic peaks by
comparing the similarity of the spectra across the peak to a spectrum from
the peak apex. The calculation is affected by the integration of the scan
chromatogram and by the scan threshold, peak coverage, and scan
wavelengths that you set in the Peak Purity Settings dialog box.
To display the purity values for the integrated peaks
1. Select a chromatogram for a scan wavelength as described in
“Displaying Scan Chromatograms” on page 149.
2. Set the integration parameters for the chromatogram as described in
“Determining Peak Areas” on page 166.
3. Right-click the chromatogram cell and choose Peak Purity to open the
Peak Purity Settings dialog box. See Figure 119.
Figure 119. Peak Purity Settings dialog box
4. In the Peak Purity Settings dialog box, select the Enable check box.
5. In the Scan Threshold box, type an appropriate scan threshold.
The scan threshold limits the portion of the peak included in the
analysis to spectral slices that have a lambda max above the scan
threshold. You can set this limit to eliminate noise from the analysis.
The limits for this box are 0 μAU to 2000000 μAU.
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6. In the Peak Coverage box, type an appropriate value for your
application. The limits for this parameter are 1 to 100% coverage.
At a setting of 100%, Xcalibur will compare all the spectral slices that
fall within the beginning and ending tick marks of the integrated peak.
If you want to limit the peak purity calculation to a specific range of
wavelengths in your scan, select the Limit Scan Wavelength check box,
and then enter a wavelength range in the Wavelength Range box.
7. Click OK to exit the Peak Purity Settings dialog box. Then, view the
effect of your peak purity settings.
• Figure 120 shows the effect of the scan wavelength on peak purity.
• You can see the effect of integration on peak purity by comparing
Figure 121 and Figure 122.
• Figure 123 shows the effect of the scan threshold setting on peak
purity.
• Figure 124 shows the effect of the peak coverage setting on peak
purity.
225 nm scan wavelength
3 uAU scan threshold
95% peak coverage
239 nm scan wavelength
3 uAU scan threshold
95% peak coverage
Figure 120. Comparison of peak purity results for two different scan wavelengths
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Benzene
Impurity
Figure 121. Chromatogram for the 225 nm scan wavelength with default integration parameters
3 uAU scan threshold
95% peak coverage
Figure 122. Chromatogram for the 225 nm scan wavelength with Auto calc integration parameters
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3 uAU scan threshold
95% peak coverage
500000 uAU scan threshold
95% peak coverage
Figure 123. Effect of scan threshold on peak purity calculation
3 uAU scan threshold
50% peak coverage
Peak Start
Peak Stop
3 uAU scan threshold
5% peak coverage
Peak Start
Peak Stop
Figure 124. Effect of peak coverage on peak purity calculations
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Appendix A
Calibration Procedures
This appendix contains the following sections:
• Verifying the Performance of the PDA Detector
• Calibrating the Autosampler
• Calibrating the LC Pump
The modules of your Surveyor Plus instrument are factory calibrated. The
PDA detector is tested for linearity, noise, and drift. The LC pump is
calibrated to produce an accurate flow rate while pumping water at
1 mL/min. Its ability to accurately proportion binary mobile phases is also
tested at a flow rate of 1 mL/min. The column oven and the tray
temperature zones of the autosampler are calibrated at 30 °C. The
positioning of its XYZ arm mechanism is calibrated for the carrier trays
supplied by Thermo Electron.
Because of the sensitivity of its optical bench, it is best to recalibrate the
Surveyor PDA Plus Detector after you install it, each time you move it,
change its flow cell, or replace either of the lamps. In addition, you might
need to increase or decrease the amount of incident light reaching the diode
array by adjusting the attenuators.
The Surveyor Autosampler Plus does not require calibration upon arrival at
its shipping destination. However, if you use custom vials or custom
microplates, you need to perform a Well Bottom Distance calibration,
which determines the actual depth of a vial or microplate well. See “Well
Bottom Distance Calibration” on page 206. If problems occur with the
column oven control, the tray temperature control, or the arm positioning,
contact a Thermo Electron service representative. Calibration procedures
for these items are included in this appendix for users who prefer to
maintain their own instruments.
Like the autosampler, the Surveyor LC Pump Plus does not require
calibration upon arrival at its shipping destination. However, over time, the
pressure readings from its built-in pressure transducer can drift, thereby
requiring re-zeroing. If you are running the LC pump at high backpressures,
you might notice an increase in its pressure pulsation. You can reduce this
pressure pulsation by adjusting the compressibility setting of the LC pump.
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Verifying the Performance of the PDA Detector
Verifying the
Performance of the
PDA Detector
This section contains the following procedures:
• Turning On the Lamps
• Adjusting the Light Output
• Performing a Wavelength Calibration
• Performing a Dark Current Calibration
Turning On the Lamps
Before you perform either calibration procedure, turn on both lamps and
allow the temperature of the detector to stabilize for approx. one hour.
To turn on the lamps
1. From the Windows desktop, select Start > All Programs > Xcalibur >
Xcalibur to open Xcalibur.
2. In the Xcalibur Roadmap - Home Page view, click the Instrument Setup
icon to display the Instrument Setup window.
3. In the Viewbar of the Instrument Setup view, click the Surveyor PDA
Plus Detector icon to display the Surveyor PDA Plus Detector
Instrument Setup View.
4. From the menu bar of the Surveyor PDA Plus Instrument Setup
window, choose Surveyor PDA Plus > Direct Control.
5. Click the Configuration tab to display the Configuration Page. See
Figure 125.
6. Note the status and usage of each lamp.
7. If they are not already On, turn on both the deuterium (D2) and
tungsten (W) lamps.
8. Allow the lamps to equilibrate for one hour before you use the detector.
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Verifying the Performance of the PDA Detector
Figure 125. Surveyor PDA Plus Direct Control – Configuration page
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Calibration Procedures
Verifying the Performance of the PDA Detector
Adjusting the Light Output
The Surveyor PDA Plus Detector has two attenuators that control the light
output from the lamps. During the lifetime of the Surveyor PDA Plus
Detector it might be necessary to adjust the attenuators to increase or
decrease the amount of light falling onto the array.
Decreasing light output to the array increases baseline noise. Increasing light
output to the array can cause saturation of the diode array. If the array is
saturated the response from the Surveyor PDA Plus Detector will be a flat
baseline.
The attenuators require adjustment when either lamp is replaced or when
the flowcell is replaced. The first time that you adjust the attenuators, you
need to create an [.spda] method. After you create the method for adjusting
the attenuators, save it with a name that you will associate with adjusting the
attenuators and store it for future use. Saving the method will simplify
future adjustments of the attenuators.
Note Before you adjust the attenuators, replace the column with a flow
restrictor, and set the pump to deliver HPLC-grade water at a flow rate
of 1 mL/min through the flowcell.
To adjust the light output from the lamps, perform the following
procedures in the order listed:
1. Preparing the LC for an Adjustment of the Attenuators
2. Accessing the Attenuators
3. Adjusting the Attenuators
Preparing the LC for an
Adjustment of the Attenuators
To prepare your LC system for an adjustment of the attenuators
1. Ensure that the lamps are On:
a. From the Windows taskbar, choose Start > All Programs >
Xcalibur > Xcalibur to open Xcalibur.
b. In the Xcalibur Home Page, click the Instrument Setup icon to
display the Instrument Setup window.
c. In the Viewbar, click the Surveyor PDA Plus button to open the
Surveyor PDA Plus Detector view.
d. Choose Surveyor PDA Plus > Direct Control.
The Surveyor PDA Plus Direct Control dialog box is displayed.
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e. Click the Configuration tab to display the Configuration page.
f. Verify that both lamps are On. If they are not On, click the Turn
On buttons for both lamps.
2. Replace the LC column with a flow restrictor.
3. Set your chromatographic pump to deliver HPLC-grade water at a flow
rate of 1 mL/min:
a. In the Viewbar of the Instrument Setup view, click the Surveyor MS
Pump or the Surveyor LC Pump icon to display the instrument
setup view for your pump.
b. Choose Surveyor MS pump > Direct Control or Surveyor LC
Pump > Direct Control.
The Direct Control dialog box for your pump is displayed.
c. Select a solvent bottle that contains HPLC-grade water and set the
flow rate to 1 mL/min.
Accessing the Attenuators
To access the attenuators
1. Open the front doors of the detector.
2. Unscrew the captive screw, and then remove the flowcell cover.
The attenuators are located on the right side of the front panel. There
are two black tabs attached to the attenuators for manual adjustments.
See Figure 126.
The left tab is the deuterium lamp attenuator and the right tab is the
tungsten lamp attenuator. Pushing the attenuator tab up increases light
output and pulling the tab down decreases light output.
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Figure 126. View of attenuators with flow cell access cover removed
Adjusting the Attenuators
To adjust the light output by adjusting the attenuators
1. Create an [.spda] method for the Surveyor PDA Plus Detector:
a. Click the Surveyor PDA Plus button in the View bar to open the
Instrument Setup view for the Surveyor PDA Plus Detector.
b. Select the Diode/Intensity option button.
c. Verify that the following parameters are specified in the Surveyor
PDA Plus Method Page - Spectra area:
• Start Diode= 2
• End Diode= 511
• Diode Step = 1
• Sample rate = 1
d. Choose File > Save to save the method. Save the method with a
filename, such as attenuator.spda.
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Note The .spda file extension is a special file extension used for all
method files based on Diode/Intensity units. These methods cannot
be used in Sequence Setup. Files with the .spda extension can only
be loaded and used in the Direct Control dialog box.
2. Load the [.spda] method to the detector:
a. In the Surveyor PDA Plus Direct Control dialog box, click the
Display tab to open the Display page.
b. In the Control area, click Load Method. Then, choose your
attenuator method file from the list.
c. Click OK.
3. Start the data stream and adjust the attenuators:
a. In the Control area, click Start.
The spectrum of light intensities is displayed in the top window. See
Figure 127. For the UV region, the diode of maximum intensity is
between diode 30 and diode 50. For the Visible region, the diode of
maximum intensity is between diode 400 and diode 500. Ignore the
spike at approximately diode number 380. This spike is due to the
deuterium lamp.
b. Adjust the attenuator with the left tab on the PDA (UV
attenuation) to achieve a maximum value of between 750000 and
775000 intensity counts in the region between diode number 30
and diode number 50.
c. Adjust the attenuator with the right tab (Visible attenuation) to
achieve a maximum value of between 750000 and 775000 intensity
counts in the region between diode number 400 and diode
number 500.
4. After you finish adjusting the attenuators, replace the flowcell access
cover, close the front doors of the detector, and replace the flow
restrictor with your LC column.
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Figure 127. Display page, showing an intensity spectrum
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Performing a Wavelength
Calibration
The alignment of the spectrum on the diode array is dependent upon the
physical alignment of various components of the optical bench. The
alignment can become offset if the detector is sharply jolted, for example, in
shipping. Such bumps and jars can slightly change the wavelength of light
reaching the photodiode array. You can use the automated wavelength
calibration to determine the wavelength accuracy of the detector and to
correct for any misalignment of the array.
To perform a wavelength calibration
1. Pump HPLC-grade methanol at 1 mL/min through the flow cell.
2. Turn On both lamps and wait 1 hour for the lamps to equilibrate:
a. From the Instrument Setup window, click the Surveyor PDA Plus
button in the View Bar to open the Surveyor PDA Plus Instrument
Setup window.
b. From the menu bar of the Surveyor PDA Plus Instrument Setup
window, choose Surveyor PDA Plus > Direct Control to open the
Surveyor PDA Plus Direct Control dialog box.
c. Click the Configuration tab to open the Configuration page.
d. Turn on the deuterium lamp by clicking Lamp On in the
Deuterium Lamp area.
e. Turn on the tungsten lamp by clicking Lamp On in the Tungsten
Lamp area.
3. After the detector has reached a stable temperature (approx. 1 hour after
you turn on the lamps), start the Wavelength Calibration Wizard and
follow the instructions on each page:
a. Click the Calibration tab to display the Calibration page. See
Figure 128.
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Figure 128. Surveyor PDA Plus Direct Control – Calibration page
b. In the Wavelength Calibration area, click Calibrate to start the
Wavelength Calibration wizard. The Wavelength Calibration
wizard contains nine pages.
The first page of the Wavelength Calibration wizard, the
preconditions page appears. See Figure 129.
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Figure 129. Wavelength Calibration wizard – page 1, listing required
preconditions
c. Read the preconditions:
• If all of the preconditions have been met, click Next to proceed
with the calibration and move on to the second page of the
wizard where you will be prompted to select a wavelength file.
See Figure 130.
• If the preconditions have not been met, click Cancel to exit the
wizard and prepare the Surveyor PDA Plus detector for
calibration.
Note The Cancel buttons on the pages of the Wavelength
Calibration Wizard can be pressed at any time a calibration is
in process to abort it.
4. In the second page of the Wavelength Calibration wizard (see
Figure 130), select a peak set from the list. The peak set should span the
wavelengths you use under normal operating conditions. Then, click
Next to display the Calibration Status page.
In Figure 130, you can see that the Holmium Oxide 5 peak set has been
chosen. This wavelength list instructs the program to calibrate the
detector at each of the five wavelengths shown.
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Note Xcalibur has four calibration files to select from. For
example, the HolmiumOxideUV file contains five wavelengths in
the UV region while the other files use sets of wavelengths from
both the UV and Visible wavelength regions. The holmium oxide
absorbance maxima are selected from a spectrum published in
“Holmium Oxide Solution Wavelength Standard from 240 to 640
nm - SRM 2034 (NIST Special Publication 260-54).”
Figure 130. Wavelength Calibration wizard – page 2, showing a list of
calibration files
5. In the third page of the Wavelength Calibration wizard, do the
following:
a. Observe the status screen that tells you the wavelength file is being
downloaded. See Figure 131.
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Figure 131. Wavelength Calibration wizard – page 3
b. After the “Click the Next button to proceed with calibration”
message appears, click Next to display the fourth page of the
Wavelength Calibration wizard shown in Figure 132.
Figure 132. Wavelength Calibration wizard – page 4, directing you to
turn the filter wheel
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6. Rotate the Holmium Oxide filter wheel to position 2 as directed in the
fourth page of the Wavelength Calibration wizard. After you turn the
wheel, the Next button becomes active. Click Next to proceed.
Note The Next button is unavailable until the filter wheel is in
position 2.
7. In the fifth page of the Wavelength Calibration wizard, observe the
status screen that tells you the calibration is being performed. See
Figure 133. After the “Click the Next button to proceed with
calibration” message appears, click Next to proceed.
The diagnostics program waits for a few seconds for the rise time filter
to equilibrate and takes a holmium oxide scan.
Figure 133. Wavelength Calibration wizard – page 5, showing the status of
the rise time filter calibration
8. Rotate the wheel back to position 1 (Open) as instructed on the sixth
page of the Wavelength Calibration wizard. See Figure 134. After you
rotate the wheel, the next button becomes active as shown in
Figure 135. Click Next to proceed.
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Figure 134. Wavelength Calibration wizard - page 6, directing you to turn the
Holmium Oxide filter wheel back to position 1
Figure 135. Wavelength Calibration wizard – page 6 with active next button
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9. In the seventh page of the Wavelength Calibration wizard, shown in
Figure 136, verify that the delta values are within ±1 nm:
• If the Delta values are acceptable, proceed to step 10.
• If the delta values are not within the range of ±1 nm, do not export
the results. Complete the calibration procedure. Then, repeat the
wavelength calibration. If, after applying a second calibration, the
Delta values are still not within the range of ±1 nm, call your
Thermo Electron service representative for assistance.
Figure 136. Wavelength Calibration wizard – page 7, showing the Delta
values
10. (Optional) Print a report of the calibration results:
a. Click Export Results to print the results to a file.
Xcalibur opens the Save As dialog box shown in Figure 137.
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Figure 137. Save As dialog box
b. Type a name in the Save As dialog box. Then click Save.
Once you have saved the file with a name of your choice, you can
view or print the contents of the file using any text editing program.
See Figure 138
Figure 138. Wavelength Calibration file, viewed in Notepad
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11. Click Apply in the Delta Values page (see Figure 136) to apply the
calibration results to the detector.
The final page of the Wavelength Calibration wizard is displayed.
See Figure 139.
12. In the eighth and final page of the Wavelength Calibration wizard, click
Finish to complete the calibration.
The calibration is saved. The date and time of the calibration are
displayed in the Wavelength Calibration area of the Calibration page.
See Figure 128 on page 182.
Figure 139. Wavelength Calibration wizard – page 8
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Performing a Dark Current
Calibration
The function of the array calibration is to measure and correct for the dark
current produced by the diodes of the photodiode array. The dark current is
the small amount of background signal that is produced by the diodes of the
array even when both lamps are turned off. Typical dark current values
range from 1500 to 3000 counts.
The environmental conditions of your laboratory can cause the dark current
of the diode array to increase over time. Therefore, we recommend that you
perform an array calibration (dark current) after any of the following events
occurs:
• After 100 hours of use or monthly, whichever comes first
• Whenever a significant temperature change occurs
• After you move the detector
• After you replace the lamp
• After you download a new firmware file
Because the dark current produced by the diodes rises as the temperature
within the detector rises, it is important to warm up the lamps for 1 hour
before you perform a dark current calibration. Warming up the lamps for
1 hour equilibrates the detector to its normal operating temperature.
Xcalibur briefly turns the lamps off as it performs the dark current
calibration routine. After it completes the dark current calibration routine,
Xcalibur turns the lamps back on.
To perform the dark current calibration
1. Pump methanol through the flow cell at 1 mL/min.
2. Turn On both lamps and wait 1 hour for the lamps to equilibrate.
3. From the menu bar of the Surveyor PDA Plus Instrument Setup
window, choose Surveyor PDA Plus > Direct Control. Then, click the
Calibration tab to display the Calibration page. See Figure 140.
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Figure 140. Calibration page, showing that the detector is currently Not calibrated
4. To start the Dark Current Calibration wizard, click Calibrate in the
Dark Current Calibration area.
The Dark Current Calibration wizard contains four pages. Xcalibur
displays the first page of the Dark Current Calibration wizard, the
Preconditions page shown in Figure 141.
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Figure 141. Dark Current Calibration wizard – page 1
5. Read the Preconditions page:
• If all of the preconditions have been met, click Next to proceed with
the calibration.
• If the preconditions have not been met, click Cancel to exit the
wizard. Then prepare the Surveyor PDA Plus Detector for
calibration and begin this procedure again.
6. From page 2 of the Dark Current Calibration wizard, observe the status
readback as the calibration proceeds. See Figure 142. After the
calibration is complete, click Next.
Figure 142. Dark Current Calibration wizard – page 2
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Xcalibur displays the third page of the Dark Current Calibration wizard
shown in Figure 143. You can export the results of the calibration from
this page.
Figure 143. Dark Current Calibration wizard – page 3
7. To print a record of the Dark Current calibration:
a. Click Export Results.
Xcalibur opens the Save As dialog box shown in Figure 144.
b. Type a name in the Save As dialog box. Then, click Save.
Once you have saved the file with a name of your choice, you can
view or print the contents of the file using any text editing program.
See Figure 145.
Figure 144. Save As dialog box
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Figure 145. Surveyor PDA Plus Dark Calibration text file
8. In the third page of the Dark Current Calibration wizard, click Apply
to apply the calibration results to the detector.
9. The Dark Current Calibration wizard proceeds to its fourth and final
page. See Figure 146.
Figure 146. Dark Current Calibration wizard – final page
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10. In the final page of the Dark Current Calibration wizard shown in
Figure 146, click Finish to complete the calibration.
The calibration is saved and the date and time of calibration are
displayed in the Dark Current Calibration area of the Calibration page
as the Last Calibration. See Figure 147.
Figure 147. Surveyor PDA Plus Direct Control – Calibration page, showing the status as
“Currently Calibrated”
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Calibrating the
Autosampler
There are four calibration options for the full-featured Surveyor
Autosampler Plus and two calibration options available for the Surveyor
Autosampler Plus Lite. This section contains the following topics that
describe three of these calibration options:
• Column Oven Calibration
• Vial Tray Metal Sensor Calibration
• Well Bottom Distance Calibration
The column oven and oven compartment temperature calibrations are
typically performed by a Thermo Electron service representative. To
perform these calibrations yourself, you need to order the Field Service
Calibration Kit (P/N 60053-62001).
The arm calibration option, which is not listed above, is performed at the
factory.
If you choose to use custom vials or custom microwell plates, you must
perform the Well Bottom Distance Calibration, which determines the
depth of the custom vial or microplate well. This calibration must be
performed each time you select a new custom tray configuration and each
time you use a new type of custom vial or custom microwell plate.
Column Oven Calibration
To perform a column oven air sensor calibration
1. Install the Oven Sensor Test Fixture (869C thermometer):
a. Open the column oven door, and then loosen the top thumbscrew
that holds the column clamp.
b. With the sensor facing down, slide the metal cable protector under
the right side of the clamp.
c. Verify that the sensor is between the upper and lower column
clamps and that it is not touching any metal.
d. Tighten the thumbscrew on the sensor, route the cable of the
sensor so as not to interfere with the door, and then close the
column oven door.
2. Set the column oven temperature of your Surveyor Autosampler Plus to
30.0° C:
a. Double-click the Xcalibur icon on your desktop to open the
Roadmap – Homepage.
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b. If the Information View is closed, click the Information View
button to open it. Click the Surveyor AS directory to display the
status pages for the Surveyor Autosampler. Verify that the Surveyor
AS status reads Ready to Download.
c. Click the Instrument Setup icon to display the Instrument Setup
window.
d. Click the Surveyor AS icon in the viewbar.
e. From the Surveyor AS menu, select Direct Control to display the
Direct Control dialog box shown in Figure 148.
Figure 148. Direct Control dialog box
f. Select Set Oven Temperature from the Command and Parameters
list.
g. Type 30.0 in the Temperature box, and then click Apply.
h. Close the Direct Control dialog box.
3. Open the Column Oven Air Sensor Wizard:
a. From the Surveyor AS menu, choose Calibration to display the
Diagnostic dialog box shown in Figure 149.
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Figure 149. Surveyor Autosampler calibration options
b. Select the Column Oven Air Sensor Calibration option button.
Then, click Next to display the Diagnostic dialog box - Internal
Standard page. See Figure 150.
Figure 150. Diagnostic dialog box – Internal Set Target page
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4. Type 30.0 in the Calibration Target Temperature box (see Figure 150).
Then, click Start Calibration to display the Diagnostic dialog box External Standard page shown in Figure 151.
Figure 151. Diagnostic dialog box – External Standard page
5. Verify that the current oven temperature is moving towards the set
point of 30.0° C:
a. Return to the Xcalibur Roadmap - Home Page.
b. Click the Surveyor AS directory in the Status window. Then, select
the Oven tab. See Figure 152.
Figure 152. Surveyor Autosampler Oven status
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6. When the current temperature readout in the Oven area reaches exactly
30.0° C (see Figure 152):
a. Type the reading from the 869C thermometer in the External
Standard Temperature box (see Figure 151).
b. Verify that the current temperature in the Oven area is still
displaying 30.0° C. Then, click Adjust in the Diagnostic - External
Standard page (see Figure 151).
c. Repeat steps 5a and 5b until the readings from the 869C
thermometer and the current temperature readout agree within
±0.2° C.
7. After the temperatures on the 869C thermometer and the current
temperature readout are within ±0.2° C of each other, click Finish at
the bottom of the External Standard page (see Figure 151).
8. Verify that the current temperature readout is stable at 30.0° C with a
maximum temperature drift of ±0.2° C (see Figure 152).
9. After you verify the stability of the column oven temperature, remove
the temperature probe and close Xcalibur.
Vial Tray Metal Sensor
Calibration
The vial tray metal temperature sensor calibration wizard is used to calibrate
the vial tray temperature control by using an external temperature sensor.
Note The vial tray metal sensor calibration is performed by a
Thermo Electron service representative. To perform this calibration
yourself, you need to order the Field Service Calibration Kit
(P/N 60053-62001).
To perform the vial tray metal sensor calibration procedure, you need the
following items that are included in the Field Service Calibration kit:
• Calibrated Omega 869C RTD thermometer
• Surveyor A/S Vial Tray Sensor
To perform a vial tray metal sensor calibration
1. To install the tray temperature sensor:
a. Open the door to the tray compartment.
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b. Install the Tray Temperature sensor (The tray temperature sensor is
a standard tray with a temperature sensor potted in a middle vial
location of the tray.) into location E of the tray compartment.
c. Route the cable of the sensor through the notch at the top on the
tray compartment so that it does not interfere with the door closure.
d. Close the door to the tray compartment.
2. Set the tray temperature of your Surveyor Autosampler to 30.0° C:
a. If the Information View is closed, click the Information View
button to open it. Click the Surveyor AS directory to display the
status pages for the Surveyor Autosampler. Verify that the Surveyor
AS status reads Ready to Download.
b. Click the Instrument Setup icon to display the Instrument Setup
window.
c. Click the Surveyor AS icon in the viewbar.
d. Select Direct Control from the Surveyor AS menu to display the
Direct Control dialog box. See Figure 153.
e. In the Command and Parameters area, select Set Tray Temperature
from the list.
f. Set the temperature to 30.0° C in the temperature box. Then, click
Apply.
g. Close the Direct Control dialog box.
Figure 153. Direct Control – Command and Parameters area
3. Start the Vial Tray Metal Sensor Calibration wizard:
a. From the Surveyor AS menu, choose Calibration.
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b. Select the Vial Tray Metal Sensor Calibration option button shown
in Figure 154, and then click Next to display the Diagnostic dialog
box – Internal Set Target page shown in Figure 155.
Figure 154. Diagnostic dialog box – Calibration options
4. Type 30.0 in the Calibration Target Temperature box. Then click Start
Calibration to display the Diagnostic dialog box – External Target page
shown in Figure 156.
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Figure 155. Diagnostic dialog box – Internal Set Target page
Figure 156. Diagnostic dialog box – External Standard page
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5. Verify that the current tray temperature is moving towards the set point
of 30.0° C:
a. Return to the Xcalibur Roadmap – Home Page.
b. Click the Surveyor AS directory in the status window, and then
select the Tray tab to display the Tray page shown in Figure 157.
Figure 157. Surveyor AS Status – Tray page
6. When the current temperature readout in the Tray area reaches exactly
30.0° C (see Figure 157):
a. Type the reading from the 869C thermometer in the External
Standard Temperature box (see Figure 156).
b. Verify that the current temperature in the Tray area is still reading
30.0° C (see step 4). Then, click Adjust in the External Standard
page (see Figure 156).
c. Repeat steps a and b until the temperatures on the thermometer and
the current temperature readout (see Figure 157) agree within ±
0.2° C
7. After the temperatures on the 869C thermometer and the current
temperature readout in the Oven area are within ± 0.2° C of each other,
click Finish at the bottom of the External Standard page.
8. Verify that the current temperature readout is stable at 30.0° C with a
maximum temperature drift of ± 0.2° C.
9. After you have verified the stability of the oven tray temperature,
remove the temperature probe and close Xcalibur.
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Well Bottom Distance
Calibration
The Well Bottom Distance wizard is used to calibrate the distance that the
needle must travel to reach the bottom of a vial or well. The XYZ arm uses
this value when you select one of the custom tray configurations. The well
bottom distance wizard consists of two dialog boxes: The Select Calibration
Method dialog box and the Well Bottom Distance dialog box.
Note Because the autosampler stores only one value for the custom well
bottom distance, you must perform a well bottom distance calibration
each time you select a new custom tray type configuration and each time
you use a different type of custom vial or custom microtitre plate.
To perform a well bottom distance calibration
1. Open the Well Bottom Distance Calibration Wizard:
a. If the Information View is closed, click the Information View
button to open it. Click the Surveyor AS directory to display the
status pages for the Surveyor Autosampler. Verify that the Surveyor
AS status reads Ready to Download.
b. Click the Instrument Setup icon to display the Instrument Setup
window.
c. Click the Surveyor AS icon in the viewbar.
d. From the Surveyor AS menu, choose Calibration to display the
Diagnostic dialog box shown in Figure 158.
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Figure 158. Diagnostic dialog box, showing calibration options
e. Select the Well Bottom Distance option button (see Figure 7-34),
and then click Next to display the Select Calibration Method page
of the Well Bottom Distance wizard. See Figure 159.
Figure 159. Well bottom Distance Calibration wizard - page 1
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2. Select one of the calibration options:
• Select the Enter The Value Manually option button to enter
previously determined value for the well bottom distance. Click
Next. Then go to step 3.
• Select the Calibrate To Find Value option button to perform an
active calibration. Click Next. Then go to step 4.
Note The Surveyor Autosampler stores only one well bottom
distance value for custom tray configurations. The number at the
top of this dialog box is the current value for the distance.
3. If you selected the Enter The Value Manually option on the Select
Calibration Method page of the wizard, you can enter a new value for
the well bottom distance in the Well Bottom Distance dialog box (see
Figure 160):
a. Enter the new value in microns.
The allowable range is 15000 to 46 990 microns (15 mm to
46.9 mm)
b. Click Set Calibration.
The new value appears at the top of the dialog box.
c. Click Finish to exit.
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Figure 160. Well Bottom Distance wizard – Manual Calibration
IMPORTANT Remove vial caps or microtitre plate lids before
performing an active well bottom distance calibration. As the needle
pierces a vial cap or a microtitre plate lid, the spring in the needle
mechanism is compressed, which can cause premature activation of the
needle sensor.
IMPORTANT Before placing a custom tray into the tray compartment of
the autosampler, check the height limitations for microplates and
high-density micro plates listed in the hardware manual for your
autosampler. Tall objects will stall the autosampler arm.
4. If you selected the Calibrate To Find Value option button on the Select
Calibration Method page of the wizard, you can perform an active
calibration using the Well Bottom Distance dialog box shown in
Figure 161:
a. Remove the cap or lid from your vial or microtitre plate.
b. Place the vial into a tray or the microtitre plate into a carrier, and
then place the tray or carrier into the tray compartment of the
autosampler.
c. Type a vial or well location in the Calibrate At Well Position
text box.
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Calibration Procedures
Calibrating the Autosampler
Note If you are calibrating the bottom distance of a vial, verify
that you have placed a vial in the selected location.
d. Click Set Calibration to activate the autosampler.
After the autosampler arm moves to the selected location, the needle
mechanism descends until it detects the bottom of the vial or well.
After it detects the bottom, the autosampler arm moves to the home
position, and the software program displays the new bottom
distance calibration value at the top of the dialog box.
5. Click Finish to accept the calibration.
Figure 161. Well Bottom Distance Calibration wizard – Active Calibration
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Calibrating the LC
Pump
Calibration Procedures
Calibrating the LC Pump
There are two calibration options for the Surveyor LC Pump that affect its
performance. These are the alpha setting, which affects the flow rate, and
the compressibility setting, which affects the pressure pulsation of the
system. The compressibility setting is set to that for water, and then the
alpha setting is factory calibrated to produce an accurate flow rate for water.
If you are pumping solvents other than water and your application is
sensitive to pressure pulsation, you might want to optimize the
compressibility setting. Changing the compressibility setting affects the
accuracy of the flow rate. Therefore, after you optimize the compressibility
setting to minimize the pressure pulsation of your system, you will want to
check the accuracy of the flow rate. If adjusting the compressibility setting
has affected the accuracy of your flow rate, you will want to adjust the alpha
setting of the LC pump.
In addition to the alpha setting and the compressibility setting, the pressure
transducer that is attached to the back of the purge manifold assembly is
also factory calibrated. If you replace the pressure transducer, you will need
to update the pressure sensor adjustment setting. Even if you never need to
replace the pressure sensor, its readout tends to drift under normal usage.
Therefore, you need to occasionally re-zero its output.
This section contains the following topics:
• Accessing the Calibration Options for the LC Pump
• Calibration Options
• Calibration Procedures
Accessing the Calibration
Options for the LC Pump
You can fine-tune the performance of the Surveyor LC Pump from the
Calibration - Surveyor LC Pump Direct Control dialog box.
To open the Calibration – Surveyor LC Pump Direct Control dialog box
1. Turn on the power to the Surveyor LC Pump.
2. Open the Instrument Setup – Surveyor LC Pump view:
a. Double-click the Xcalibur desktop icon or choose Start > All
Programs > Xcalibur > Xcalibur.
b. Click the Instrument Setup icon on the Home Page Roadmap View
to display the Instrument Setup window.
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Calibration Procedures
Calibrating the LC Pump
c. Click the Surveyor LC Pump icon in the View bar to display the
Surveyor LC Pump view.
3. From the Surveyor LC Pump view, choose Surveyor LC Pump >
Direct Control > Calibration to open the Calibration – Surveyor LC
Pump Direct Control dialog box shown in Figure 162.
Figure 162. Calibration page for the Surveyor LC Pump
Calibration Options
The Xcalibur data system provides the following calibration options
(see Figure 162) for the Surveyor LC Pump Plus. These options are
described in this topic.
• Compressibility
• Pressure Recorder Full Scale
• Flow Rate Adjustment (Alpha)
• Pressure Sensor Adjustment
• Pressure Transducer Zero
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A
Compressibility
Calibration Procedures
Calibrating the LC Pump
The compressibility of a liquid is a measure of its resistance to a decrease in
volume caused by an increase in pressure. Compared to gases, most liquids
are relatively incompressible. Water, a commonly used mobile phase
solvent, is even less compressible than most organic solvents because of its
extensive hydrogen bonding and cluster structure.
Effect of the Compressibility Setting
Even though liquids are relatively incompressible, the compressibility of
your mobile phase can affect your chromatography if you are running the
LC pump at the upper limit of its pressure range. You can use the
compressibility setting for the Surveyor LC Pump to compensate for the
compressibility of your mobile phase. However, changing the
compressibility setting simultaneously affects both the pressure pulsation of
the Surveyor LC Pump and the flow rate of the mobile phase.
The compressibility effect is caused by the portion of the cam cycle in which
the pistons are compressing the mobile phase. The compressibility portion
of the cam cycle extends from the time at which the primary piston starts
discharging until the time at which the secondary piston reaches full
intake—approximately 80° of the cam cycle. To minimize the pressure
pulsation and maintain a constant flow rate, the on-board CPU
compensates for this compression by making fine adjustments to the speed
of the stepping motor. If the compressibility setting is incorrect, the LC
pump will not be able to effectively minimize the pressure pulsation.
Figure 163 shows the effect of the compressibility setting on the pressure
pulsation. The pressure trace was recorded as water was pumped at a flow
rate of 3 mL/min. Three runs were recorded and the compressibility setting
was changed between the runs. The compressibility settings for the three
runs were 0.45 GPa-1, 1.25 GPa-1, and 3.0 GPa-1.
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Calibration Procedures
Calibrating the LC Pump
Figure 163. Overlaid LC pump pressure traces, showing the effect of the compressibility setting
Table 10 shows a comparison of water pumped at three different
compressibility settings. The correct compressibility setting for water is
0.45 GPa-1. The pressure pulsation as well as the actual flow rate increased
as the compressibility setting increased.
Table 10. Measured flow rate and pressure pulsation vs. compressibility setting
Compressibility
Setting
%Pressure Pulsation
Actual Flow Rate
(mL/min)
% Rel Diff
0.45 GPa-1
± 0.2%
3.01
+ 0.3%
1.25 GPa-1
± 0.4%
3.05
+ 1.8%
± 1%
3.18
+ 5.9%
3.0 GPa
-1
Compressibility Ratios for Common Solvents
Most mobile phases are made up of a mixture of solvents. Therefore, you
need to determine the correct compressibility setting for a given mobile
phase empirically. Table 11 lists the compressibility ratios for a few
common solvents.
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Calibration Procedures
Calibrating the LC Pump
Table 11. Compressibility ratios for common solvents
Pressure Recorder Full Scale
Solvent
Compressibility Ratio (GPa-1)
Water
0.45
Acetonitrile
1.20
Methanol
1.25
Hexane
1.60
The pressure recorder full-scale calibration option sets the scaling factor for
the pressure trace if you choose to monitor the pressure with a chart
recorder. The pressure recorder terminals are located on the back of the
Surveyor LC Pump. See Figure 164. Integer values from 1 to 10 are within
the allowable range.
Full scale = Set value × 4.9 MPa (50 kgf/cm2) (full scale voltage = 1 mV)
For example:
When the set value = 1, the full scale is 4.9 MPa (50 kgf/cm2)
When the set value = 10, the full scale is 49.0 MPa (500 kgf/cm2)
4.9 MPa = 50 kgf/cm2 = 49 bar = 711 psi
Note The value for the scaling factor is stored until you change it or
until you turn off the power to the Surveyor LC Pump.
Pressure
Terminals
NC
FW DOWNLOAD
PRESSURE PRESSURE+
NC
NC
+5V @ 15OmA
GND
PUMP ON
GND
INJECT HOLD
PUMP READY
PROG. START
NC
+5V @ 150mA
GND
UNIT ID
ENET
Figure 164. Terminals located on the back of the Surveyor LC Pump Plus
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Calibration Procedures
Calibrating the LC Pump
Flow Rate Adjustment (Alpha)
The alpha value for your Surveyor LC Pump Plus is factory calibrated based
on a flow rate of 1.000 mL/min for distilled water. A sticker containing the
factory calibration value is located inside the pump on the motor casing.
You can optimize the alpha value for your application by updating the alpha
setting. The allowable range for alpha values is 1 to 9.9. If you are pumping
water, decreasing the alpha value by 1, decreases the flow rate by 1%.
Whereas, increasing the alpha value by 1, increases the flow rate by 1%.
Pressure Sensor Adjustment
Reset the Pressure Sensor Adjustment parameter when the pressure sensor is
replaced. Each pressure sensor is labeled with a data sticker that contains a
“0.XXXX” value for the pressure sensor adjustment setting. Multiply this
value by 10000 and then update the pressure sensor adjustment setting for
your Surveyor LC Pump Plus.
Pressure Transducer Zero
The pressure readout for the Surveyor LC Pump Plus is produced by a cell
type potentiometer. This type of device tends to drift by small increments.
Therefore, you will occasionally need to re-zero the readout. To update the
pressure transducer zero value, turn off the pump flow and open the drain
valve knob before clicking the pressure transducer zero button in the
software program that operates your pump.
Calibration Procedures
You fine-tune the performance of the Surveyor LC Pump Plus from the
Calibration - Surveyor LC Pump Direct Control dialog box (Figure 162 on
page 212). For instructions on accessing this dialog box, see “Accessing the
Calibration Options for the LC Pump” on page 211.
This topic contains the following calibration procedures for fine-tuning the
performance of the Surveyor LC Pump Plus:
• Updating the Compressibility Setting
• Optimizing the Compressibility Setting
• Scaling the Output of the Pressure Recorder Terminals
• Updating the Flow Rate Adjustment Setting
• Updating the Pressure Sensor Adjustment Setting
• Re-zeroing the Pressure Transducer
Updating the Compressibility
Setting
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If you have already determined the best compressibility setting for your
application, update the compressibility setting of the Surveyor LC Pump
Plus.
Thermo Electron Corporation
A
Calibration Procedures
Calibrating the LC Pump
To update the compressibility setting
1. Open the Calibration page for the Surveyor LC Pump as described in
“Accessing the Calibration Options for the LC Pump” on page 211.
2. Type a new value into the Compressibility setting box. Then click Set.
Optimizing the Compressibility
Setting
To optimize the compressibility setting for you application
1. Set up your system:
a. Set up a device to record the pressure output from the back panel
pressure terminals. See “Pressure Recorder Full Scale” on page 215.
b. Scale the output of the pressure terminals as described in “Scaling
the Output of the Pressure Recorder Terminals” on page 219.
2. Download the mobile phase parameters specified in your application:
a. If it is not already on, turn on the power to the Surveyor LC Pump
Plus.
b. Open the Instrument Setup - Surveyor LC Pump view as described
in step 2 on page 211.
c. From the Surveyor LC Pump view, choose Surveyor LC Pump >
Direct Control > Operation to open the Operation - Surveyor LC
Pump Direct Control dialog box shown in Figure 165.
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Calibration Procedures
Calibrating the LC Pump
Figure 165. Surveyor LC Pump – Direct Control – Operation page
d. In the Method area, enter the specified flow rate in the Flow Rate
box and enter the specified mobile phase composition in the
Component A through Component D boxes
e. Click Download to send these parameters to the on-board CPU of
the Surveyor LC Pump.
f. Click Pump On to turn on the pump flow.
3. Record the pressure trace.
4. If the pressure pulsation is too high for your application, change the
compressibility setting. Then, repeat steps 2 and 3. Continue to change
the compressibility setting until the level of pump pulsation is adequate
for your application.
Note Changing the compressibility setting will affect the flow rate.
5. After you change the compressibility setting to minimize the pressure
pulsation, check the flow rate. If necessary, adjust the flow rate
adjustment setting as described in “Updating the Flow Rate Adjustment
Setting” on page 219.
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Scaling the Output of the
Pressure Recorder Terminals
Calibration Procedures
Calibrating the LC Pump
To set the full-scale output of the pressure recorder terminals
1. Open the Calibration page for the Surveyor LC Pump Plus as described
in “Accessing the Calibration Options for the LC Pump” on page 211.
2. Enter a value from 1 to 10 in the Pressure Recorder Full-Scale box
(Figure 162 on page 212). Then, click Set. For information on scaling
the output of the pressure recorder terminals, see “Pressure Recorder
Full Scale” on page 215.
Updating the Flow Rate
Adjustment Setting
If you change the compressibility setting for the Surveyor LC Pump, you
might also need to change its flow rate adjustment setting. Changing the
flow rate adjustment setting can compensate for minor deviations in the
flow rate of approximately ±5% from the expected value.
To update the flow rate adjustment setting
1. Open the Calibration page for the Surveyor LC Pump as described in
“Accessing the Calibration Options for the LC Pump” on page 211.
2. Enter a new value in the Flow Rate Adjustment (alpha) box (see
Figure 162 on page 212):
• If the actual flow rate is below the set value, increase the alpha value.
If you are pumping water, increasing the alpha value by 1, increases
the flow rate by 1%.
For example, if the pump is set to deliver a flow rate of 1 mL/min
and the actual flow rate is 0.96 mL/min, which is 4% below the set
rate, raise the current setting by 4. If the current setting is 5.0, enter
a new setting of 9.0.
• If the actual flow rate is above the set value, decrease the alpha value.
If you are pumping water, decreasing the alpha value by 1, decreases
the flow rate by 1%.
For example, if the pump is set to deliver a flow rate of 1 mL/min
and the actual flow rate is 1.04 mL/min, which is 4% above the set
rate, lower the current setting by 4. If the current setting is 5.0, enter
a new setting of 1.0.
3. Click Set.
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Calibration Procedures
Calibrating the LC Pump
Updating the Pressure Sensor
Adjustment Setting
Reset this parameter when the pressure sensor is replaced. Each pressure
sensor is labeled with a data sticker that contains a “0.XXXX” value for the
pressure sensor adjustment setting. Multiply this value by 10,000 and enter
the result in the Pressure Sensor Adjustment setting box.
To update the pressure sensor adjustment setting
1. Open the Calibration page for the Surveyor LC Pump as described in
“Accessing the Calibration Options for the LC Pump” on page 211.
2. Enter the value on the data sticker “0.XXXX” multiplied by 10000 in
the Pressure Sensor Adjustment setting box (see Figure 162 on
page 212).
3. Click Set.
Re-zeroing the Pressure
Transducer
The offset error of the pressure transducer is the value that is displayed when
it should be zero, such as when the purge manifold knob is set to the open
position. You can correct for this offset error by re-zeroing the pressure
readout of the pressure transducer when the purge manifold knob is open.
To re-zero the output of the pressure transducer
1. Stop the flow of solvent from the LC pump:
a. From the Instrument Setup window, choose Surveyor LC Pump >
Direct Control > Operation.
b. Click Pump Off.
2. Open the drain valve to ensure that the transducer is actually sensing
zero system pressure. See Figure 166.
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Open
Calibration Procedures
Calibrating the LC Pump
Closed
Figure 166. Drain valve in open and closed positions
3. Open the Calibration page of the Direct Control dialog box by
choosing Surveyor LC Pump > Direct Control > Calibration.
4. Click Zero in the Pressure Transducer area (see Figure 162 on
page 212).
5. When you are finished zeroing the pressure transducer, close the drain
valve by turning the knob clockwise until you feel resistance.
Note Applying excessive force to the drain valve knob reduces the
life span of its O-ring.
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Index
A
accessories
FEP tubing 2, 2
reservoir vials 7
stainless steel tubing 3
standard vials 8
wash bottle assembly 9
active
pinned cells 141
unpinned cells 141
air, removing from solvent lines 52
alpha values for flow rate adjustment 216, 219
areas of peaks, displaying 156
attenuators of the PDA 17
autosampler
calibrating 197
LED states 21
loading trays 125
method parameters 148
setting up sample preparation 98
status states viewed from Status pages 44
using custom vials or microplates 206
autozero function for the UV/Vis detector 97
Avalon integration algorithm 153
B
Bandwidth box 90
bandwidth filter 92
baseline
zeroing 18
baseline drift, reducing 121
baseline noise, reducing 52, 88
busy status message for autosampler 44
C
cables
Ethernet 4, 19, 24
serial communication 19, 24
calibration
column oven 197
dark current 191
tray temperature 201
Thermo Electron Corporation
wavelength 181
well bottom distance 206
cell states 140, 142
cells
adding to real-time display 132
inserting in Qual Browser 159
working with the cell grid 140
Channel list 93
Channels area 91
channels, discrete wavelength 91
checking
the solvent lines for air 52
the status of the Surveyor devices from Xcalibur 42
chemicals
filtering 125
preparing 125
chromatograms
cursor actions 142
displaying for UV data 165
displaying peak names, retention times, heights, areas 156
integrating peak areas 167
column LC
damaging 66
equilibrating 122
column oven
autosampler Temp LED states 21
control described 16
setting temperature 81
column oven test fixture, installing 197
columns, spreadsheet, arranging 118
Comm LED states 21
communicating with Xcalibur 19, 24
compressibility
defined 213
updating 216
values 214
concentric syringe
bore usage for sample preparation 102
configuring 30
flushing 56
configuring your Surveyor LC system 25
creating
calibration curves 116
instrument methods 63
sample preparation routines 98
sequences 114
Surveyor Plus Getting Started with Xcalibur 223
Index: D
cursor actions, clicking and dragging 142
custom trays
calibration of well bottom distance 206
selecting options 28
D
dark current calibration 191
data acquisition
analyzing acquired data 135
starting 126
viewing real-time 130
data system
Comm LED states 21
data acquisition 20
deactivate
Verify Door is Closed control 31
Wait For Temperature Ready control 31
dead volume for transfer tubing, configuring 31
Degas LED states 21
degassing unit, built-in 3
Delay box 93
delay volume
LC pump 5
MS pump 3
preheat tubing behind column oven 16
delta values for PDA calibration
shown in Xcalibur 188
detectors
LED states 21
method parameters report 148
devices
adding to the instrument configuration 26
configuring the autosampler 28
configuring the MS pump 34
configuring the PDA detector 37, 37
start instrument 127
turning on/off 50
Diagnostics Program
performing a dark current calibration 191
performing a wavelength calibration 181
Reference Calibration page 61
Wavelength Calibration page 61
dilutions, automating 103
diode voltage leakage 191
Direct Control dialog box
autosampler 56
LC pump 53
direct control status message for autosampler 44
discrete wavelengths for the PDA detector 91
224
Surveyor Plus Getting Started with Xcalibur
display options
chromatogram cell 154
map cell 162
spectrum cell 157
door of autosampler, Verify Door is Closed option 31
drain valve knob for LC pump 52
dual wavelength program 97
E
equilibrating the column during a sequence run 70
error status message for autosampler 44
errors
clearing an LC pump error 53
status message for autosampler 44
Ethernet cables 19
F
file extensions
meth for instrument methods 109
raw for data files 136
sld for sequences 120
spda for method files based on intensity units 179
fill position 13
filter frit in LC pump 6
filter wheel in PDA 17
firmware
signals for versions of LC pump firmware 20
upload version 34
verifying for the Surveyor LC Pump 58
fixed loop injection 15
flashing LEDs, meaning 21
flow rate
adjustment setting 219
changes between timepoints for MS pump 67
programming for MS Pump 67
range for LC pump 4
range for MS pump 3
flush bottle location 9
flush speed, setting 80
flush volume, setting 79
flush/wash source, setting 79
flushing
the sample loop 57
the solvent lines 52
the wash tubing and the syringe 56
font size of display, changing 146
fraction collector, triggering 92
Thermo Electron Corporation
Index: G
full loop injection
description 15
selecting 81
setting volume 78
G
gradient delay volume
LC pump pulse dampener 5
preheat tubing 16
gradient profile
graphical display for LC pump 74
graphical display for MS pump 67
gradient pump program
creating for LC pump 74
creating for MS pump 70
gradient start signal from pump 20
gradient table
parameters for LC pump 75
parameters for MS pump 67
grinding sound, caused by high flush speeds 80
H
height of chromatographic peaks, labeling 156
I
inactive cells 140
initialization process for syringe 40
initializing status message 43
inject position 14
injection mode
described 14
setting 81
injection port of autosampler (figure) 12
injection valve, description 13
injection volume
equation for full loop injections 15
setting 78
inserting cells into the grid 159
instrument method report 148
instrument methods
creating 63
saving 109
viewing 148
Instrument Setup window, opening 64
integration
auto calculation 167
Avalon 167
Surveyor Plus Getting Started with Xcalibur
isocratic pump program
creating for LC pump 73
creating for MS pump 69
L
labeling
chromatograms 156
contents of reservoir vials and wash bottle 81
instrument methods 109
lamps
deuterium 17, 18
lamp warm-up message 43
LED states 21
safety interlock 18
tungsten 18
tungsten-halogen 17
turning on 174
wavelength range 17
Lamps LED states 21
launching Xcalibur 41
layout files for PDA data, creating 149
LC pump
clearing pump errors 53
components 5
configuring 35
firmware versions, verifying 58
in-line filter frit 6
purging 6, 52
LEDs, status on right door 21
Level box 93
loading the Surveyor Autosampler 125
Lock Display button 130
loop loading speed 80
M
maintenance
adjusting the attenuators 176
maintenance log, enabling and disabling 31
manual bleeder valve of MS pump 60
method for instrument control, creating 63
method report 148
microplates
description 8
selecting options 28
MS pump
delay volume 3
Direct Control dialog box 124
priming 59
serial communications 24
Thermo Electron Corporation
225
Index: N
N
names of peaks in chromatograms, displaying 156
naming
data files 116
instrument methods 109
sequences 119
needle
height from bottom, setting 78
tubing assembly 7, 12
needle assembly, description 11
needle tubing assembly, description 12
New Sequence Template dialog box (figure) 115
no waste injection
description 14
setting volume 78
normalization parameters 154
pressure transducer
replacing 216
zeroing 216, 220
priming MS pump 59
processing methods 116
pulse dampener outlet for MS pump 60
pump ready signal 20
purge valve 3
purge valve, MS pump 54
purging the Surveyor LC Pump 52
Q
Qual Browser
accessing 136
cursor actions 142
displaying a chromatogram and spectrum in the same window
159
O
layout files, creating 149
setting the display options for UV chromatograms 154
On, Off, Standby shortcut menu 50
On/Off switches for Surveyor devices (figure) 40
R
P
partial loop injection
description 15
selecting 81
PCR plate, configuring 29
PDA detector
configuring 36
functional description 17
LED states 21
peaks
displaying areas 166
integrating 166
peak coverage 170
purity 169
pmd file extension 116
Position Arm to Access Tray direct command 125
Power
LED states 21
turning on/off 40
preheating tubing 16
Prep Operations list 100
pressure
minimum for LC pump 72
recorder terminals 215
sensor adjustment 220
units for displaying backpressure 36
pressure recorder terminals, scaling the output 219
226
Surveyor Plus Getting Started with Xcalibur
ready to download message 43
real-time data
locking 130
reviewing 131
removing air from the solvent lines 52
report, instrument method 148
Reservoir Content Page 81
resolution, spectral 91
retention times
displaying 156
effect of column oven on reproducibility 85
rise time filter
for PDA data 88
for the UV/Vis detector 96
rules for sample preparation 102
Run LED states 21
S
safety interlock, lamps 18
sample loops
available sizes 13
size versus maximum injection volume 14, 15
Sample Preparation page, opening 98
sample rate 90, 91
Thermo Electron Corporation
Index: T
sample trays
figure showing conventional trays 117
loading into or removing from tray compartment 125
selecting options 28
temperature control 16
saving
instrument methods 109
layout files 163
sequences 119
Savitsky-Golay bandwidth filtering 92
scan threshold 169
sequences
creating 114
saving 119
turning off the lamps 129
turning off the pump flow 129
serial communication cables 19
signal polarities for autosampler configuration 32
solvent composition
linearity for MS pump 67
programming for LC pump 74
programming for MS pump 67
solvent lines
four lines for mobile phase solvents 2
wash bottle 2
solvent platform location 2
solvent reservoir bottles 2
spda file type 179
Spectra box 89
spectra for UV data, displaying 156
spectrum
cursor actions 142
displaying a spectrum 159
for a specific time point 164
spectrum maximum 151
stack address 19, 23
stack number 36, 37
standard syringe
configuring 30
size 10
standby, shortcut menu item 50
status
checking the status of the LC devices 42
Info View 44
LEDs for Surveyor devices 21
switches
magnetic door 8
On/Off 40
synchronizing the Surveyor LC devices 20
syringe speed, setting 79
syringe valve, description and figure 9
syringe, flushing 56
Surveyor Plus Getting Started with Xcalibur
T
temperature 16
autosampler Temp LED 21
control 16
time
gradient program for LC pump 74
gradient program for MS pump 67
timed event
adding 94
Channel list box 93
Delay box 93
Level box 93
Type list 92
Timed Events page
description 82
displaying 82
total scan 151
transfer tubing, figure 12
tray door, Verify Door is Closed option 31
tray temperature
autosampler LED states 21
control 16
setting 81
tray temperature sensor, installing 201
trays. See sample trays. 28
triggering a fraction collector 92
tubing
behind column oven 16
degasser 3
solvent lines 2
wash bottle 9
Type list box for external events 92
U
unit ID
figure 23
relation to communication protocols 19
uploading firmware version 34
UV/Vis detector
configuring 37
direct controls 122
programming 96
status 49
wavelength modes 18
V
vacuum Degas LED states 21
Thermo Electron Corporation
227
Index: W
valves
manual bleeder for MS pump 60
purge 3
syringe 2-position rotary valve 9
Valco 6-port injection valve 13
variable volume injections
setting volume 78
types 15
Verify Door is Closed option, choosing 8, 31
vial bottom sensing
auto 31
enabling and disabling 31, 31
Vial Not Found message 125
vial tray
described 16
temperature calibration 201
viewing
device status 44
instrument method report 148
real-time data acquisition 130
viscous flush solvents 80
volume
flush 56
full loop injections 15
gradient delay, pulse dampener in LC pump 5
gradient delay, pulse dampener of MS pump 3
no waste injection mode 14, 14
partial loop injections 15
preheat tubing 16
sample loops 13
sample preparation operations 102
standard needle tubing and needle tubing extension 9
transfer tubing 12
tubing in chambers of vacuum degassing unit 3
X
Xcalibur
Autosampler Direct Control dialog box 56
controlling a fraction collector 92
Home Page, displaying 64, 98
Instrument Setup Window, accessing 64
launching 41
LC pump Direct Control dialog box 53
MS pump Direct Control dialog box 55
Qual Browser, opening 136
Sequence Setup window, accessing 114
XYZ arm, description 11
Z
zeroing
pressure transducer 220
the baseline on a wavelength change 18, 97
W
Waiting for Trigger status message, possible cause 32
wash bottle location 9
wash volume
note 79
setting 79
wavelength accuracy, calibrating 181
wavelength scans for the PDA 89
Well Bottom Distance
calibrating 206
caution 209
note 208
well ordering, selecting 29
well position, selecting 29
228
Surveyor Plus Getting Started with Xcalibur
Thermo Electron Corporation
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