Xcalibur®
Xcalibur®
Getting Productive:
Quantitative Analysis
XCALI-97103 Revision C
For Research Use Only
Not for use in Diagnostic Procedures
June 2006
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Contents
Preface ............................................................................................. ix
About This Guide ......................................................................ix
Related Documentation .............................................................ix
Safety and Special Notices...........................................................x
Contacting Us.............................................................................x
Assistance .................................................................................x
Changes to the Manual and Online Help................................xi
Thermo Electron Corporation
Chapter 1
Introduction..........................................................................................1
About Quantitative Analysis........................................................2
Quantitation Techniques ............................................................3
Using External Standards for Quantitation ..............................5
Using Internal Standards for Quantitation ...............................6
Overview of the Quantitation Features in Xcalibur 2.0 ...............8
Instrument Setup .....................................................................9
Processing Setup ....................................................................10
Sequence Setup ......................................................................11
Quan Browser ........................................................................12
Acquiring and Quantitatively Processing Data
with Xcalibur 2.0 ...................................................................13
Chapter 2
Processing Setup ..............................................................................15
The Processing Setup Window..................................................16
Features of the Processing Setup Window ..............................16
Applying Changes to a Page ...................................................18
Customizing Processing Setup................................................20
The Quan View ........................................................................21
Working Interactively with the Chromatogram and
Spectrum Previews .................................................................22
Opening a Raw File ...............................................................22
Previewing Processing ............................................................22
Setting Processing Parameters ................................................23
Using the Cursor....................................................................24
Using the Toolbar ..................................................................28
Customizing the Previews ......................................................29
Xcalibur: Getting Productive with Quantitative Analysis
v
Contents
Identification ............................................................................30
Identification Parameters .......................................................31
Entering the Identification Parameters for Your
Processing Method..............................................................36
Detection ..................................................................................41
Peak Integration.....................................................................43
Peak Detection for LC ...........................................................49
Peak Detection for GC...........................................................50
Advanced Detection Parameters .............................................58
Data Flags ..............................................................................61
Peak Identification Options ...................................................62
Calibration................................................................................64
Assigning an ISTD.................................................................65
Assigning a Target..................................................................66
Isotope Correction .................................................................69
Setting Calibration and Quantitation Flags ............................72
Levels ........................................................................................74
Entering the Calibration or the QC Levels .............................75
Using the Standard Dilution Option .....................................76
System Suitability......................................................................78
Resolution..............................................................................79
Symmetry...............................................................................80
Peak Classification .................................................................81
Peak Purity................................................................................84
Enable Peak Purity .................................................................85
Limit Scan Wavelength ..........................................................85
Reports .....................................................................................86
Sample Reports ......................................................................87
Summary Reports ..................................................................88
Programs...................................................................................89
Chapter 3
vi
Xcalibur: Getting Productive with Quantitative Analysis
Automating Analysis........................................................................ 91
The Sequence Setup View .........................................................92
About Sequences .......................................................................94
Arranging the Columns..........................................................96
Changing User Labels ............................................................97
Creating a New Sequence..........................................................98
Importing a Sequence ............................................................98
Creating a Sequence with the New Sequence
Template Wizard ................................................................99
Creating a Sequence Manually .............................................106
Thermo Electron Corporation
Contents
Modifying a Sequence .............................................................108
Filling Down Columns ........................................................108
Inserting a Row....................................................................110
Deleting a Row ....................................................................110
Going to a Sequence Row ....................................................111
Transferring Row Information .............................................111
Printing a Sequence..............................................................112
Checking Disk Space ...........................................................113
Exporting a Sequence...........................................................115
Changing the List Separator Character.................................116
Running Samples ....................................................................117
Opening the Run Sequence Dialog Box ...............................117
Setting General Run Options...............................................119
Changing Acquisition Options.............................................119
Selecting a Startup or Shutdown Method.............................120
Specifying Pre- and Post-Run Acquisition Programs ............120
Choosing Processing Actions................................................121
Starting the Run...................................................................121
Reprocessing Samples..............................................................122
The Acquisition Queue ..........................................................124
Sample Information Dialog Box...........................................125
Managing Tasks ...................................................................126
Chapter 4
Thermo Electron Corporation
Reviewing Quantitation in Quan Browser .................................129
About Quan Browser ..............................................................131
How Quan Browser Works..................................................131
Getting Started in Quan Browser .........................................134
The Quan Browser Window ...................................................136
The Title Bar .......................................................................137
The Toolbar and Menu Bar .................................................137
Component List...................................................................139
Results Grid .........................................................................139
Chromatogram View............................................................139
Companion View.................................................................139
The Results Grid.....................................................................141
Bracket/Group in Use ..........................................................142
Calibration File ....................................................................142
Results Grid Columns..........................................................142
Working Directly With The Grid ........................................143
Xcalibur: Getting Productive with Quantitative Analysis
vii
Contents
Chromatogram View...............................................................147
Chromatogram View Shortcut Menu...................................147
Viewing Peak Information ...................................................148
Qualifier Peak Information ..................................................155
Spectrum Candidate Information.........................................156
Setting User Peak Detection Parameters...............................158
Changing Display Options...................................................165
Calibration Companion View .................................................166
Calibration Companion View Shortcut Menu .....................166
Adjusting Calibration Settings..............................................167
Spectrum Companion View....................................................175
Reports ...................................................................................176
The Reports Dialog Box.......................................................176
Selecting Samples for Reports...............................................177
Quan Browser Procedures .......................................................179
Editing a Sequence...............................................................179
Reviewing Samples...............................................................180
Reviewing a Chromatogram.................................................182
Modifying Detection and Identification...............................183
Integrating Chromatogram Peaks Manually .........................184
Modifying Calibration Parameters .......................................185
Index ................................................................................................. 187
viii
Xcalibur: Getting Productive with Quantitative Analysis
Thermo Electron Corporation
Preface
About This Guide
Welcome to Xcalibur® 2.0, the Thermo Electron mass spectrometry data
system!
This Getting Productive: Quantitative Analysis manual describes how to use
your Thermo Electron system and Xcalibur for quantitative analysis.
It describes how to:
•
Set up a method for automatic quantitative processing.
•
Create a sequence or batch of samples for analysis and processing under
full software control.
•
Review and rework your data using Xcalibur’s quantitative reviewing
utility, Quan Browser.
It is assumed that you have read your instrument’s Getting Started and are
familiar with the basic features of Xcalibur such as Home Page and
Instrument Setup.
Related
Documentation
Thermo Electron Corporation
In addition to this guide, Thermo Electron provides the following
documents for Xcalibur 2.0:
•
Administrator’s Guide: Configuring Xcalibur Software for Compliance with
21 CFR Part 11
•
Getting Productive: Processing Setup and the Analysis of Quantitation Data
•
Getting Productive: Qualitative Analysis
•
Getting Productive: Designing and Generating Custom Reports with
XReport
•
Getting Productive: Creating and Searching Libraries
•
Help available from within the software
Xcalibur: Getting Productive with Quantitative Analysis
ix
Preface
Safety and Special
Notices
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.
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.
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:
1-800-685-9535
Fax:
1-561-688-8736
techsupport.finnigan@thermo.com
x
Xcalibur: Getting Productive with Quantitative Analysis
Thermo Electron Corporation
Preface
Contact Customer Service
In the US and Canada for ordering information:
Phone:
1-800-532-4752
Fax:
1-561-688-8731
International contacts for ordering information:
Visit www.thermo.com/finnigan for the current listing.
Changes to the Manual
and Online Help
Thermo Electron Corporation
To suggest changes to this guide or to the 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
techpubs.finnigan-lcms@thermo.com
Xcalibur: Getting Productive with Quantitative Analysis
xi
Chapter 1
Introduction
Xcalibur 2.0 is a complete quantitative and qualitative analysis software
package that enables you to acquire data specifically for analytes of interest,
to perform confirmatory library searches, and to determine the
concentration of analytes in samples. Xcalibur 2.0 interfaces with the
XReport reporting package to print individual sample reports and sequence
summary reports for analyses. For more information on XReport, see the
Getting Productive: Designing and Generating Custom Reports with XReport
manual.
This introductory chapter describes some of the basic principles and
terminology of quantitation1 and provides a brief overview of quantitation
with Xcalibur 2.0. This chapter contains the following sections:
•
About Quantitative Analysis
•
Quantitation Techniques
•
Overview of the Quantitation Features in Xcalibur 2.0
•
Acquiring and Quantitatively Processing Data with Xcalibur 2.0
1For
further information on the principles of quantitation, see the following: Mass Spectrometry:
Principles and Applications; de Hoffman, E., Charette, J., Stroobant, V.; Wiley: New York, 1996 and
Introduction to Mass Spectrometry, 3rd ed.; Watson, J.T., Lippincott-Raven: Philadelphia, PA, 1997.
Thermo Electron Corporation
Xcalibur: Getting Productive with Quantitative Analysis
1
1
Introduction
About Quantitative Analysis
About Quantitative
Analysis
Quantitative analysis is the process of measuring the amount of a particular
component in a sample.
Quantitative analysis consists of the following steps:
•
Preparing samples
•
Developing a suitable chromatographic method
•
Calibrating the detector’s response
•
Analyzing the samples
•
Reviewing and reporting the results
It is beyond the scope of this manual to describe sample preparation and
chromatographic method development. This manual assumes that you have
achieved these important prerequisites to high quality quantitation. See the
Getting Started and Hardware manuals for your instrument for guidance in
these areas.
2
Xcalibur: Getting Productive with Quantitative Analysis
Thermo Electron Corporation
1 Introduction
Quantitation Techniques
Quantitation
Techniques
To carry out quantitation, evaluate the response of the detector to known
amounts of the target component. Response is based on either the height of
the chromatogram peak, or more commonly, the area under the peak’s
profile (see Figure 1). In both cases, take into account and properly
integrate the baseline of the detected peak.
RT 0.68
100
90
Relative Abundance
80
70
60
50
40
Peak Start
30
Peak End
20
10
0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Time [min]
Figure 1.
Integrated chromatographic peak
Instrument response is generally measured with several samples commonly
called standards or calibration standards. These standards must cover a
suitably wide range of concentrations or amounts and should bracket the
range of expected concentrations in the unknown. Responses to these
standards are plotted in a graph called a calibration curve. Ideally, this curve
should correspond to the equation of a straight line with a slope equal to
one to ensure the highest degree of precision.2 Only the portion of the curve
that shows an essentially linear relationship between amount and response
should be considered valid.
Fitting an equation to the calibration curve with a user chosen method (for
example, a least squares regression) provides a response factor - a
comparative measure of the response of the detector to a component. It is
2Mass
Spectrometry: Principles and Applications; de Hoffman, E., Charette, J., Stroobant, V.; Wiley:
New York, 1996; p 162.
Thermo Electron Corporation
Xcalibur: Getting Productive with Quantitative Analysis
3
1
Introduction
Quantitation Techniques
based on the amount of sample injected and the resulting peak area or peak
height. Consequently, the response factor gives a quantitative measure of
how responsive or sensitive the detector is to a certain component.
Quantitation of samples containing unknown amounts of the target
component is achieved by first calculating the peak area or height and then
computing and applying the appropriate response to the equation derived
from the calibration curve. This process provides an estimate of the amount
of the unknown component. The precision of the measurement depends on
the quality and, to a lesser extent, the quantity of the calibration data.
The detection limit of the quantitation method is the lowest concentration
of analyte in a sample that can be detected but not necessarily quantitated as
an exact value. The lower and upper quantitation limits are the lowest and
highest concentrations of analytes in a sample that can be measured with an
acceptable level of accuracy and precision, respectively. In an analytical
method, the highest concentration calibration standard defines the upper
quantitation limit. The quantitation range is the range of concentration
between the lower and upper quantitation limits (including these limits)
that can be reliably and reproducibly quantified with acceptable levels of
accuracy and precision through the use of a concentration-response
relationship.
There are two basic quantitation techniques:
•
External standard quantitation
•
Internal standard (ISTD) quantitation
The chosen method determines the calculation method, both for the
generation of the calibration curves and for subsequent quantitation.
This section contains the following topics:
4
•
Using External Standards for Quantitation
•
Using Internal Standards for Quantitation
Xcalibur: Getting Productive with Quantitative Analysis
Thermo Electron Corporation
1 Introduction
Quantitation Techniques
Using External Standards
for Quantitation
An external standard is a separate sample that contains the compound of
interest at a known concentration in solution. In the external standard
quantitation technique: Xcalibur analyzes a series of standards and
constructs a calibration curve by plotting the magnitude of the detector
response as a function of the external standard concentration. Xcalibur then
analyzes the unknown sample and determines the concentration by
matching the magnitude of the detector response with that on the
calibration curve (see Figure 2).
Use external standards if various components in a sample are being analyzed
and if all compounds of interest can be assayed by using a single set of
external standards. This approach offers time- and cost-effective
quantitation for applications using high precision autosamplers and
traditional UV/Vis detectors. However, for some types of analyses, this
method cannot achieve the highest level of precision and accuracy.
Depending on the instrumentation, variations in analyte and solution
stability, injection reproducibility, and matrix interference can lead to lower
precision levels in the external standard method than in the internal
standard method.
Response for Target Compound
500000
Response for
Unknown Sample
400000
300000
200000
Amount in
Unknown Sample
100000
0
0
20
40
60
80
100
Amount of Target Compound
Figure 2. Calibration curve generated by using an external standard
Thermo Electron Corporation
Xcalibur: Getting Productive with Quantitative Analysis
5
1
Introduction
Quantitation Techniques
Using Internal Standards
for Quantitation
An internal standard (ISTD) is a component that is added to a sample to act
as a response reference for one or more non-ISTD components in the
sample. The concentration or amount of an ISTD in any standard or
unknown sample typically remains constant.
Because quantitative mass spectrometric analysis usually involves multiple
steps, the total error in the analysis results from the accumulation of the
errors at each step. In general, sample handling errors account for a larger
fraction of the total error than do detector errors. Fortunately, the internal
standard method can reduce both sources of error. For example, internal
standards can correct for variations in a component’s peak area that are
caused by the following:
•
Injection unreproducibility
•
Changes in analyte solution volume
•
Matrix and coeluter interference (both suppression and enhancement)
•
System instability
•
Variations in the source conditions
For maximum precision, the ISTD component should be added as early as
possible to the start of the sample workup, particularly in those quantitative
methods that require sample manipulations such as extraction, cleanup, and
dilution. Since the ISTD and non-ISTD components are analyzed together,
the internal standard quantitation approach has the advantage that it
corrects for injection and other sample handling errors. The ISTD must
behave chemically in an identical or similar manner to the target compound
through the extraction, cleanup, and analytical processes.
The ISTD component can also be added as the last step of sample
preparation prior to the sample’s use to compensate for fluctuations in the
reproducibility of the sample injection.
In general, ISTDs are used in a quantitation experiment as follows:
1. Xcalibur analyzes series of standard solutions containing known
concentrations of the target compound and ISTD. Then Xcalibur plots
the ratio of the target compound and the ISTD detector responses as a
function of the corresponding ratio for the two quantities present in the
solution.
2. A fixed amount of the ISTD is added to each sample prior to any
manipulation. After the samples are prepared and analyzed, the quantity
6
Xcalibur: Getting Productive with Quantitative Analysis
Thermo Electron Corporation
1 Introduction
Quantitation Techniques
of the target compound present in an unknown sample can be obtained
from the calibration curve (see Figure 3).
Ideally, an ISTD should be closely related to the target component in terms
of its physical and chemical properties. If the ISTD is used only to
compensate for injection reproducibility or changes in the analyte solution
volume, it must possess a similar retention (k) to the target component, but
it does not need to be chemically similar to the target component. It must
be pure, not present in the sample, and inert towards the components of the
sample. ISTD components are typically analogs, homologues or isomers of
the target non-ISTD component. An ideal ISTD is a structural or
isotopically-labeled analog of one of the target components. Stable
isotope-labeled ISTDs act almost identically to the analyte throughout
sample manipulation and with regard to ionization tendencies and
fragmentation. Internal standards labeled with two or more deuterium (D)
atoms are frequently used for LC/MS.
Response for Target Compound / Response for ISTD
There can be any number of ISTD components in a sample, but each
non-ISTD component can be calibrated against only one ISTD.
1.4
1.2
1.0
0.8
0.6
0.4
Amount in
Unknown
0.2
0.0
Figure 3.
Thermo Electron Corporation
Response Ratio for
Unknown Sample
0.0
0.5
1.0
1.5
2.0
2.5
Amount of Target Compound
Calibration curve generated by using an internal standard
Xcalibur: Getting Productive with Quantitative Analysis
7
1
Introduction
Overview of the Quantitation Features in Xcalibur 2.0
Overview of the
Quantitation Features
in Xcalibur 2.0
Xcalibur 2.0 is divided into six main views: Instrument Setup, Sequence
Setup, Processing Setup, Qual Browser, Quan Browser, and Library Browser
(see Figure 4). Use the Instrument Setup, Sequence Setup, Processing Setup,
and Quan views to develop instrument methods and processing methods for
the quantitative analysis of your unknowns.
This manual does not cover the Instrument Setup, Qual Browser, or Library
Browser views. For information on qualitative analysis, see your Getting
Productive: Qualitative Analysis manual.
This section describes the following views:
•
Instrument Setup
•
Processing Setup
•
Sequence Setup
•
Quan Browser
Figure 4.
8
Xcalibur: Getting Productive with Quantitative Analysis
Roadmap - Home Page
Thermo Electron Corporation
1 Introduction
Overview of the Quantitation Features in Xcalibur 2.0
Instrument Setup
The Instrument Setup view enables you to develop instrument methods for
the instrument that you have created in the Xcalibur Instrument
Configuration program. An instrument method is a set of experimental
parameters and operating settings for a specific instrument system, such as
an HPLC, GC/MS, or LC/MS system. Use the Instrument Setup view to
create new instrument methods or modify existing instrument methods.
The Instrument Setup view contains separate pages for each device, such as
an autosampler, LC pump, GC, PDA detector, or mass spectrometer. Select
these pages using the Xcalibur Instrument Configuration program. Display
the Instrument Setup view by clicking the Instrument Setup icon on the
Roadmap - Home Page.
Figure 5.
Thermo Electron Corporation
Instrument Setup view, showing the Surveyor AS - Surveyor AS
Method page
Xcalibur: Getting Productive with Quantitative Analysis
9
1
Introduction
Overview of the Quantitation Features in Xcalibur 2.0
Processing Setup
Use the Processing Setup view to create a post-acquisition data processing
method. Xcalibur uses a processing method to identify, detect, and integrate
components in a chromatogram, to generate calibration curves, to quantify
unknowns, and to produce reports.
Also, use Processing Setup to create processing methods capable of
interpreting data acquired using either a GC or LC separation technique.
Processing Setup also provides three integration algorithms for determining
the area or height of chromatographic peaks and a variety of calibration
curve types to fit the data.
The Processing Setup view is discussed in detail in Chapter 2: Processing
Setup.
Figure 6.
10
Xcalibur: Getting Productive with Quantitative Analysis
Processing Setup - Identification page
Thermo Electron Corporation
1 Introduction
Overview of the Quantitation Features in Xcalibur 2.0
Sequence Setup
Use the Sequence Setup view to set up a sequence of samples for acquisition
and batch reprocessing. A sequence defines each sample as a standard,
unknown, QC, or blank, and identifies its position on an autosampler tray
when appropriate. A sequence also identifies the instrument method to be
used for data acquisition and the processing method to be applied during
automatic processing. The Sequence Setup view is described in detail in
Chapter 3: Automating Analysis.
This topic contains the following subtopics:
Sample Types
•
Sample Types
•
Sample Brackets
Each quantitative analysis consists of a number, or sequence, of samples.
The sequence represents the order of sample analysis. A quantitation
sequence contains:
•
One or more standards
•
One or more unknown samples
For more demanding applications, also use optional quality control (QC)
samples and blank samples.
Standards
A calibration standard is a sample containing known amounts of all target
components. The purpose of a standard is to measure the response of the
detector to the target components so that a calibration curve can be
generated for each component.
Unknowns
An unknown sample is one containing unknown amounts of the target
components.
QCs
Thermo Electron Corporation
A QC sample contains a known amount of one or more specific target
compounds. QC samples are placed in the sequence so that quantitation
results can be tested for quality assurance purposes. After the QC sample is
analyzed, the measured quantity is compared with the expected value and an
acceptability range. The quantitation of a QC sample is classified as passed
if the difference between the observed and expected quantities is within the
user defined tolerance. A QC sample is classified as failed if the difference
between the observed and expected quantities is outside the defined
tolerance.
Xcalibur: Getting Productive with Quantitative Analysis
11
1
Introduction
Overview of the Quantitation Features in Xcalibur 2.0
Blanks
A blank sample contains no target components but might contain an ISTD
when the internal standard quantitation technique is being used. The
analysis of a blank sample can confirm that there are no residual
components in the solvent system that can cause erroneous results.
Sample Brackets
Xcalibur contains several bracketing techniques that help create customized
calibration routines. For example, you can inject a set of standards and build
a calibration curve before injecting unknowns. Xcalibur determines the
concentration of the analyte in the unknowns based on this initial
calibration curve or you can inject a set of standards both before and after
injecting unknowns. In the second method, bracket the sample solutions
with standard solutions. Xcalibur builds the calibration curve based on a
weighted average of the calibration sets and determines the concentrations
of the analyte in the unknowns.
Create specialized bracketing routines with the New Sequence Template
dialog box. The bracketing routines available in Xcalibur are discussed in
detail in “Choosing a Bracket Type” on page 102.
Quan Browser
Use the Quan Browser view to examine the quantitative results by applying
a processing method to the acquired data files, adjusting the processing
parameters, generating new calibration data, and recalculating the
quantitation results. Quan Browser is described in Chapter 4, “Reviewing
Quantitation in Quan Browser”.
Figure 7.
12
Xcalibur: Getting Productive with Quantitative Analysis
Quan Browser view, showing the results grid, chromatogram view,
and calibration view
Thermo Electron Corporation
1 Introduction
Acquiring and Quantitatively Processing Data with Xcalibur 2.0
Acquiring and
Quantitatively
Processing Data
with Xcalibur 2.0
With Xcalibur 2.0, quantitative analysis usually involves the following steps.
The order of some of these steps is not rigid. For example, you can acquire
and process a set of data files using a sequence that contains both an
instrument method and a processing method, and you can print reports
without previewing them first.
1. Create an instrument method.
Xcalibur 2.0 uses an instrument method to store a specific set of
parameters used to operate the autosampler, LC pump or MS pump, gas
chromatograph, mass spectrometer, PDA detector, and so on.
This manual does not cover the process of creating an instrument
method.
2. Create an acquisition sequence.
An acquisition sequence identifies the position of the samples in an
autosampler tray (if appropriate), the instrument method used to
control the HPLC, GC/MS, or LC/MS instrument, and the directory
and filenames for the acquired data files.
See “Creating a New Sequence” on page 98 for instructions on creating
a sequence to acquire raw data files.
3. Run the sequence to acquire the raw data file(s).
Run either one sample or a series of samples from the current sequence.
See “Running Samples” on page 117 for instructions on running
sequence and acquiring raw data files.
4. Create a processing method.
Create processing methods in the Processing Setup view. Xcalibur 2.0
uses a processing method to identify, detect, and integrate components
in a chromatogram, generate calibration curves, quantify unknowns,
and produce reports. Xcalibur 2.0 contains several built-in report
templates. Report templates have an .xrt file extension.
See Chapter 2: Processing Setup for instructions on using the
Processing Setup view to create a processing method.
Thermo Electron Corporation
Xcalibur: Getting Productive with Quantitative Analysis
13
1
Introduction
Acquiring and Quantitatively Processing Data with Xcalibur 2.0
5. Create a processing sequence by adding the processing method to the
original “acquisition” sequence.
A processing sequence contains a processing method, consists of a list of
sample data files, and includes information on sample type and
calibration or QC level.
6. Reprocess a representative raw file or the entire sequence with the
processing method by using the Batch Reprocess feature in the sequence
Setup view.
Reprocessing a raw file produces a result file. Result files have an .rst file
extension. See “Reprocessing Samples” on page 122 for instructions on
batch reprocessing a sequence.
Tip The built-in report templates are generic and might not produce
the results you expect. Preview a report in the XReport reporting
package before printing reports for an entire sequence
7. After Xcalibur 2.0 processes the raw data files, you can evaluate the peak
detection settings, the integration settings, and the calibration curve for
each component in Quan Browser. As you evaluate the results of the
processing method, modify some of its parameters in Quan Browser. If
the processing method contains a report template, print reports from
Quan Browser.
8. Preview a report for a representative data file from the XReport
reporting package.
To produce customized reports, open a representative result file [.rst] in
the XReport reporting package and create a report template.
9. Once you are satisfied with the way a report displays your data, add the
report to the processing method if you have not already done so and
batch reprocess the sequence to generate printed reports.
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Chapter 2
Processing Setup
This chapter describes the Processing Setup window and explains how to use
it to create a quantitative processing method for automated batch analysis. It
defines the parameters required for identifying, integrating, and
quantitating the peaks in chromatograms. In addition, it explains how to
add a report template and additional programs to the processing method.
Because the focus of this manual is quantitative analysis, the Quan view of
Processing Setup is described in detail. For a quick tutorial on how to create
a processing method for the quantitative analysis of data, see the Xcalibur
Getting Productive: Processing Setup and the Analysis of Quantitation Data
manual. For information on the Processing Setup - Qual view, see the
Xcalibur Getting Productive: Qualitative Analysis manual.
This chapter contains the following sections:
Thermo Electron Corporation
•
The Processing Setup Window
•
The Quan View
•
Working Interactively with the Chromatogram and Spectrum Previews
•
Identification
•
Detection
•
Calibration
•
Levels
•
System Suitability
•
Peak Purity
•
Reports
•
Programs
Xcalibur: Getting Productive with Quantitative Analysis
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2
Processing Setup
The Processing Setup Window
The Processing Setup
Window
Use the Processing Setup window to create a processing method for
automated batch analysis. It contains dialog boxes for entering the
parameters required for qualitative and quantitative data processing,
reporting, and running additional programs, such as file copying
procedures.
This section contains the following topics:
Features of the Processing
Setup Window
16
•
Features of the Processing Setup Window
•
Applying Changes to a Page
•
Customizing Processing Setup
The Processing Setup window (see Figure 8) consists of the following:
•
A title bar containing the application name, Processing Setup; the active
view: Quan, Qual, Reports, or Programs; the active page; the name of
the opened processing method; and the selected type of calibration
method: Int Std (internal standard) or Ext Std (external standard)
•
A menu bar
•
A toolbar
•
A view bar containing graphical buttons leading to the four views of
Processing Setup: Quan, Qual, Reports and Programs
•
The selected view – Quan and Qual views are multi-paged
•
A status bar showing information about activities within
Processing Setup
•
The Components list, available in the Quan view
•
The Chromatogram and Spectrum previews, available in the
Identification and Detection pages of the Quan view and in the
Identification, Spectrum Enhancement, and Peak Purity pages of the
Qual view
Xcalibur: Getting Productive with Quantitative Analysis
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2 Processing Setup
The Processing Setup Window
Title Bar
Menu Bar
Toolbar
View Bar
Selected View
(Quan view)
Chromatogram
Preview
Components
List
Spectrum
Preview
Status Bar
Figure 8. Processing Setup window - Quan View - Identification page
Display or hide the View Bar, Components list (in Quan View), Toolbar,
and Status Bar by choosing the appropriate View menu command:
•
Choose View > View Bar to display or hide the View Bar
•
Choose View > Components List to display or hide the Components
list
•
Choose View > Toolbar to display or hide the Toolbar
•
Choose View > Status Bar to display or hide the Status Bar
To maximize the display of a Processing Setup view, hide all four of these
features.
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2
Processing Setup
The Processing Setup Window
Applying Changes to a
Page
Each Processing Setup page features OK and Cancel buttons. These are
enabled only if you change one or more parameters on the page. Until you
make a change, these buttons are grayed out.
After you change or edit a parameter, do one of the following:
•
Click OK to apply the changes to the current processing method.
Xcalibur reports any validation errors.
•
Click Cancel to undo all changes made to the page and revert to the
previously applied values.
Note These actions do not affect the saved version of the processing
method. Modify the saved version by using the File > Save command
Select Options > Enable Warnings to display the Apply Changes dialog
box shown in Figure 9.
In the Apply Changes dialog box, do one of the following:
•
Click Yes to apply changes.
•
Click No to discard any changes and proceed with the selected action.
•
Click Cancel to stop the intended action and return to the current page
without applying or discarding changes.
•
To avoid displaying the Apply Changes dialog box, select the Don’t Tell
Me About This Again check box.
Figure 9.
Apply Changes dialog box
Choose Options > Enable Warnings to re-enable this and all other warning
dialog boxes.
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2 Processing Setup
The Processing Setup Window
When the Apply Changes dialog box is active and you attempt one of the
following actions without applying or discarding changes, Xcalibur does not
permit you to proceed until you apply or undo the page modifications:
Thermo Electron Corporation
•
Switch to another page
•
Switch to another component
•
Switch to another View, using either the buttons in the View Bar or the
options on the View menu
•
Change the chromatography type in the Chromatography Options
dialog box (Options > Chromatography By)
•
Change the calibration type in the Calibration Options dialog box
(Options > Calibration By)
•
Click the Close button on the title bar
•
Choose File > Open
•
Choose File > <most recently used file list>
•
Choose File > Save
•
Choose File > Save As
•
Choose File > Exit
•
Choose File > Import Method
•
Choose File > New
•
Choose Options > Standard Dilution (from the Levels page)
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2
Processing Setup
The Processing Setup Window
Customizing Processing
Setup
By default, Xcalibur loads the most recently used processing method [.pmd]
into the Processing Setup window at startup. You can change this option or
configure Xcalibur to open a raw file [.raw] into the Chromatogram and
Spectrum previews when a processing method is opened.
To adjust these options, choose the Options > Settings menu command to
open the Settings dialog shown in Figure 10.
Figure 10. Settings dialog box
In the Startup Mode area, do one of the following:
•
Click the Load Last Processing Method option to load the most
recently used processing method when you start a Processing Setup
session.
•
Click the Create New Processing Method option to start a new
processing method when you begin a Processing Setup session.
In the Auto-open raw file area, do one of the following:
20
•
Click the On option to automatically open a raw file with each
processing method. Xcalibur populates the chromatogram and spectrum
cells with the raw file associated with the processing method when it was
last saved.
•
Click the Off option to not open a raw file when you open a processing
method.
Xcalibur: Getting Productive with Quantitative Analysis
Thermo Electron Corporation
2
The Quan View
Processing Setup
The Quan View
Use the Processing Setup - Quan View to enter the quantitative analysis
parameters of the processing method.
The Quan view (see Figure 8 on page 17) consists of the following
tabbed pages, which are described in detail in subsequent sections of this
chapter:
•
Use the Identification page to name components and specify retention
time and peak identification criteria.
•
Use the Detection page to control peak detection and integration in the
chromatogram plot.
•
Use the Calibration page to select the type of calibration applied to the
data.
•
Use the Levels page to enter the concentrations of calibration standards
and QC (quality control) standards.
•
Use the System Suitability page to specify the pass or fail criteria for
chromatographic peaks.
•
Use the Peak Purity page to specify peak purity parameters for Surveyor
PDA data. The Surveyor PDA is a photodiode array detector, capable of
scanning the UV-Vis range from 190 nm to 800 nm.
A Components list (if enabled) appears to the right of each page in Quan
view. This list contains all of the component names defined in the active
processing method. A processing method might contain different
Identification, Detection, Calibration, Levels and System Suitability page
parameters for each listed component.
To view the Components List, choose View > Components List.
To view the parameters for a particular component, click its name in the
Components list.
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Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
Working Interactively
with the
Chromatogram and
Spectrum Previews
Processing Setup - Quan View displays the Chromatogram and Spectrum
previews in the lower portion of the Identification and Detection pages.
Using a representative raw file, these previews help to do the following:
•
Preview the results of peak detection and integration in the
Chromatogram Preview.
•
Set some of the Identification and Detection parameters interactively.
This section contains the following topics:
Opening a Raw File
•
Opening a Raw File
•
Previewing Processing
•
Setting Processing Parameters
•
Using the Cursor
•
Using the Toolbar
•
Customizing the Previews
To open a raw file (.raw file extension)
1. Choose File > Open Raw File or click the Open Raw File button on
the toolbar.
2. Select a relevant raw file in the Open Raw File dialog box and click
Open.
Note When a processing method is saved when a raw file is present, the
raw file name is saved in the processing method. The associated raw file
opens automatically whenever the processing method is opened if you
have selected the On option in the Auto-Open Raw File area in the
Settings dialog box.
Previewing Processing
Using a suitable raw file that contains MS data, PDA data, or both, use the
Chromatogram and Spectrum previews to evaluate peak identification,
detection, and integration parameters.
Xcalibur processes the raw file using the parameters of the Identification and
Detection pages. The Chromatogram preview is centered on the Expected
Retention Time value of a selected component and its display width is based
on its View Width value. It shades all detected peaks and indicates the start
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2 Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
and end of each peak with a blue baseline. Initially, the spectrum shown in
the Spectrum preview is the one corresponding to the apex scan of the first
detected peak in the chromatogram. If no peak has been detected in the
chromatogram, the Chromatogram preview shows the whole raw file and
the Spectrum preview shows the spectrum for the first scan in the raw file.
Re-scale the chromatogram or spectrum previews by using:
•
Cursor actions (see Using the Cursor on page 24)
•
Buttons on the toolbars
•
Zoom menu commands, either from the top-level menu, or from the
shortcut menu (see Using the Toolbar on page 28)
Edit the parameters on the Identification and Detection pages manually by
typing in a value or interactively by performing a cursor action in either the
Chromatogram or Spectrum previews. When you manually or interactively
edit values on the Identification or Detection pages, Xcalibur removes the
shading and baselines from all detected peaks in the Chromatogram
preview. This indicates that the previews do not match the information
currently shown on the page.
After you change the value of a parameter, do one of the following:
•
Click OK to perform the peak detection processing again using the
current parameters.
•
Click Cancel to discard all changes made to the page.
Xcalibur shades all detected peaks and adds their baselines to indicate the
peak start and end positions.
Setting Processing
Parameters
Create a quantitative processing method by manually entering values for all
the required Quan view parameters. In the Identification and Detection
pages, take advantage of the interactive features of Processing Setup. This
involves the use of the chromatogram and spectrum previews together with
a raw file that is representative of the analysis requirements.
Use the previews to set the following:
Thermo Electron Corporation
•
The expected retention time for the component
•
The mass or wavelength ranges
•
Spectrum Qualifier table for GC data (see Spectrum Detection on
page 50)
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Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
Using the Cursor
Within the Chromatogram and Spectrum previews, use the cursor in three
ways:
•
Click in the cell to select a point
•
Drag a line parallel to any axis to select a range
•
Drag a line in any diagonal direction to select 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 1). When one of the cells is
pinned, the cursor action in any of the inactive cells is always applied to the
pinned cell.
Table 1.
Effect of cursor action in an active cell
Cursor Action
Effect
Drag parallel to X-axis
Rescales graph showing selected X range only, same Y range
Drag parallel to Y-axis
Rescales graph showing selected Y range only, same X range
Dragged area
Rescales graph showing both the selected X and Y ranges
The effect of these actions depends on the state of the preview. There are
three hierarchical states for the Chromatogram and Spectrum previews:
•
Inactive
•
Active and unpinned
•
Active and pinned
Only one of the previews can be active at any one time. The inactive view
has no border. The active preview is highlighted with a gray border. The
pinned preview is highlighted with a gray border and contains a green pin in
its upper-right corner (see Figure 11).
Inactive
Active
Pinned
Figure 11. Inactive, active, and pinned Spectrum previews
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2 Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
To make a preview active or active and pinned
1. Verify that the other preview is not pinned. Click anywhere within a
preview to make it active.
Xcalibur highlights it with a gray border.
2. Click its pin icon to fix it as the active preview.
Cursor actions in an active or active and pinned preview cause the preview
to be scaled according to the dimensions of the dragged line or area.
Cursor actions have a variety of effects when one of the previews is pinned.
Table 1 lists the effects of clicking and dragging in the previews on the
Identification page. Table 2 lists the effects of clicking and dragging in the
previews on the Detection page.
Table 2.
Effect of cursor action in the previews on the Quan View Identification page
Pinned
Preview
Cursor Action in
inactive Preview
Effect
Spectrum
Click the Chromatogram
preview.
Detector type: All
Drag across a time range in the
Chromatogram preview.
Detector type: All
Click the Spectrum preview.
Detector type: MS
Spectrum
Chromatogram
The retention time selected is entered
in the Expected (min) box. The Spectrum
preview displays the mass spectrum
corresponding to that retention time.
The retention time of the highest point
of the dragged range is entered into the
Expected (min) box. Spectrum preview
displays the mass spectrum of that
retention time.
The selected mass is entered into the
Mass (m/z) box as an addition to any
existing value(s). When the Trace type is
TIC, it is changed to Mass Range.
Detector type: PDA
The selected wavelength is entered into
the Wavelength (nm) box as an addition
to any existing value(s). When the Trace
type is Total Scan, it is changed to
Wavelength Range.
Thermo Electron Corporation
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Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
Table 2.
Effect of cursor action in the previews on the Quan View Identification page, continued
Pinned
Preview
Cursor Action in
inactive Preview
Effect
Chromatogram
Drag across an m/z range in the
Spectrum preview.
Detector type: MS
The selected mass range is entered into
the Mass (m/z) box as an addition to any
existing value(s). When the Trace type is
TIC, it is changed to Mass Range.
Detector type: PDA
The selected wavelength range is
entered into the Wavelength (nm) box
as an addition to any existing value(s).
When the Trace type is Total Scan, it is
changed to Wavelength Range.
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2 Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
Table 3.
Effect of cursor action in the previews on the Quan View Identification page
Pinned
Preview
Cursor Action in
inactive Preview
Effect
Spectrum
Click Chromatogram preview.
Detector type: MS
Spectrum preview displays the mass
spectrum of the selected retention time.
When Peak Detection is set to Spectrum,
the spectrum table is populated with all
ions in the displayed spectrum (replacing
any existing values); subject to the Low
Intensity Cutoff threshold set in the
Spectrum Options dialog box (Options >
Spectrum command).
Spectrum
Drag across a time range in
Chromatogram preview.
Detector type: MS
Spectrum preview displays the mass
spectrum of the retention time of the
highest point in the dragged range.
When Peak Detection is set to Spectrum,
the spectrum table is populated with all
ions in the displayed spectrum (replacing
any existing values); subject to the Low
Intensity Cutoff threshold set in the
Spectrum Options dialog box (Options >
Spectrum command).
Chromatogram
Thermo Electron Corporation
Drag across an m/z range in
Spectrum preview.
Detector type: MS
When Peak Detection is set to Spectrum,
the spectrum table is populated with all
ions in the dragged range (replacing any
existing values); subject to the Low
Intensity Cutoff threshold set in the
Spectrum Options dialog box (Options >
Spectrum command).
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2
Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
Using the Toolbar
Use the buttons on the toolbars to re-scale a Chromatogram or Spectrum
preview. The toolbar contains the following buttons:
•
Normalize Y
•
Zoom out Y
•
Zoom in Y
•
Auto Range Y
•
Zoom in X
•
Zoom out X
•
Display all data on X axis
•
Reset scaling to full scale for both X and Y axes
The Zoom menu contains equivalent commands. Display the toolbar as a
shortcut menu by right-clicking on the appropriate preview or re-scale the
chromatogram using the cursor.
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2 Processing Setup
Working Interactively with the Chromatogram and Spectrum Previews
Customizing the Previews
To customize the display of a chromatogram or spectrum preview
1. Verify that neither preview is pinned. Click anywhere within a preview
to make it active.
A gray border appears around the active preview.
2. Choose Options > Display Options or right-click the preview and
choose Display Options from the shortcut menu.
The Display Options dialog box contains five tabbed pages for changing the
plotting style, colors, axes, labels and normalization method (see Figure 12).
For more information about display options, see Xcalibur’s online Help.
Figure 12. Display Options dialog box, showing the label options for a
chromatogram
Thermo Electron Corporation
Xcalibur: Getting Productive with Quantitative Analysis
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2
Processing Setup
Identification
Identification
Use the Quan View - Identification page to name a component and specify
retention time, detector type, and detection and integration criteria.
Xcalibur uses the parameters on this page to generate a chromatogram from
a raw file and identify each component peak within the chromatogram.
This section contains the following topics:
•
Identification Parameters
•
Entering the Identification Parameters for Your Processing Method
Figure 13. Quan View - Identification page
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2
Identification Parameters
Processing Setup
Identification
Use the Identification page to enter and select the following parameters:
Name
Use the Name combo box to enter the names of the components in the
sample and lists all the component names for the active processing method.
To display the component identification settings for a component on the
list, click the name of the component in the Name combo box or click on a
component name in the Components pane.
Detector Type
Use the Detector list to select the type of detector used to acquire the data.
The available selections are MS, Analog, A/D Card, PDA, or UV.
Peak Detection
Use the Peak Detection list to select the type of peak detection algorithm.
The available selections are Genesis, ICIS, or Avalon.
The ICIS peak detection algorithm has been designed for MS data and has
superior peak detection efficiency at low MS signal levels. This is the
Xcalibur default peak detection algorithm. The Genesis peak detection
algorithm is the original Xcalibur peak detection algorithm. This algorithm
has been provided for backward compatibility with Xcalibur 1.0 studies.
The Avalon peak detection algorithm supports detectors other than MS
because it detects negative chromatographic peaks, and shoulders more
accurately than Genesis or ICIS.
For new processing methods, change the default detection algorithm at any
time for each type of detector. From the Roadmap view of the Home Page,
choose Tools > Configuration and select the appropriate option on the
Detection page (see Figure 14).
Figure 14. Xcalibur Configuration dialog box
Filter
Thermo Electron Corporation
The Filter field is active if you select an MS detector type. Use this combo
box to specify a scan filter. A scan filter causes processing to be applied to a
subset of the scans in a raw file.
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2
Processing Setup
Identification
When you load a raw file, Xcalibur lists the scan filters associated with it in
the Filter combo box. Xcalibur creates scan filters from the instrument
method during data acquisition. Select a scan filter from the list. Xcalibur
applies the scan filter to the data in the raw file and displays the resulting
filtered chromatogram data in the Chromatogram preview after you
click OK.
You can also type your own filter in the scan filter format. For information
about scan filter formats, consult the Xcalibur online Help.
Trace
Raw files can contain data for more than one chromatogram. Use the Trace
fields to specify the type of chromatogram to use.
The Trace options depend on your selection of Detector Type:
•
For MS scans, select Mass Range, TIC or Base Peak.
•
For Analog data, select from four channels (labeled Analog 1-4).
•
For data from an A/D Card, select from four channels (labeled A/D Card
Ch. 1-4).
•
For PDA data, select Wavelength Range, Total Scan, or Spectrum
Maximum.
Use the first Trace list to select a basic chromatogram. Use the second list to
select a logical operator, + or -, to be used in combination with the third list.
Use the third list to add a valid chromatogram type to or subtract a valid
chromatogram type from the chromatogram type specified in the first Trace
list.
Most often, you will probably use a single trace such as a TIC. A second
trace is useful for subtracting contributions to a chromatogram from a
solvent or other noise. Table 4 lists the various MS trace combinations and
gives examples of their use. Table 5 lists the various trace combinations for
non-MS detectors and gives examples of their use.
Table 4.
32
MS Traces and combinations
Trace
Use
TIC
Compiles a chromatogram from all the ions in each MS scan.
Mass Range
Compiles a chromatogram from a single mass or a range of masses
in each scan. This can be a list of masses or ranges separated by
commas and summed.
Base Peak
Compiles a chromatogram from the most abundant ion within the
specified mass range.
Xcalibur: Getting Productive with Quantitative Analysis
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2
Table 4.
MS Traces and combinations, continued
Trace
Use
TIC - Mass Range
Use this check box to ‘clean up’ a TIC by subtracting a range of
background contamination and thereby allowing less abundant
masses to have a more significant effect on the chromatogram. For
example, subtract dominant solvent or contaminant peaks in the
mass range from 50 to 150 form TIC data acquired from 50 to 1000.
TIC - Base Peak
Use in situations where the most intense spectral peak throughout
the run is due to a contaminant. Subtracting the base peak from the
TIC removes the contribution from the contaminant.
Mass Range - Mass
Range
Removes a variety of background, solvent or contaminant peaks from
a chromatogram, for example, in data acquired from m/z 50 to 900;
solvent contamination is evident below m/z 150 and there are
intense contaminant peaks in the intermediate range m/z 500 to
600. Use Mass Range 1 = 150 to 900; Mass Range 2 = 500 to 600.
Base Peak Mass Range
Rarely used, for example, when the most intense peaks in the
spectrum are m/z 130 at one point in the chromatogram and m/z 140
at another. If there are no sample masses in this range, BPI– (125 to
145) could remove the effect of these peaks.
Mass Range + Mass
Range
Similar uses to Mass Range – Mass Range above. Considering the
same example as above, identical results could be obtained using
this trace combination with:
Mass Range 1 = 150 to 499; Mass Range 2 = 601 to 900.
Base Peak + Mass
Range
Useful if the Base Peak trace does not show up every chromatogram
peak of interest. The mass range of interest can then be added to
enhance the spectrum.
Table 5.
Thermo Electron Corporation
Processing Setup
Identification
Basic non-MS traces
Trace
Use
Analog x
(1 to 4)
For monitoring any external detector, such as an FID detector, that
provides an analog signal.
A/D Card Channel
(1 to 4)
For monitoring any external detector that provides a digital signal.
You can also specify channel combinations.
Wavelength Range
View the summed absorbance for a range of PDA wavelengths.
Total Scan
View the summed absorbance for all the PDA wavelengths.
Spectrum Maximum
View a plot of the maximum absorbance for each timepoint in the
run.
Analog x + Analog y
As Analog x - Analog y above. You could add two channels
corresponding to the wavelengths of two compounds of interest
(ranges cannot be set on some detectors, only single channels).
Wavelength Range +
Wavelength Range
You could add two channels corresponding to the wavelengths of
two compounds of interest (ranges cannot be set on some detectors,
only single channels).
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Processing Setup
Identification
Table 5.
Mass (or Wavelength)
Basic non-MS traces, continued
Trace
Use
Analog x - Analog y
For some external detectors that give out an analog signal, such as
UV detectors, it is possible to monitor more than one channel
(typically two) and to set channels to a range, for example, 220 to
500 nm. These outputs are simple analog voltages (typically 0 to 1V).
You could acquire two channels from the same detector, one a range
and one a single wavelength or smaller range (for example, at a
contaminants' specific wavelength). Subtract one from the other, for
example, (220 to 500) – (260 to 280) nm.
Total Scan Wavelength Range
Use this option to subtract a single wavelength or small range (for
example, at a contaminants' specific wavelength) from the total
scan.
Wavelength Range Wavelength Range
Acquire two channels from the same detector, one a range and one
a single wavelength or smaller range (for example, at a
contaminants' specific wavelength). Subtract one from the other, for
example, (220 to 500) - (260 to 280) nm.
The Mass box is available only if you select an MS detector type in the
Detector Type box (Figure 13). The Wavelength box is available only if you
select a PDA detector type in the Detector Type box.
For an MS detector type, use the Mass box to specify the mass or mass range
for trace combinations featuring Mass Range or Base Peak traces (for
example, Mass Range, TIC - Base Peak, TIC - Mass Range). When you use
Base Peak ± Mass Range or Mass Range ± Mass Range trace combinations, an
additional Mass (m/z) box appears for you to specify the second mass range.
For the PDA detector type, use the Wavelength box in the cases where the
specified Trace combination features Spectrum Maximum or Wavelength
Range to specify the wavelength or wavelength range for the chromatogram.
When you use a trace combination such as Wavelength Range + Wavelength
Range (see Table 5), an additional Wavelength box appears for you to specify
the second wavelength range.
The Mass/Wavelength boxes hold up to 50 ranges. These should be
separated using the List separator character, normally a comma. This can be
found on the Number tab of Regional Settings in the Control Panel of
Microsoft® Windows® XP Professional.
Note You must provide a mass or wavelength range for each enabled
Mass Range or Wavelength Range box. When a Mass Range or
Wavelength Range box is blank, Xcalibur does not permit you to save the
parameters or change to another page until you have provided a range (or
have switched to a different trace combination that does not involve
Mass/Wavelength Ranges).
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2
Expected (min)
Processing Setup
Identification
Use the Expected (min) box to enter the expected retention time for the
selected component. Xcalibur identifies the selected component as the
highest peak with an apex within the expected retention time range.
Window (sec)
Use the Window box to enter a retention time window for the elution of the
selected component. The value should be equal to, or in excess of, the peak
width. The valid range is 1.0 to 999.0 seconds.
Use as RT Reference
Use the RT Reference check box to choose the actual retention time (RT) of
the selected component to adjust the expected retention time of one or more
of the remaining components.
Note When the expected retention time of the RT Reference
component falls out of its retention time window during a sequence,
Xcalibur cannot adjust the expected retention times of its dependent
components.
View Width
Use the View Width box to enter the current view width (in minutes) for
the chromatogram preview. The valid range is 0.1 to 999 minutes. A view
width can be specified for each component. Xcalibur displays a
chromatogram time axis with a range defined by the component retention
time of a component ± [view width/2]. When the view width exceeds the
chromatogram acquisition time, Xcalibur uses the acquisition time for the
view width.
Adjust Using
Use the Adjust Using check box to adjust the expected retention time (RT)
of the selected component based on the actual retention time of a RT
Reference component (see Use as RT Reference). Select the RT Reference
component from the adjacent list. At least one RT Reference component
must be available for the check box to be active.
Note When the expected retention time of the RT Reference
component falls out of its retention time window during a sequence,
Xcalibur cannot adjust the expected retention times of its dependent
components.
Keys
Thermo Electron Corporation
Use the Keys text field to enter comments about the component’s analysis.
The text field holds up to 30 characters and is case sensitive for alphabetic
characters (for example “abc” is recognized as being different from “Abc”).
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Processing Setup
Identification
Entering the Identification
Parameters for Your
Processing Method
To enter the identification parameters for each component in the sample
to quantify
1. Enter the name of a component:
a. From the Name combo box, select <New>.
b. Type the name of a component.
c. Press ENTER or click OK.
The new component appears in the Components list.
2. Select the type of detector(s) used to acquire the open raw file from the
Detector Type list.
The available selections are MS, Analog, A/D Card, PDA, or UV. The
raw file(s) that you plan to process with this processing method can
contain chromatograms from more than one type of detector. However,
to quantitate the data from two or more detectors for a particular
component, create two or more unique names for the component.
3. Select the integration algorithm to use from the Peak Detection list:
•
For MS data, select ICIS or Genesis.
•
For Analog, A/D Card, PDA, or UV data, select Avalon.
4. (Optional) When you are working with MS data, select an appropriate
filter for the selected component from the Filter list. The filter list is
dependent upon the open raw file.
5. Select a trace from the Trace list. The available selections are described in
Trace on page 32. If you select a mass range or a wavelength range,
specify the range (see Figure 15 or Figure 16).
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Processing Setup
Identification
Mass Range
Selected
Mass Range
Specified
Figure 15. Example of selecting a Mass Range for MS data
Wavelength Range
Selected
Wavelength
Range Specified
Figure 16. Example of selecting a logical operator for PDA data
6. (Optional) When you are working with MS or PDA data, if you selected
a range for the trace in step 5 above, enter the appropriate range(s) in the
Mass box or the Wavelength box for the MS data or PDA data,
respectively.
To change the range or to add a new range
1. Type the range in the box. The valid range is dependent upon the
configured detector. The format is [Low Mass/Wavelength] - [High
Mass/Wavelength]. For example, for the range m/z 123 through 456,
type 123 – 456.
2. Open a representative raw file in the Chromatogram and Spectrum
previews. Pin the Chromatogram preview.
3. Drag the required mass range on the Spectrum preview (or click to select
a single mass-to-charge ratio value). The mass range or wavelength range
is added to the Mass text box or the Wavelength text box, respectively.
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Processing Setup
Identification
The Mass/Wavelength boxes hold up to 50 ranges. Ranges must be
separated using the List separator character. The default setting for the
List separator in English is a comma.
4. To verify the List separator setting on the PC, choose Regional and
Language Options > Regional Options from the Control Panel of
Microsoft® Windows® XP Professional. Click Customize to open the
Customize Regional Options dialog box. Click the Numbers tab and
verify the selection in the List separators box.
Note You must provide a mass or wavelength range for each enabled
Mass Range or Wavelength Range box. When a Mass Range or
Wavelength Range box is blank, Xcalibur does not permit you to
save the parameters or change to another page until you have
provided a range (or switched to a different trace combination
which does not involve Mass/Wavelength Ranges).
Mass Range
Specified
Mass Range Selection
Chromatogram
Preview Pinned
Figure 17. View of the Chromatogram and Spectrum Previews, showing the Chromatogram preview pinned
and a mass range selection in the Spectrum preview
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Processing Setup
Identification
5. Enter the expected retention time for the component by manually
typing an appropriate value in the Expected (min) box or by
interactively working in the previews:
a. Open a representative raw file if you have not already done so.
b. Pin the Spectrum preview. Do one of the following:
•
Drag the cursor horizontally across the component peak in the
Chromatogram preview. Xcalibur updates the Expected (min)
field with the time of the apex scan.
•
Click the chromatogram. The clicked time is transferred to the
Expected box whether or not it is the apex of the peak.
6. Enter an appropriate retention time window for this component by
typing a value in the Window (sec) box. Base the value of the retention
time window on the width of the peak.
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Processing Setup
Identification
7. (Optional) To allow adjustments to the expected retention time value of
this component:
•
Select the Use as RT Reference check box to track and use the
actual retention time of this component to adjust the value of the
expected retention time of other components in the chromatogram.
•
Select the Adjust Using check box and select a reference component
from the adjacent list to adjust the value of the expected retention
time of this component based on the actual retention time of a
reference component.
8. (Optional) Enter additional descriptive information about the selected
component in the Keys text box.
9. To identify another component repeat steps 1 through 10.
10. To delete a component from the Components list:
a. Click its name in the Components list.
b. Choose Options > Delete Component (for example,
Options > Delete Anthracene) and confirm the deletion.
Proceed to the next section.
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Detection
Processing Setup
Detection
Use the Detection page, shown in Figure 18, to specify peak integration and
detection criteria.
The parameters in the Peak Detection area are determined by the selection
in the Chromatography Options dialog box. Xcalibur detects the type of
instrument (LC or GC) connected (when it is run for the first time) and
makes this the default type. The parameters in the Peak Integration area are
the same for the GC and LC Chromatography modes.
This section contains the following topics:
Thermo Electron Corporation
•
Peak Integration
•
Peak Detection for LC
•
Peak Detection for GC
•
Advanced Detection Parameters
•
Data Flags
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Processing Setup
Detection
Figure 18. Quan view - Detection page for LC
Figure 19. Peak Detection area for GC/MS
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Processing Setup
Detection
To change the chromatography mode, choose Options > Chromatography
By to open the Chromatography Options dialog box. Click either the GC
or the LC option and click OK (see Figure 20).
Figure 20. Chromatography Options dialog box
Peak Integration
Xcalibur 2.0 provides three peak detection algorithms: ICIS for low signal to
noise MS data, Avalon for non-MS data, and Genesis for backward
compatibility with Xcalibur 1.0 data files.
Specify the Peak Detection algorithm for a component in the Quan view Identification page and select the values for the available integration
parameters on the Quan view - Detection page.
This topic contains the following subtopics:
Thermo Electron Corporation
•
Genesis Peak Integration Parameters
•
ICIS Peak Integration Parameters
•
Avalon Peak Integration Parameters
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Processing Setup
Detection
Genesis Peak Integration
Parameters
The Genesis Peak Integration area shown in Figure 21 contains the
following options for peak integration:
Figure 21. Genesis Peak Integration area
The two graphical display boxes (entitled Min and Max) at the right of the
Genesis Peak Integration area depict the effect of small and large values for
the selected option as a visual reminder of how the option operates on data.
For example, the boxes in the margin show the large and small values for the
peak integration parameters and illustrate their effects on a simple data
representation, not the actual data.
Smoothing Points
Use the Smoothing Points box to specify the degree of data smoothing to be
performed on the active component peak prior to peak detection and
integration. The valid range is any odd value from 1 (no smoothing)
through 15 (maximum smoothing).
S/N Threshold
Use the S/N Threshold box to set the signal-to-noise threshold for peak
integration. Xcalibur does not integrate peaks with signal-to-noise less than
this value. Peaks with signal-to-noise greater than this value are integrated.
The valid range is 0.0 to 999.0.
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Detection
Enable Valley Detection
Select the Enable Valley Detection check box to use the Xcalibur valley
detection approximation method to detect unresolved peaks. This method
drops a vertical line from the apex of the valley between unresolved peaks to
the baseline. The intersection of the vertical line and the baseline defines the
end of the first peak and the beginning of the second peak.
Expected Width (sec)
Use the Expected Width (sec) box to enter the expected peak width (in
seconds). This value controls the minimum width that a peak is expected to
have if valley detection is enabled.
With valley detection enabled, any valley points nearer than the [expected
width]/2 to the top of the peak are ignored. When a valley point is found
outside the expected peak width, Xcalibur terminates the peak at that point.
Xcalibur always terminates a peak when the signal reaches the baseline,
independent of the value set for the expected peak width. The valid range is
0.0 to 999.0 seconds.
Constrain Peak Width
Select the Constrain Peak Width check box to constrain the peak width of
a component during peak integration of a chromatogram. Set values that
control when peak integration is turned on and off by specifying a peak
height threshold and a tailing factor.
Peak Height
Use the Peak Height box to enter the percent of the total peak height
(100%) that a signal needs to be above the baseline before integration is
turned on or off. This box is active only when the Constrain Peak Width
check box is selected. The valid range is 0.0 to 100.0%.
Peak Tailing Factor
Use the Peak Tailing Factor box to control how Xcalibur integrates the tail
of a peak. This factor is the maximum ratio of the trailing edge to the
leading side of a constrained peak. This box is active only when the
Constrain Peak Width check box is selected. The valid range is 0.5
through 9.0.
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Detection
ICIS Peak Integration Parameters
The two graphical display boxes (entitled Min and Max) at the right of the
ICIS Peak Integration area depict the effect of small and large values for the
selected option as a visual reminder of how the option operates on data. For
example, the boxes in the margin show the large and small values for the
peak integration parameters, and illustrate their effects on a simple data
representation, not the actual data.
The ICIS Peak Integration area on the Detection page contains the
following integration options:
Smoothing Points
Use the Smoothing Points box to enter the amount of smoothing that
Xcalibur applies before integration. The valid range is any odd value from
1 (no smoothing) through 15 (maximum smoothing).
Baseline Window
Use the Baseline Window box to enter the number of scans to review to find
a local minima. The valid range is 1.0 to 500. The default value is 40 scans.
Area Noise Factor
Use this box to specify the noise level multiplier used to determine the peak
edge after the location of a peak candidate. The valid range is 1 through
500. The default multiplier is 5.
Peak Noise Factor
Use the Peak Noise Factor box to specify the noise level multiplier used to
determine the potential peak signal threshold. The valid multiplier range is
1 through 1000. The default multiplier is 1
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Detection
Peak Width Constraints
Enable the Constrain Peak Width check box to constrain the peak width of
a component during peak integration of a chromatogram. Set values that
control when peak integration is turned on and off by specifying a peak
height threshold and a tailing factor.
Peak Height
Use the Peak Height (%) box to enter the percentage of the total peak height
that a signal needs to be above the baseline before integration is turned on or
off. The valid range is 0 to 100.0%.
Tailing Factor
Use the Tailing Factor box to enter the maximum ratio of the trailing edge
to the leading side of a constrained peak. The valid range is 0.5 to 9.0.
Avalon Peak Integration
Parameters
The Avalon peak integration algorithm specified on the Identification page
is used for non-MS data (see Figure 22).
Figure 22. Avalon Peak Integration area, showing the default parameters
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Processing Setup
Detection
The Avalon Peak Integration area in the Detection page contains the
following:
Smoothing Points Box
Use the Smoothing Points box to enter the degree of data smoothing to be
performed on the selected component peak prior to peak detection and
integration. The valid range is any odd value from 1 (no smoothing)
through 15 (maximum smoothing). To smooth the component peak data
prior to integration, type a value in the Smoothing Points box.
Events List
To detect peaks, Avalon uses the settings for initial events and user-defined
timed events that are in the Event list.
To calculate values for initial events, open a raw file and make the
chromatogram view active. Click Auto Calculate Initial Events to update
the values in the event list. Change the settings in the event list by clicking
Advanced to display the Avalon Event List dialog box, which contains an
editable event list. Highlight the row to change and enter any revised
settings in the boxes below the list. Click Change to update automatically
the event list both here and on the Detection page and to update
automatically the chromatogram display.
The Time column contains either the term initial value or a value of time in
minutes. The Event column contains descriptions of the detection
parameters for initial events and timed eventuates Value column contains
values associated with initial events and timed events. For more details about
using the Avalon Event List dialog box to edit integration events, see
“Avalon Event List” on page 60.
The default events and their effects are listed in Table 6.
Table 6.
48
Default Avalon peak integration events
Event
Effect
Start Threshold
Controls the intensity threshold for the start of a chromatographic
peak.
End Threshold
Controls the intensity threshold for the end of a chromatographic
peak.
Area Threshold
Controls the area cutoff. Any peaks with a final area less than the area
threshold is not detected. This control is in units of area for the data.
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Table 6.
Processing Setup
Detection
Default Avalon peak integration events, continued
Event
Effect
P-P Threshold
Controls how much peak overlap must be present before two or more
adjacent peaks create a peak cluster. Peak clusters have a baseline
drop instead of valley to valley baselines, specified as a percent of
peak height overlap.
Bunch Factor
Controls the bunching of chromatographic points during integration
and does not affect the final area calculation of the peak. The Bunch
Factor must be an integer between 1 and 6; a high bunch factor groups
peaks into clusters.
Negative Peaks
Automatically resets after a negative peak has been found.
Tension
Controls how closely the baseline should follow the overall shape of
the chromatogram. A lower tension traces the baseline to follow
changes in the chromatogram more closely. A high baseline tension
follows the baseline less closely over longer time intervals. Set in
minutes.
Auto Calculate Initial Events Button
The Auto Calculate Initial Events button is active with the event list of the
Avalon peak detection algorithm only if a raw file is open. When you click
this button, Avalon automatically estimates the initial values for the
detection of peaks based on the data in the current raw file and displays
those initial values in the event list. Use this button to force Avalon to search
for the best values of initial events that detect peaks in the data. Click this
button to leave any timed event in the event list unchanged.
Auto Calculate Initial Events determines initial values for the following
events only: Start Threshold, End Threshold, Area Threshold,
P-P [Resolution] Threshold, Bunch Factor, Negative Peaks, and Tension.
Use the same event list to specify timed events for these events.
Peak Detection for LC
Thermo Electron Corporation
The ICIS, Genesis, and Avalon Peak Detection areas contain options for
determining how Xcalibur detects peaks within the retention time window.
The following parameters are available for the LC mode:
Highest Peak
Use the Highest Peak option to identify the active component with the
highest peak in the retention time window. This is the default LC
chromatography mode option.
Nearest Peak
Use the Nearest Peak option to identify the selected component with the
peak having a retention time nearest to the Expected value.
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Processing Setup
Detection
Minimum Peak Height
Peak Detection for GC
This is a signal-to-noise threshold for peak integration (available in ICIS
and Genesis). Xcalibur does not integrate peaks with signal-to-noise less
than this value, only integrating peaks with signal-to-noise greater than this
value. The valid range is 0.0 to 999.0.
In addition to the peak detection options available for LC, the Spectrum
option is available for GC. Use the Spectrum option to use a reference
spectrum for component identification. Xcalibur attempts to match the
reference spectrum with a series of unknown spectra and calculates a score
for each comparison.
In GC Chromatography mode, Ion Ratio Confirmation is available with the
Highest Peak and Nearest RT options.
Spectrum Detection
The spectrum detection mode is designed specifically for use in gas
chromatography (GC), where peak widths are typically about 6 seconds and
often significantly less. It can therefore be difficult to define a precise
retention time window for a specific peak. When you use a large retention
time window, it is possible that Xcalibur identifies several peaks within it. In
LC, peaks are significantly wider and the definition of a retention time
window is generally a simple matter.
Spectrum detection relies on your providing a reference spectrum for the
component peak. If this is available, spectrum detection is preferable to
using Highest Peak or Nearest RT modes for GC users.
This topic contains the following subtopics:
50
•
Spectrum Detection Parameters
•
How It Works
•
Using Spectrum Detection
•
Editing the Peak Identification Table
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Processing Setup
Detection
Spectrum Detection Parameters
The reference spectrum can consist of up to 50 ions. There are three
match-criteria thresholds: Forward, Reverse, and Match (see Figure 23).
Figure 23. ICIS Peak Detection area with spectrum detection enabled
Xcalibur uses the reference spectrum to locate the target component within
the chromatogram and uses the thresholds to filter potential candidates.
Normally when using this mode, set the Identification page for an MS
detector type with a single Mass value specified.
How It Works
The Spectrum Detection algorithm involves the following steps:
1. Xcalibur calculates the component’s predicted retention time range,
using the parameters specified on the Identification page to calculate the
expected retention time and window.
2. Xcalibur compares the reference spectrum with the chromatogram and
compares the spectral scans (the number of spectra depends on the scan
speed) across the component’s retention time range with the reference
spectrum and calculates Forward and Reverse match factors.
3. Xcalibur computes the peak detection function. Xcalibur uses the
Forward and Reverse match values, together with the intensity of the
component’s mass, for each spectrum within the retention time range.
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Processing Setup
Detection
4. Xcalibur carries out peak detection and integration on the
chromatogram plot and peak detection function. Using the parameters
on the Detection page, Xcalibur detects peaks in both the component’s
mass chromatogram plot and the peak detection function. If enabled,
smoothing is applied before detection.
5. Xcalibur compares peaks in the two plots and calculates match values.
Xcalibur selects potential candidates for the component peak. When a
peak apex in the peak detection function is within two scans of a peak
apex of the chromatogram plot, the peak is identified as a candidate.
Xcalibur computes a match value for each candidate, taking into
account how close a candidate is to the component’s predicted retention
time and the width of the component’s retention time range.
6. Xcalibur filters the results, discarding any candidate with one or more of
its Forward, Reverse or Match values below the specified threshold
values.
7. Xcalibur selects the top three candidates. It chooses the highest Match
value candidate as the found peak and stores information about any
second- and third-best candidate. View this information in Quan
Browser or in printed reports.
Using Spectrum Detection
To use the Spectrum Detection mode
1. Open the Chromatography Options dialog box by choosing Options >
Chromatography By.
2. Select GC detection mode by clicking the GC option. Click OK.
3. In the Detection page, click the Spectrum option in the Peak Detection
area.
Xcalibur displays the Spectrum detection options. For semi-automated
mass spectral peak entry, Xcalibur discards any ions with intensities
below the Low Intensity Cutoff percentage parameter in the Spectrum
Options dialog box (see Figure 24).
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Processing Setup
Detection
4. To adjust the low intensity cutoff, select Options > Spectrum to open
the Spectrum Options dialog box (see Figure 24).
Figure 24. Spectrum Options dialog box
5. Enter mass/charge [m/z] and intensity data for up to 50 mass spectral
peaks in the Spectrum peak identification table either manually or
semi-automatically using a raw file containing good quality spectral data
of the component.
To enter data manually:
a. Select an m/z table box and enter the value for an ion characteristic
of the component.
b. Select the Intensity percentage table box and enter a value for the
relative intensity of the ion.
c. Repeat this procedure for all the ions in the reference spectrum (up
to a maximum of 50).
To enter data using a raw file:
a. Pin the Spectrum preview.
b. Drag the cursor across the appropriate component peak in the
Chromatogram preview. Xcalibur displays the spectrum from the
scan at the peak apex in the Spectrum preview.
c. Pin the Chromatogram preview and drag the cursor across the
required Spectrum range. The ion m/z and Intensity values are
copied to the peak identification table, overwriting any existing
values.
d. Perform manual adjustments to the peak identification table values,
as described above for manual data input.
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Processing Setup
Detection
6. Select Thresholds for spectrum matching:
a. Use the Forward box to enter a Forward comparison threshold
b. Use the Reverse box to enter a Reverse comparison threshold
c. Use the Match box to enter a Match comparison threshold
7. Click OK to save the settings.
Editing the Peak Identification Table
Use the available shortcut menu to insert, delete, clear, or move rows in the
table.
To insert a row
1. Click the row number above the position.
2. Right-click and select Insert Row from the shortcut menu.
To delete a row or range of rows
1. Click the row number of the row to delete. To delete a range of rows,
drag the cursor to the final row in the range.
2. Right-click and select Delete Rows from the shortcut menu, or press
DELETE.
Ion Ratio Confirmation
Ion Ratio Confirmation is for use only with Highest Peak and Nearest RT
peak detection in Xcalibur’s GC Chromatography mode.
Using Ion Ratio Confirmation, Xcalibur can confirm the identity of a target
peak using qualifier ions. This feature can be useful when several peaks are
present in the retention time window. This situation often occurs in gas
chromatography where narrow peak widths and large numbers of peaks
make it difficult to target a component using a retention window alone.
It is particularly useful in applications using SIM acquisition (rather than
full scan) to achieve high sensitivity for high accuracy quantitative results. In
this case, Spectrum peak detection is not appropriate because limited mass
spectral peak data are available.
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Processing Setup
Detection
Ion Ratio Confirmation Parameters
Use the qualifier ion table (see Figure 25) to enter mass-to-charge [m/z]
values for up to five qualifier ions. For each qualifier ion, provide a Target
Ratio and target ratio tolerance [Window ±%].
Figure 25. Qualifier ion table in the Ion Ratio Confirmation area
How it Works
The Ion Ratio Confirmation algorithm involves the following steps:
1. Xcalibur generates a mass chromatogram for the quantitation mass(es).
Using the parameters you specified in the Trace, Filter and Mass (m/z)
fields, Xcalibur generates a mass chromatogram for the quantitation
mass(es).
2. Xcalibur carries out peak detection. Using the parameters you specified
on the Identification and Detection pages, Xcalibur carries out peak
detection. If no peak is found, the component is flagged as not found
and no ion ratio confirmation is carried out.
3. Xcalibur generates a mass chromatogram for each specified qualifier ion.
Using the same Identification and Detection parameters, Xcalibur
generates a mass chromatogram for each qualifier ion and detects peaks.
When these chromatograms do not feature a peak or if the retention
time of the qualifier ion peak apex lies outside the Qualifier Ion
Coelution window (centered on the quantitation peak), Xcalibur rejects
the quantitation peak and terminates the ion ratio confirmation
procedure.
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Processing Setup
Detection
4. Xcalibur calculates the ratio of each qualifier ion peak to the
quantitation peak.
•
If you are using area response, Xcalibur integrates each qualifier ion
peak to determine its area. It calculates the ratio of the qualifier ion
peak area to the quantitation peak area and compares this ratio with
the specified target ratio. When the calculated ratio is outside of the
target ratio by more than the specified tolerance (Window ±%),
Xcalibur rejects the quantitation peak and sets the IRC Flag for the
quantitation peak to false.
•
If you are using Height response, Xcalibur calculates the ratio of the
qualifier ion peak height to the target peak height. Xcalibur
compares this ratio with the specified target ratio. If the calculated
ratio is outside the target ratio by more than the specified tolerance
(Window ±%), Xcalibur rejects the quantitation peak and sets the
IRC Flag for the quantitation peak to false.
Xcalibur repeats these four steps for each qualifier ion. All qualifier ratios
must be within the target ratio tolerances for the IRC Flag to be set to true.
Using Ion Ratio Confirmation
1. Open the Chromatography Options dialog box by choosing Options >
Chromatography By.
2. Click the GC option and click OK.
3. In the Detection page, click the Highest Peak or Nearest RT option as
required.
4. Select the Ion Ratio Confirmation check box.
5. Enter details of the qualifier ions for the current component:
a. Select an m/z box and type the value for an ion characteristic of the
component.
b. Select the Target Ratio% box and type a value for the Target ratio.
c. Select the Window ±% box and enter a value for the relative
intensity tolerance applied to the Target Ratio percentage.
d. Repeat this procedure for all the ions (up to a maximum of 5).
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Detection
6. Select a Window% calculation option:
•
Click the Absolute option to use the target ratio tolerances in the
Window ±% column as absolute percentages of the target ratio.
•
Click the Relative option to use the target ratio tolerances in the
Window ±% column as relative percentages of the target ratio.
7. Enter a value for the Qualifier Ion Coelution window in minutes.
When the retention time of any qualifier ion peak apex lies outside the
Qualifier Ion Coelution window (centered on the quantitation peak),
Xcalibur rejects the quantitation peak. Quantitation peaks with
matching qualifier ion peaks (within the Coelution window) are tested
by Xcalibur for ion ratio confirmation according to the selected method.
8. Click OK to save the settings.
Editing the Qualifier Ion Table
A shortcut menu is available for you to insert or delete rows in the table.
To insert a row
1. Click the row number above the position.
2. Right-click and select Insert Row from the shortcut menu.
To delete a row or range of rows
3. Click the row number of the row to delete. To delete a range of rows,
drag the cursor to the final row in the range.
4. Right-click and select Delete Row from the shortcut menu, or press
DELETE.
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Processing Setup
Detection
Advanced Detection
Parameters
Xcalibur’s default options provide suitable chromatographic peak detection
for most applications. In certain circumstances, you might need to change
some of these parameters. Show advanced options by clicking Advanced on
the Detection page.
This topic contains the following subtopics:
ICIS Advanced Parameters
•
ICIS Advanced Parameters
•
Avalon Event List
Xcalibur makes ICIS Advanced Parameters available if you are using the
ICIS peak detection algorithm. Click Advanced on the Detection page to
open the ICIS Advanced Parameters dialog box (see Figure 26).
Figure 26. ICIS Advanced Parameters dialog box
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Processing Setup
Detection
Use this dialog box to select the following detection criteria for
chromatographic peaks:
Note The default values are suitable for most analysis requirements.
Change these settings only if standard chromatogram detection and
integration options do not provide the desired result.
Noise Method
Xcalibur uses this advanced parameter to determine how the noise level of
the data is determined by the ICIS peak detection algorithm.
Use the INCOS Noise option to use a single pass algorithm to determine
the noise level. This is the default noise method.
Use the Repetitive Noise option to use a multiple pass algorithm to
determine the noise level. In general, this algorithm is more accurate in
analyzing the noise than the INCOS Noise method.
Use the RMS noise option to use a root mean squared (RMS) algorithm to
determine the noise level. By default, Xcalibur uses Peak To Peak for the
noise calculation. Xcalibur automatically selects RMS if you determine the
noise region manually.
Peak Parameters
The Xcalibur ICIS peak detection algorithm uses the following advanced
peak detection parameters.
Use the Min Peak Width box to enter the minimum number of scans in a
peak. The valid range is 0 to 100 scans. The default value is 3 scans.
Use the Multiplet Resolution box to enter the minimum separation in scans
between the apexes of two potential peaks. This is a criterion to determine if
two peaks are resolved. The valid range is 1 to 500 scans. The default value
is 10 scans.
Use the Area Tail Extension box to enter the number of scans past the peak
endpoint to use in averaging the intensity. The valid range is 0 to 100 scans.
The default value is 5 scans.
Use the Area Scan Ratio box to enter the number of scans on each side of
the peak apex to be allowed. The valid range is 0 to 100 scans. The default
value of 0 scans specifies that all scans from peak start to peak end are to be
included in the area integration.
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Processing Setup
Detection
Avalon Event List
When using the Avalon peak integration algorithm, click Advanced on the
Detection page to open the Avalon Event List dialog box shown in
Figure 27. Use this dialog box to add additional integration events to the
events list and edit the values of the integration events.
To add an event to the list, select an event from the Event list in the
Advanced Events List dialog box. Type values for the event in the Time and
Value boxes. Click Add.
To edit the time and value of an event, highlight the event in the Event list
in the Advanced Events List dialog box. Type new values for the selected
event in the Time box and the Value box. Click Change to automatically
update the event list both here and on the Detection page and to
automatically update the chromatogram display.
To delete an event, highlight the event in the Event list in the Advanced
Events List dialog box. Click Delete to automatically update the event list
both here and on the Detection page and to automatically update the
chromatogram display. You cannot delete events for Initial Value
timepoints.
Figure 27. Avalon Events List dialog box
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Data Flags
Processing Setup
Detection
Use the Data Flags dialog box, shown in Figure 28, to set flags for peak area
and peak height thresholds. Flags are recorded as true or false in the result
file. If you set a value to zero, the flag is always false. Flags are reported in
Quan Browser and in printed or exported Reports.
Figure 28. Data Flags dialog box
To open the Data Flags dialog box, click Flags on the Detection page. Flags
are reported as true if they exceed these threshold values:
Use the Area Threshold box to enter a value for the Area Threshold Data
flag. This is an absolute value of peak area (counts).
Use the Height Threshold box to enter a value for the Height Threshold
Data flag. This is an absolute value of peak height (counts).
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Processing Setup
Detection
Peak Identification
Options
Choose the Options > Identification menu command to open the
Identification Options dialog box shown in Figure 29. This dialog box
contains the parameters used by Xcalibur to estimate baseline noise and to
correct retention time assignments for the void volume of the
chromatographic column.
Figure 29. Identification Options dialog box
Void Time
Use the Void Time area to obtain corrected retention times for each peak.
Void time is the time taken by a non-retained compound to elute from the
column.
To obtain the corrected retention time for each peak either:
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•
Click the Value (min) option and enter a value for the void time (this is
subtracted from the elution time for all recorded peaks), or
•
Click the First Peak option and set the void time to that of the first
detected peak. Xcalibur subtracts this time from the elution time for all
remaining peaks.
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Baseline
Processing Setup
Detection
Xcalibur calculates baseline noise in an iterative process using the filtered
and smoothed mass chromatogram. The noise calculation process draws a
line through the baseline composed of a number of points with a noise ratio
that is less than a specified tolerance.
Xcalibur uses the calculated baseline noise value throughout the peak
characterization process to determine whether or not baseline adjusted
intensities or heights of measurements are significant. The value is judged
significant if it is greater than the product of noise and S/N threshold
(Genesis Detection page only). Likewise, when values are less than this
product, they are considered baseline values.
The parameters defining this process are:
Baseline and Noise Window
Xcalibur applies this parameter to each peak and calculates the baseline and
baseline noise within this window (valid range 0.1 to 1000). To ensure an
accurate noise calculation, enter a value that includes the base width of the
peak and an appreciable amount of baseline. If the window is too small, the
baseline is positioned up the sides of the peak.
Baseline Noise Tolerance
This parameter controls how the baseline is drawn in the noise data. The
higher the baseline noise tolerance value, the higher the baseline is drawn
through the noise data. The valid range is 0.0 to 100.0.
Minimum Number of Scans in Baseline
This parameter defines the minimum number of scans that Xcalibur uses in
the baseline calculation. A larger number includes more data in determining
an averaged baseline. The valid range is 2 to 100.0.
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Processing Setup
Calibration
Calibration
Use the Calibration page to assign either target compound type or ISTD
type to each of the components defined on the Identification page:
When you select Target compound, the Target Compounds area is enabled.
Use this area to:
•
Assign an ISTD to the compound
•
Carry out an Isotope Contribution Correction
•
Select a calibration curve type
•
Select the response type
•
Specify units
When you select ISTD:
•
The ISTD area becomes active
•
The Target Compounds area is grayed
•
The Levels page becomes unavailable
•
In the ISTD area, you provide the amount and units for the ISTD
component.
ISTD options are available only if you have selected the Internal Standard
option in the Calibration Options dialog box (see Figure 30). This is the
default option for Xcalibur.
Figure 30. Calibration Options dialog box
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Processing Setup
Calibration
To change the calibration mode
1. Choose Options > Calibration By.
2. In the Calibration Options dialog box, click either the Internal
Standard option or the External Standard option as required.
3. Click OK to save the setting.
Note
1. When you click External Standard, any ISTD components in the
active processing method are converted to Target Compounds. The
ISTD option and area are grayed. The Amount and Unit
information is lost.
2. When you click Internal Standard, Xcalibur prompts you to assign at
least one of the components as an ISTD when saving the processing
method.
Assigning an ISTD
To define a component as an ISTD
1. Click a component in the Components list located at the far right of the
Processing Setup window. If this list is not visible, choose View >
Components list.
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Processing Setup
Calibration
2. Click the ISTD option in the Component type area to enable the ISTD
area (see Figure 31).
Figure 31. Calibration page for an ISTD component type
3. Type a value in the Amount box to specify the amount of the internal
standard into each sample.
4. Type a label in the Units box to specify the units for the value of the
internal standard entered in step 3 above. This label can be displayed in
a report. It is not used to perform any unit conversions.
Assigning a Target
To define a Target Compound
Note When creating an internal standard method, define at least one
component to be an ISTD before defining any other components as
target compounds.
1. Click a component in the Components list located at the far right of the
Processing Setup window. If this is not visible, choose View >
Components list.
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Calibration
2. Click the Target Compound option in the Component type area
(Figure 32). The Target Compounds area becomes active.
Figure 32. Calibration page for a Target Compound type.
3. Select an Internal Standard (ISTD) for the Target from those listed in
the ISTD combo box.
4. To make calibration corrections for isotope contributions of the internal
standard to the target compound or the target compound to the internal
standard, click Isotope%. This opens the Correction For Isotope
Contribution dialog box shown in Figure 33.
Note Check that the values in the Correction For Isotope
Contribution dialog box are set to zero if the processing method
does not require isotope contribution correction.
5. Select a Calibration Curve type from those listed in the Calibration
Curve combo box. The available options are defined in Table 7.
6. When you have selected a Linear or Quadratic calibration curve, select
one of the calibration point Weighting options: Equal, 1/X, 1/X2, 1/Y,
1/Y2, or 1/s2. Xcalibur applies the weighting when it calculates the
least-squares regression calibration curve. See the Xcalibur online Help
for more details.
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Processing Setup
Calibration
Table 7.
Available calibration curve types
Curve Type
Definition
Linear
A linear polynomial curve of the following mathematical form:
Y = mX + B, where m is the slope of the curve and B is the intercept
point on the Y-axis.
Quadratic
A quadratic polynomial curve of the following mathematical form:
Y = AX2 + BX + C, where A, B, and C are the polynomial coefficients.
Linear Log-Log
A linear polynomial curve of the following mathematical form:
log10[Y] = m log10[X] + B, where m is the slope of the curve and B is
the intercept point on the Y-axis.
Quadratic Log-Log
A quadratic polynomial curve of the following mathematical form:
log [Y] = log [X2] + B log [X] + C, where A, B, and C are the
polynomial coefficients.
Average RF
A calibration curve where the slope of the calibration curve is
constructed from the average response factor of all levels. This
calibration curve always passes through the origin.
Point-to-Point
A curve where straight lines are drawn between averaged replicate
data at each calibration level.
Cubic Spline
A calibration curve where a cubic polynomial curve is fitted between
each pair of calibration levels such that the slopes of the separate
cubic polynomial curves match at common calibration curve points.
Locally Weighted
A calibration curve that is constructed from individual line segments.
At multiple points across the calibration region, a weighted linear
regression is performed. The point slopes are then connected to
form a continuous line. At any point on the curve, the calculated
amount is based on the nearest weighted linear regression.
7. When you have selected a Linear, Quadratic, Point-to-Point or Cubic
Spline curve type, select how to treat the origin in the calibration curve
calculation by selecting one of the following:
•
Click the Ignore option to ignore the origin in the calibration curve
calculation.
•
Click the Force option to force the calibration curve through the
origin.
•
Click the Include option to include the origin as an extra data
point. This option is not available for the Point-to-Point or Cubic
Spline curve types.
8. Type a label in the Units box for the selected component. This unit label
appears on graphs and reports.
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Calibration
9. Select the peak response method:
•
Click the Area option to quantitate based upon the integrated area
of component peaks.
•
Click the Height option to quantitate based upon the calculated
height of component peaks.
Repeat this procedure for all the components in the active method to define
as target compounds.
Isotope Correction
Use the Correction For Isotope Contribution dialog box shown in Figure 33
to correct for an impurity in the internal standard compound that elutes at
the same time as the target compound or an impurity in the target
compound that elutes at the same time as the internal standard, or both.
Figure 33. Correction For Isotope Contribution dialog box
Access the dialog box by clicking Isotope% in the Target Compounds area
on the Detection page. This is only available for components defined as
Target Compounds.
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Processing Setup
Calibration
The Correction for Isotope Contribution dialog box contains the following
options:
Contribution of ISTD to Target
Compound
To make a correction to the target compound arising from a contribution
due to the ISTD
Note Check that the values in the Correction For Isotope Contribution
dialog box are set to zero if you do not require isotope contribution
correction.
1. Analyze the ISTD reagent using the processing method to be used for
quantitation of the target compound. Use the respective peak areas or
heights to determine the following ratio:
ISTDimpurity/ISTDpure
Where:
ISTDimpurity is the response due to the impurity compound in the
internal standard reagent that elutes at the same time as the target
compound.
ISTDpure is the response of the pure internal standard compound.
2. Enter this value in the Contribution of ISTD to Target Compound (%)
text box. Xcalibur uses this ratio as the value x in the following impurity
correction expressions:
ISTDcorr = [ISTDobs - y TCobs]/[1-yx]
TCcorr = [TCobs - x ISTDobs]/[1-yx]
Where:
ISTDcorr is the corrected amount of internal standard.
ISTDobs is the apparent amount of ISTD, as measured by Xcalibur at
the retention time for the ISTD. This peak consists of ISTDcorr +
TCimpurity.
TCcorr is the corrected amount of the target compound.
TCobs is the apparent amount of target compound, as measured by
Xcalibur at the retention time for the target compound. This amount
consists of
TCcorr + ISTDimpurity.
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Contribution of Target Compound
to ISTD
Processing Setup
Calibration
To make a correction to the Target Compound arising from a contribution
due to the ISTD
1. Analyze the target compound using the processing method to be used
for quantitation of the target compound without the ISTD present. Use
the respective peak areas or heights to determine the following ratio:
TCimpurity/TCpure
Where:
TCimpurity is the response due to the impurity compound in the target
compound that elutes at the same time as the ISTD.
TCpure is the response of the pure target compound.
2. Enter this value in the Contribution of Target Compound to ISTD (%)
text box.
Xcalibur uses this ratio as the value y in the following impurity
correction expressions:
ISTDcorr = [ISTDobs - y TCobs]/[1-yx]
TCcorr = [TCobs - x ISTDobs]/[1-yx]
Where:
ISTDcorr is the corrected amount of internal standard.
ISTDobs is the apparent amount of ISTD, as measured by Xcalibur at
the retention time for the ISTD. This peak consists of ISTDcorr +
TCimpurity.
TCcorr is the corrected amount of the target compound.
TCobs is the apparent amount of target compound, as measured by
Xcalibur at the retention time for the target compound. This amount
consists of TCcorr + ISTD impurity.
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Processing Setup
Calibration
Setting Calibration and
Quantitation Flags
Use the Calibration page to set limits for the calibration and quantitation
flags. Xcalibur reports these flags in result files, printed reports, and Quan
Browser. To set limits for the flags, click Flags to open the Calibration and
Quantitation Flags dialog box (see Figure 34).
Figure 34. Calibration And Quantitation Flags dialog box
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Processing Setup
Calibration
Calibration Flag
Use the R-Squared box to enter a threshold to test the goodness of fit of the
calibration curve. Xcalibur calculates a coefficient of determination
(R-squared) whenever it computes a calibration curve. When the value is
less than the R-squared threshold, the R-squared flag in the result file is set
to true; otherwise it is set to false.
Quantitation Flags
The Quantitation Flags area contains the following limit boxes:
Thermo Electron Corporation
•
Use the Detection Limit box to enter a threshold for the limit of
detection. When the quantified component concentration is less than
the Detection Limit threshold, the Detection Limit flag in the result file
is set to true; otherwise it is set to false.
•
Use the Linearity Limit box to enter a threshold for the linearity limit.
When the quantified component concentration is greater than the
Linearity Limit threshold, the Linearity Limit flag in the result file is set
to true; otherwise it is set to false.
•
Use the Quantitation Limit box to specify a flag threshold for the limit
of quantitation. When the quantified component concentration is less
than the Quantitation Limit threshold, the Quantitation Limit flag in
the result file is set to true; otherwise it is set to false.
•
Use the Carry Over Limit box to enter a threshold for the carryover
limit. When the quantified component concentration is greater than the
Carryover Limit threshold, the Carryover Limit flag in the result file is
set to true; otherwise it is set to false.
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Processing Setup
Levels
Levels
Use the Levels page to define the concentrations of target compounds in the
calibration standard samples (see Figure 35). A Level is a text label for each
of the defined amounts. After setting up a sequence, Cal Levels are
associated with calibration standard samples and QC Levels are associated
with QC samples.
The Levels page is not available for components defined as ISTDs in
the processing method since they are spiked into samples at a fixed
amount as specified by the Amount box of the ISTD area on
the Calibration page.
Figure 35. Levels page
Enter the concentrations of the calibration standards manually or use the
semi-automated standard dilutions option.
This section contains the following topics:
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•
Entering the Calibration or the QC Levels
•
Using the Standard Dilution Option
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Entering the Calibration or
the QC Levels
Processing Setup
Levels
To enter the calibration levels or the QC levels
1. Click a target component in the Components list located at the far right
of the Processing Setup window. The Levels page is not available for
ISTD components.
2. Enter information about calibration standards into the Calibration
Levels table:
•
Type appropriate labels, such as standard 1, standard 2, and so on, in
the Cal Level boxes.
•
Type a value for the concentration of the standard sample in the
Amount boxes.
Also, use the Standard Dilution dialog box (see Figure 36) to set
calibration levels for all the target components. See the next topic:
Using the Standard Dilution Option.
3. Enter QC Level data. Type the following information in the QC Level
table:
•
Use the QC Level boxes to enter QC level labels.
•
Use the Amount boxes to enter the amount of the component added
at each level (in a QC sample).
•
Use the QC% Test boxes to enter the percent tested at each QC
level. Xcalibur measures the quantity of the QC component in the
same manner as unknown components. The measured quantity is
then compared with a user defined expected quantity and a user
defined percent test.
4. Repeat the procedure for all target components.
5. Click OK to save the settings.
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Processing Setup
Levels
Using the Standard
Dilution Option
Use the Standard Dilution dialog box shown in Figure 36 to enter
calibration level information for all target components simultaneously. At
the top of the dialog, the Target Compound Components readback line
displays the total number of target components defined in the processing
method out of the total number of all components in the method.
The Selected Components readback line displays the selected number of
non-ISTD components for Standard Dilution out of the total number of all
components in the method.
To use the Standard Dilution dialog box to enter Calibration Levels
information for all the target components
1. From the Levels page, choose Options > Standard Dilution to open
the Standard Dilutions dialog box.
Figure 36. Standard Dilution dialog box
2. Use the Amount boxes in the Base Amounts table to enter the base
amount for each component. Provide a value for each listed component.
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Processing Setup
Levels
3. Enter information in the Dilution Factors table:
•
Use the Cal Level boxes to enter up to 50 calibration levels. To enter
a calibration level, type the new name in the appropriate Cal Level
box (32 characters maximum). To delete a Cal Level row, click the
numbered tile to the left of the row. Xcalibur highlights the row.
Press DELETE.
•
Use the Dilution boxes to enter the stock dilution factors for each
calibration level. To enter a dilution factor, type the value in the
appropriate Dilution box. The value must be greater than
0.00000001 and less than or equal to 1.
4. Click OK to save the new settings and close the dialog box.
Xcalibur calculates the calibration levels for all the target compound
components defined in the method using the parameters from the Standard
Dilution dialog box. It does this by:
•
Transferring the Cal Level values to the Cal Level column of the
Calibration Levels table for each component.
•
Multiplying the Dilution factor with the Base Amount value. The result
is transferred to the corresponding Amount box in the Calibration
Levels table for the component.
Xcalibur repeats this procedure for all Calibration Levels and all
components.
Note Xcalibur doesn’t update the Standard Dilution dialog box with any
changes made directly on the Levels page. When you use the dialog box
to set up a method, use it to modify the method subsequently. Any
manual changes made to the Levels page are lost if you subsequently use
the Standard Dilution dialog box.
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Processing Setup
System Suitability
System Suitability
Use the System Suitability page, shown in Figure 37, to enable a number of
automated chromatographic checks that assign a pass or fail qualification to
a target peak. These checks are based on an analysis of the quantitation peak
and, if ion ratio confirmation is enabled, all qualifier ion peaks within the
retention time window. Warning flags are reported in Sample and Summary
report, and in Quan Browser.
Figure 37. Processing Setup - Quan view - System Suitability page
The tests are divided into three groups:
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Resolution parameters
•
Symmetry parameters
•
Peak classification parameters
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Processing Setup
System Suitability
Flagging is used to:
•
Fail a compound and therefore a sample
•
Monitor trends occurring in successive sample injections
Table 8 summarizes the system suitability flags associated with the system
suitability parameters. Use any of the three System Suitability test groups by
selecting the Enable check box associated with it.
Table 8.
Resolution
System Suitability flags
Parameter
Flag
Meaning
Resolution
R
Target peak(s) fail to meet the resolution threshold
Symmetry
S
Target peak(s) fail to meet the symmetry threshold
Peak width
W
Peak width is greater than the maximum allowed
peak width
Tailing
T
Target peak(s) exceed the tailing failure threshold
Column overload
O
Target peak(s) exceed the specified asymmetry
factor at the specified peak height
Baseline clipping
B
Target peak(s) fail the baseline-clipping test
Minimum signal to noise ratio
N
Target peak(s) fail the minimum signal-to-noise
ratio test
The resolution test uses a single parameter, the resolution threshold.
Xcalibur uses this parameter to check the resolution of quantitation peaks
against a threshold value. Resolution testing is based on a comparison of the
peak height with the adjacent valley height within the quantitation window.
Resolution threshold is defined as the ratio:
100 × V/P
Where:
V is the horizontal asymptote extended from the target peak’s apex to the
lowest point in the valley between the target peak and a neighboring peak.
P is the height of the target peak.
The default value for the resolution threshold is 50% and the valid range is
0 to 100%. Compounds are flagged (R) if any of the target peaks fail to
meet the resolution threshold.
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Processing Setup
System Suitability
Symmetry
Use the Symmetry settings to specify system suitability checks for the
symmetry of quantitation peaks. Symmetry is determined at a specified peak
height and is a measure of how even-sided (symmetrical) a peak is about a
perpendicular dropped from its apex.
The test uses two parameters:
•
Use the Peak Height box to specify the Peak Height % at which
Xcalibur measures the symmetry of target peaks. Enter any value within
the range 1% to 100%.
•
Use the Symmetry Threshold box to specify the Symmetry Threshold
value. Enter any value within the range of 0% to 100%. The default
value is 90% at 50% peak height. Xcalibur determines symmetry at the
peak height specified in the Peak Height % box.
A realistic practical tolerance for capillary GC data might be 70% at 50%
peak height.
For the purposes of the test, a peak is considered symmetrical if:
(Lesser of L and R) × 100 / (Greater of L and R) > Symmetry Threshold%
Where:
L is the distance from the left side of the peak to the perpendicular dropped
from the peak apex, measured at Peak Height % of the peak height.
R is the distance from the right side of the peak to the perpendicular
dropped from the peak apex, measured at Peak Height % of the peak height.
Measurements of L and R are taken from the raw file without smoothing.
Compounds are flagged (S) if any of the target peaks fail to meet the
symmetry threshold.
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Peak Classification
Detect Peak Width
Processing Setup
System Suitability
Use the Peak Classification Parameters area to specify parameters for five
classification checks:
The settings in the Detect Peak Width area allow you to specify system
suitability checks for the width of quantitation peaks.
The peak width test uses three parameters:
Detect Tailing
•
Use the Peak Height % box to specify the Peak Height % at which
Xcalibur tests the width of target peaks. Enter any value within the
range 0% to 100%. The default value is 50%.
•
Use the Min Peak Width box to specify the minimum peak width, at the
specified peak height, for the peak width suitability test. The default
value is 1.8. Set any value in the range 0 to 30 seconds. Compounds are
flagged (W) if the peak width is less than the minimum peak width.
•
Use the Max Peak Width box to specify the maximum peak width, at
the specified peak height, for the peak width suitability test. The default
value is 3.6. Set any value in the range 0 to 30 seconds. Compounds are
flagged (W) if the peak width is greater than the maximum peak width.
Use the Detect Tailing area to specify system suitability checks for the tailing
of peaks.
The test uses two parameters:
•
Use the Peak Height box to specify the Peak Height % to measure the
tailing of target peaks. Enter any value within the range 1% to 100%.
•
Use the Failure Threshold box to specify the failure threshold for the
tailing test. The valid range is 1 to 100.
Tailing is calculated at the value defined in the Peak Height % box. For the
purposes of the test, a peak is considered to be excessively tailed if:
R / L > Failure Threshold %
Where:
L is the distance from the left side of the peak to the perpendicular
dropped from the peak apex, measured at Peak Height % of the peak
height.
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Processing Setup
System Suitability
R is the distance from the right side of the peak to the perpendicular
dropped from the peak apex, measured at Peak Height % of the peak
height.
Xcalibur takes measurements of L and R from the raw file without
smoothing and flags compounds (T) if any of the target peaks exceed the
tailing failure threshold.
Detect Column Overload
Use the Detect Column Overload area to specify system suitability checks
for column overloading.
The test uses two parameters:
•
Use the Peak Height box to specify the Peak Height % to measure
column overloading. Enter any value within the range 1% to 100%.
•
Use the Failure Threshold box to specify the failure threshold value for
the column overload test. The valid range is 1 to 100.
A peak is considered to be overloaded if:
L / R > Failure Threshold %
Where:
L is the distance from the left side of the peak to the perpendicular
dropped from the peak apex, measured at Peak Height % of the
peak height.
R is the distance from the right side of the peak to the perpendicular
dropped from the peak apex, measured at Peak Height % of the
peak height.
Xcalibur takes measurements of L and R from the raw file without
smoothing and flags compounds (O) if any of the target peaks exceed the
failure threshold.
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Detect Baseline Clipping
Processing Setup
System Suitability
This test uses the Number of peak widths for noise detection parameter to
check quantitation peaks for baseline clipping.
Xcalibur considers a peak to be baseline clipped if there is no signal (zero
intensity) on either side of the peak within the number of peak widths
specified in the Number Of Peak Widths For Noise Detection box. The
default value is 1.0 and the permitted range is 0 to 100. The range is
truncated to the quantitation window if the specified number of peak
widths extends beyond the window’s edge.
Xcalibur flags compounds (B) if any of the target peaks fail the
baseline-clipping test. Baseline clipping is often indicative of problems with
the MS detector and associated electronics.
Detect Minimum Signal-to-Noise
Ratio
Use the Signal to Noise Ratio box to specify the minimum allowable
signal-to-noise ratio for a peak within the quantitation window. The default
value is 3 and the permitted range is 0 to 100. Xcalibur calculates the
signal-to-noise ratio within the quantitation window using only the baseline
signal. Any extraneous, minor, detected peaks are excluded from the
calculation.
Xcalibur flags compounds (N) if any of the target peaks fail the
signal-to-noise test.
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Processing Setup
Peak Purity
Peak Purity
Use the Peak Purity page, shown in Figure 38, to enable or disable peak
purity parameters for PDA chromatograms in the active cell. Unresolved
peaks in data generated from a PDA (UV) detector can indicate the presence
of analyte impurities. The Peak Purity computation detects changes in peak
shape during a run, assesses peak purity from the changes in the shape of a
peak and computes a correlation factor from the collected data. The
correlation factor is a measure of the purity of the scan at the apex of a single
chromatogram peak, when compared with the scans at other times within
the same peak.
Figure 38. Peak Purity page in the Quan view
The peak purity feature is active in Processing Setup or Qual Browser only
when both of the following conditions are true:
•
A raw data file for a PDA analysis is open
•
PDA is selected from the Detector Type list in the Quan view of
Processing Setup or PDA is selected from the Detector list in the
Chromatogram Ranges dialog box of Qual Browser
Determine suitable peak purity parameters for raw data by processing the
raw file in Qual Browser; Xcalibur displays the correlation factor in the
active chromatogram view of Qual Browser. Include this correlation factor
in a processing method by using the Quan view or the Qual view of
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Processing Setup
Peak Purity
Processing Setup. Finally, produce a Peak Purity report by using the Reports
view of Processing Setup when you include the correlation factor in a
processing method.
Enable Peak Purity
Use the Enable check box to enable or disable Peak Purity parameters for the
PDA chromatograms in the active cell. To enable Peak Purity parameters
and calculate Peak Purity results, select the Enable check box. Peak
detection occurs automatically prior to the peak purity calculation.
Scan Threshold
Use the Scan Threshold box to specify a minimum value of intensity for
wavelength scans in milliabsorbance units (mAU). A Peak Purity
computation using scan threshold starts with a scan at the apex of the peak
and collects wavelength data from scans on both sides of the apex until the
scan threshold is reached.
Use scan threshold for either symmetrical or asymmetrical peaks. The
default value for scan threshold is 3 mAU. The range of possible values
is 0 to 1000 mAU (or 1 AU). In a sample with high background or noise,
you might start with a value for scan threshold of 40 mAU.
Peak Coverage
Use the Peak Coverage box to specify a maximum percent value of the width
of the integrated peak. A Peak Purity computation using peak coverage starts
with the scan at the apex of the peak and collects wavelength data from scans
on both sides of the apex until the percent peak coverage is reached. Use
peak coverage for symmetrical peaks. The default value for peak coverage is
95% of the integrated peak width.
Limit Scan Wavelength
Use the Limit Scan Wavelength check box to enable or disable the
wavelength range box. Select the check box to limit the number of
wavelengths to include in the Peak Purity computation and enter a range in
the Wavelength Range box.
Use the Wavelength Range box to specify a range of UV scans (in
nanometers) that includes the wavelengths of the peak(s) of interest. A Peak
Purity computation using wavelength range starts with the scan at the apex
of a peak and collects wavelength data from scans on both sides of the apex
until all the wavelengths in the range are included. Use wavelength range for
either symmetrical or asymmetrical peaks. The default wavelength range is
the full width of the scan.
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Processing Setup
Reports
Reports
Xcalibur’s automated reporting creates comprehensive, high quality, printed
documentation. The XReports reporting package uses Microsoft Word to
create custom report templates, using a palette of Report Objects for
insertion at any point in a page. From XReports, customize reports to suit
personal requirements. Reports are specified in the Reports view (see
Figure 39).
The Reports view of Processing Setup lists:
•
Sample Reports for each sample.
•
Summary Reports for a sequence of bracketed samples or non-bracketed
samples.
Xcalibur is equipped with a number of standard templates.
Figure 39. Processing Setup - Reports view
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Sample Reports
Processing Setup
Reports
The Sample Reports list consists of the seven columns described in Table 9.
Table 9.
Description of Sample Report columns
Column Heading
Use
Enable
Use this check box to enable or disable a report.
STD
Use this check box to produce a report for a Standard sample type.
QC
Use this check box to produce a report for a QC sample type.
Unk
Use this check box to produce a report for an Unknown sample type.
Other
Use this check box to produce a report for all other sample types.
Save As
Use this check box to select an export option for the report. Xcalibur
saves the exported file with the sample file name and the appropriate
extension in the Data folder where result files are stored. The valid file
types are:
• None - print only, no exported file
• Text - ASCII text file (*.txt)
• Doc - Word XP file (*.doc)
• HTML file - HTML (*.html)
Report Template
Displays the full path name of the template to be used by Xcalibur in
the generation of the sample report.
Specify a Report Template Name in three ways:
•
Click the cell and type the full path and filename.
•
Double-click the cell and browse to the file.
•
Click the cell first. Then right-click the cell and select Browse from the
shortcut menu.
To change any of the report sample type fields (Enable, Std, QC, Unk, or
Other), click the appropriate cell to display a check box. Enable or disable
the option as required.
A shortcut menu is available within the grid. Right-click within a row to
access additional commands to:
Thermo Electron Corporation
•
Open XReports to edit a report template
•
Delete the selected row or rows
•
Insert a row above the selected row or rows
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Processing Setup
Reports
Summary Reports
The summary reports list consists of the three columns described in
Table 10. Edit cells and rows in the same manner as described above in
“Sample Reports” on page 87.
Table 10. Description of Summary Report columns
Column Heading
Use
Enable
Use this check box to enable or disable a report.
Save As
Use this check box to select an export option for the report. Xcalibur
saves the exported file with the sample file name and the appropriate
extension in the Data folder where result files are stored. The valid
file types are:
• None - print only, no exported file
• Text - ASCII text file (*.txt)
• Doc - Word XP file (*.doc)
• HTML file - HTML (*.html)
Report Template
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Displays the full path name of the template to be used by Xcalibur in
the generation of the sample report.
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Programs
Processing Setup
Programs
Use the Programs view of Processing Setup, shown in Figure 40, to list
programs or macros to be run by Xcalibur after the analysis of a sample and
the processing of the resulting data. Xcalibur runs the programs in the listed
order.
Figure 40. The Programs view in Processing Setup
Programs are defined by the nine headings described in Table 11.
Table 11. Program Columns
Thermo Electron Corporation
Column
Use
Enable
Determines whether Xcalibur runs the specified program during
post-processing.
Std
Determines whether Xcalibur runs a program after a Standard sample
analysis.
QC
Determines whether Xcalibur runs a program after a QC sample
analysis.
Unk
Determines whether Xcalibur runs the program after an Unknown
sample analysis.
Other
Determines whether Xcalibur runs the program after any other type of
sample analysis.
Action
Displays Run Program or Run Microsoft Excel Macro options.
Program or Macro
Name
Lists the full pathname of the program or Excel macro
Sync
Determines whether the selected program is to be run synchronously.
When you select Yes, Xcalibur waits for the program to terminate
before starting any other processing task. When you select No,
Xcalibur continues with other processing tasks without waiting for the
program to terminate.
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Processing Setup
Programs
Table 11. Program Columns, continued
Column
Use
Parameters
Specifies any command parameters for the selected program. Use the
following macro parameters in the Parameters column.
To specify a Program or Macro
1. Click the Enable field of the appropriate row. Do one of the following:
•
Click the Program or Macro Name cell and type the full path name.
•
Double-click the cell to identify the program using a standard
Browse dialog box.
•
Click the cell first. Right-click it and choose Browse from the
shortcut menu.
2. To change any of the program sample type fields (Std, QC, Unk, or
Other), click the appropriate cell to access a check box: select or deselect
the option as required.
A shortcut menu is available within the grid. Right-click within a row to
access additional commands to:
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•
Browse to a program or macro file (enabled only when a Program or
Macro Name cell has been selected).
•
Delete the selected row or rows.
•
Insert a row above the selected row or rows.
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Chapter 3
Automating Analysis
This chapter describes how to create a sequence for automated
instrument control and data acquisition or data reprocessing from the
Sequence Setup view. In Xcalibur, a sequence is a sequential list containing a
variety of sample types. Each row of the list corresponds to one sample
injection.
Use the Sequence Setup view to do the following:
•
Create a sequence for the automated acquisition of one or more samples
•
Run a sequence containing one or more samples
•
Reprocess a batch of previously acquired raw data files
Use the Sequence Setup - Acquisition Queue page to control and prioritize
sequences. Each time you start a processing action in Sequence Setup,
Xcalibur also starts a process queue service in the background. When
Xcalibur finishes an acquisition, it sends the data to the process queue for
processing. Xcalibur processes samples and sequences using a first-in
first-out queue priority.
This chapter specifically describes how to set up and use a sequence for
automated quantitative analysis and contains the following sections:
Thermo Electron Corporation
•
The Sequence Setup View
•
About Sequences
•
Creating a New Sequence
•
Modifying a Sequence
•
Running Samples
•
Reprocessing Samples
•
The Acquisition Queue
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The Sequence Setup View
The Sequence Setup
View
Sequence Setup is one of the view options on Xcalibur’s Home Page. To
select Sequence Setup, do one of the following:
•
Choose View > Sequence Setup View from Home Page
•
Click the Sequence Setup button on the Roadmap view.
Sequence Setup - Home Page has four toolbars:
•
Use tools on the View toolbar to select the Home Page views (see
Table 12).
•
Use tools on the Sequence Editor toolbar to edit sequences in the
Sequence Setup view (see Table 13).
•
Use tools on the Roadmap toolbar to control acquisitions and to
access Instrument Setup and Processing Setup.
•
Use tools on the Plot toolbar to control real time plots during data
acquisition.
Display or hide a toolbar by choosing the appropriate View menu
command. Within Sequence Setup, it is useful to display the View and
Sequence Editor toolbars.
Table 12. View Toolbar
Button
Purpose
Use the Home Page view button to open the Home page view.
Use the Sequence view button to open the Sequence template.
Use the Real Time Plot view button to view chromatogram and spectrum data
for the current sample during a real time run.
Use the Information view button to monitor Run Manager status, Xcalibur
components status, and the acquisition queue.
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The Sequence Setup View
Table 13. Sequence Toolbar (partial list)
Button
Purpose
Use the Fill Down button to open the Fill Down dialog box to copy sequence
information from one selected sample row to one or more other sample rows.
Use the Browse File Name button to select a file. Depending on where you
have placed the cursor, clicking this button opens one of the following:
• the Select Data File dialog box, to select a path and file name that already
exist on your computer or network
• the Select Instrument Method dialog box, to select an instrument control
method
• the Select Processing Method dialog box, to select a processing method for
qualitative analysis, quantitative analysis, or both
Use the User Labels button to open the User Labels dialog box and change the
heading captions of five information boxes on the Sequence Setup view.
Use the Column Arrangement button to open the Column Arrangement dialog
box and display or hide specific sequence columns.
Use the Transfer Row Information button to open the Transfer Row Information
dialog box and match samples by either Sample ID or by Position.
Use the Disk Space button to open the Disk Space dialog box and determine
how much available disk space you have on your disk drive(s).
Use the Run Sequence button to open the Run Sequence dialog box and collect
data for one or more consecutive samples selected from the active sequence.
Use the Batch Reprocess button to open the Batch Reprocess Setup dialog box
and reprocess data for samples selected from the active sequence.
The Start Analysis button starts the next run in the sequence queue.
The Stop Analysis button immediately terminates the current sample run.
Xcalibur creates a raw file for the terminated sample that contains the raw
data already collected. Xcalibur then enters the Paused state, as indicated by
the flashing red message Paused to the right of the Sequence box in the
Current area in the Status view of the Home Page window. To start the next
sample in the sample list queue after you perform a Stop Analysis command,
use the Start Analysis command or click the (depressed) Pause/Resume
Analysis button in the toolbar.
The Pause/Resume Sequence Queue button pauses the sample list processing
when the button appears in the up (not depressed) position. Xcalibur changes
the appearance of the button from the up (not depressed) to the down
(depressed) state. If a sample list has been downloaded or if sample data
acquisition has started, Xcalibur completes the current sample and creates a
raw file for the sample. Xcalibur then enters the Paused state, as indicated by
the flashing red Paused to the right of the Sequence box in the Current area in
the Status view of the Home Page window. Resumes sample list processing
with the next sample in the sample list queue. The button changes in
appearance from the down (depressed) to the up (not depressed) state to
indicate that Xcalibur is resuming sample list processing.
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About Sequences
About Sequences
Each row in the sequence table describes a single sample acquisition. For
quantitation, a sequence can contain standards, QC check samples, blanks
and unknowns.
By default, the Sequence Setup - Homepage displays the Sequence template
shown in Figure 41. The available parameters are described by the column
headings. The sequence parameters available in Xcalibur are listed in
Table 14.
Figure 41. Default Sequence template
To open an existing sequence, click Open or choose File > Open to display
the Open dialog box.
To create a new sequence, click New or choose File > New to display the
New Sequence Template dialog box.
To save the current sequence, click Save or choose File > Save.
Table 14 lists the available sequence parameters.
Display some or all of the sequence parameters in the Sequence template,
label five custom user parameters, and rearrange the displayed columns.
This section contains the following topics:
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•
Arranging the Columns
•
Changing User Labels
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About Sequences
Table 14. Sequence Parameters
Thermo Electron Corporation
Column Name
Available Parameters
Sample Type
Unknown, Blank, QC (quality control) and depending on the type of
bracketing selected: Standard Clear, Standard Update, Start Bracket,
End Bracket, Standard Bracket
File Name
Name of the data file that contains the raw data acquired during the
sample run.
Sample ID
An identifier unique to the sample. This field can also be used to
import a barcode identifier.
Path
The path to the raw file that Xcalibur creates for the sample data.
Xcalibur creates this file with extension .raw.
Inst Meth
The path and file name of the Instrument method to be used for data
acquisition. Create an Instrument method from the Instrument Setup
view. Instrument methods contain instrument control parameters.
Proc Meth
The path and file name of the processing method to be used to
process the acquired data. Create a processing method from the
Processing Setup view. Processing methods contain the information
required to detect, identify, and quantitate unknowns. You do not
need a processing method to acquire data.
Position
The sample’s vial number. The format of the entry depends on the
configured autosampler, for example, Surveyor AS could have A:1
or A:B5.
Inj Vol
The volume of sample to be injected in microliters.
Dil Factor
Dilution factor used to prepare the sample.
Level
The level, if defined, for sequence rows corresponding to Calibration
or QC samples.
Company
User-defined topic with a default heading of Company.
Phone
User-defined topic with a default heading of Phone.
Comment
An additional field for any other information about the sample or
analysis procedure.
Sample Name
Text description of the sample.
Sample Wt
A reporting feature (not used in quantitation calculations).
Sample Vol
A reporting feature (not used in quantitation calculations).
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About Sequences
Arranging the Columns
To change the number or the arrangement of the columns in Sequence
Setup
1. Choose Change > Columns or click the Column Arrangement button
on the toolbar to open the Column Arrangement dialog box shown in
Figure 42. The columns currently displayed are listed in the Displayed
Columns pane in the order that they appear.
Figure 42. Column Arrangement dialog box
2. To display a column that is currently hidden:
a. Select the column heading in the Available Columns list.
b. Click Add.
The column title is moved from the Available Columns list to the
Displayed Columns list.
3. To hide a column that is currently displayed:
a. Select the column heading in the Displayed Columns list.
b. Click Remove.
The column title is moved from the Displayed Columns list to the
Available Columns list.
4. To change the order of the Displayed Columns:
a. Select the column heading to move.
b. Click Move Up or Move Down.
5. Click OK to save the changes and close the dialog box. Xcalibur displays
the columns in Sequence Setup in the new arrangement.
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Changing User Labels
Automating Analysis
About Sequences
Define the caption labels of the five user defined columns.
To change a heading caption in the User Labels dialog box
1. Select Change > User Labels or click the User Labels button (see
Figure 43).
Figure 43. User Labels dialog box
2. Enter the new heading caption in the heading box to replace the current
heading caption. To use no heading, delete the text and leave the box
blank.
3. Repeat for each of the five heading captions to change.
4. Click OK to save your new captions.
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Automating Analysis
Creating a New Sequence
Creating a New
Sequence
Importing a Sequence
There are three ways to create a new sequence. You can:
•
Import a sequence from a text file
•
Provide Xcalibur with some basic details in the New Sequence Template
wizard and allow it to create the sequence
•
Type the sequence manually in Sequence Editor
Xcalibur reads and imports comma separated text files with a .csv file
extension. This file format can be created by a text editor, such as Microsoft
Notepad, or a spreadsheet program, such as Microsoft Excel.
To import a sequence:
1. Choose File > Import Sequence to open the Import Sequence dialog
box (see Figure 44).
Figure 44. Import Sequence dialog box
2. Click Browse to select the file for importing or type the path and file
name directly into the Import from File box.
3. Select the sequence columns to be included in the imported file from
the Select Columns to Import area.
4. Click All to select all the column options.
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Creating a New Sequence
5. Click Clear to deselect all the column options.
6. Click OK to import the selected columns of the specified sequence.
Xcalibur displays the imported file in Sequence Setup.
Xcalibur generates an invalid file message if you attempt to import a file
with an incorrect extension or a file in which the separator character is
different from the character currently set in the International dialog box.
See “Exporting a Sequence” on page 115.
Creating a Sequence with
the New Sequence
Template Wizard
Creating a sequence by using the New Sequence Template wizard is
particularly useful when you are running large numbers of similar samples
or when you are running bracketed, calibration, or QC samples.
To display the New Sequence Template wizard, click the New Sequence
button on the toolbar or select File > New (see Figure 45).
Figure 45. New Sequence Template dialog box
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Creating a New Sequence
In the New Sequence Template wizard, enter information about the samples
to inject by performing the procedures provided in this topic in the order
listed:
1. Entering General Information
2. Specifying Samples
3. Choosing a Bracket Type
4. Specifying Standards, Blanks, and QCs
5. Completing the Sequence
Entering General Information
In the General area (see Figure 46) of the New Sequence Template dialog
box, make the following entries or selections:
•
Type a base file name for the raw files in the Base File Name box.
Xcalibur applies this name to all of the raw files that it creates using the
new sequence. Xcalibur then determines an incremental numeric suffix
for the base name starting at 01. To have Xcalibur start with a different
number, type the number in the Starting Number box.
Figure 46. General area
100
•
Type a path to the directory to store the raw files in the Path box or click
Browse to locate the drive and directory.
•
Select an existing instrument method in the Instrument Method box or
click Browse to locate an appropriate instrument method.
•
Select an existing processing method in the Processing Method box or
click Browse to locate the file. You do not need a processing method to
acquire raw data files.
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Automating Analysis
Creating a New Sequence
Tip The Calibration File box is grayed out until you select the None
bracket type option. For information on the Calibration File box, see
“None” on page 102.
Specifying Samples
In the Samples area, shown in Figure 47, make the following entries and
selections:
•
Type a numeric value for the total number of samples to be injected in
the Number of Samples box. This value includes blanks, unknowns,
calibration standards, and QC check samples.
•
Type a numeric value for the number of replicate injection per sample in
the Injections Per Sample box.
•
Type a name to identify the samples in the Base Sample ID box.
Xcalibur adds a suffix to the base sample ID starting with 01.
•
Select the type of tray for the autosampler from the Tray Type list. You
cannot make a selection for the Surveyor Autosampler from this list.
•
Type the location of the vial for the first injection in the sequence in the
Initial Vial Position box. The entry format depends on the configuration
of your autosampler.
•
Select the Re-Use Vial Positions check box to use the same vial for
replicate samples.
•
To select specific vials, click Select Vials to display the Vial Selection
dialog box (see Figure 48). Use this dialog box to create a sequence of
samples from individually selected vials on any of the configured trays.
Select or deselect a vial by clicking it. Deselect all selected vials
(highlighted in blue) by clicking Cancel Selection in the New Sequence
Template dialog box.
Figure 47. Samples area
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Creating a New Sequence
Figure 48. Vial Selection dialog box
Note The Select Vials feature is only available for certain autosamplers,
such as the Surveyor Autosampler.
Choosing a Bracket Type
In the Bracket Type area, select one of the calibration bracket options:
None
If you select the None option, the Calibration File box is active. If you have
a stored calibration file for samples in this sequence, click Browse to the
right of the Calibration File box to select your calibration file [.cal]. The
samples in the sequence are processed in the order they are submitted.
Xcalibur orders your Unknown, Blank (optional), Calibration (optional),
and QC (optional) samples in the following repetitive sequence:
102
1. Calibration Blank Sample
4. QC Sample(s)
2. Calibration Sample(s)
5. QC Blank Sample
3. Calibration Blank Sample
6. Unknown Sample(s)
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Automating Analysis
Creating a New Sequence
Open
Use the Open option to specify that the sequence contains one open
bracket. Place samples and calibrants in any order. Calibration samples are
processed before Unknown and QC samples.
Xcalibur orders your Unknown, Blank (optional), Calibration (optional),
and QC (optional) samples in the following repetitive sequence:
1. Blank Sample
6. Unknown Sample(s)
2. Calibration Sample(s)
7. Calibration Blank Sample
3. Blank Sample
8. Calibration Sample(s)
4. QC Sample(s)
9. Calibration Blank Sample
5. QC Blank Sample
Non-Overlapped
Use the Non-Overlapped option to specify that the sequence contains one
or more non-overlapped brackets.
Xcalibur orders your Unknown, Blank (optional), Calibration (optional),
and QC (optional) samples in the following repetitive sequence:
1. Calibration Blank Sample
6. Unknown Sample(s)
2. Calibration Sample(s)
7. Calibration Blank Sample
3. Calibration Blank Sample
8. Calibration Sample(s)
4. QC Sample(s)
9. Calibration Blank Sample
5. QC Blank Sample
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Creating a New Sequence
Overlapped
Use the Overlapped option to specify that the sequence contains one or
more overlapped brackets.
Xcalibur orders your Unknown, Blank (optional), Calibration (optional),
and QC (optional) samples in the following repetitive overlapping-bracket
sequence:
1. Calibration Blank Sample [Bracket 1]
2. Calibration Sample(s) [Bracket 1]
3. Calibration Blank Sample [Bracket 1]
4. QC Sample(s) [Bracket 1]
5.
QC Blank Sample [Bracket 1]
6. Unknown Sample(s) [Bracket 1]
7. Calibration Blank Sample [Bracket 1, 2]
8. Calibration Sample(s) [Bracket 1, 2]
9. Calibration Blank Sample [Bracket 1,2]
10. QC Sample(s) [Bracket 2]
11. QC Blank Sample [Bracket 2]
12. Calibration Blank Sample
13. Calibration Blank Sample [Bracket 2, 3]
14. Calibration Sample(s) [Bracket 2, 3]
15. Calibration Blank Sample [Bracket 2, 3]....
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Creating a New Sequence
Specifying Standards, Blanks,
and QCs
To add standards, blanks, and QCs to your sequence list
1. Select the Add Standards check box in the Calibration area (see
Figure 49).
Figure 49. Calibration area
2. Do the following:
a. In the Number of Calibration Sets box, type a numeric value for the
number of calibration sets to add to the sequence list.
b. In the Injections Per Level box, type a numeric value for the number
of injections per calibration level.
3. Select the Add Blanks check box to add blanks to the beginning of a set
of bracketed samples.
4. Select the Fill In Sample ID For Standards check box to automatically
fill in the Calibration Sample ID in the new sequence. This information
is defined in the processing method for each calibration standard level.
5. Select the Add QCs check box in the QC area to add quality control
samples to the Sequence list. Do the following:
•
Select the After First Calibration Only option to run a quality
control sample only after the first group of calibration samples in the
new sequence. Subsequent calibration samples sets are not followed
with a quality control sample.
•
Select the After First Calibration Only option to add a quality
control sample after every calibration sample set in the new
sequence.
6. Select the Add Blanks check box in the QC area to add one blank after
each set of quality control samples.
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Creating a New Sequence
7. Select the Fill In Sample ID for QCs check box to automatically fill in
the Quality Control (QC) Sample ID in the new sequence. This
information is defined in the processing method for each QC level.
Completing the Sequence
Click OK to save the changes and close the New Sequence Template dialog
box. Xcalibur now generates a sequence based on the information you have
provided.
Creating a Sequence
Manually
To create a sequence manually, define the following parameters for each
sample row in the sequence as described in Table 15.The remaining
columns not described in Table 15 are simple text fields used for reporting
purposes:
•
User Labels 1-5 (On installation these have defaults: Study, Client,
Laboratory, Company, Phone)
•
Comments
•
Sample Name
•
Sample Weight
•
Sample Volume
The columns might be hidden. They are not essential for the running of a
sample or sequence. To enter text information under any of these column
headings, click the relevant grid cell and type the required information.
Table 15. Defining sequence parameters
106
Column
Action
Sample Type
Double-click the Sample Type cell and select: Unknown, Blank, QC, or Std
Bracket from the list.
File Name
Enter a file name for storing the sample data.
Sample ID
Enter a Sample identification number.
Path
Enter the directory path to store the sample’s raw file.
Inst Meth
Enter the path and filename of an instrument method file or double-click in
the box and browse to the appropriate file using the Select Directory
dialog box. Instrument methods have a .meth file extension.
Proc Meth
Enter the path and filename of the processing method file or double-click
in the box and browse to the appropriate file using the Select Directory
dialog box. A process method is required if the sample type is QC, Std
Clear or Std Update or to run a process on the raw file. Processing
methods have the a .pmd file extension.
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Creating a New Sequence
Table 15. Defining sequence parameters, continued
Column
Action
Cal File
To add a Calibration file to the sequence, create a Sequence list using the
None bracket type. In the Cal File cell, type the path and filename of the
calibration file to be used to process the samples in the current sequence
using the None bracket type or double-click in the Calibration File box to
open the Select Calibration File dialog box to find and select the path and
file name.
Position
In the Position cell, type the position of the vial to inject the sample. The
allowable vial positions depend on your autosampler configuration.
Inj Vol
In the Inj Vol cell, type the injection volume (in microliters). If you do not
enter an injection volume, Xcalibur uses the default injection volume set in
the Instrument method. The allowable injection volume range depends on
the configured autosampler and the type of injection specified in the
Instrument method.
Level
Double-click in the Level box to open the Select Level dialog box. After
creating and selecting a processing method with Calibration or QC levels
select a level and click OK. Specify a level if the sample type is QC, Std
clear, or Std Update.
ISTD Corr Amt
Type a non-zero numeric value in this box to modify the amount of internal
standard for this sample. If the value in this box is not 0.000, the value is
used in an algorithm to correct for the case where the internal standard
amount(s) specified in the active processing method are correct, but
where the amount of internal standard present in this sample is different
than the amount specified in the processing method.
Dil Factor
To change the dilution factor, double-click the Dil Factor box. The cursor
changes to the vertical bar cursor. Enter the correct factor. The cursor
changes back to the original cursor when you click any other area of the
view.
If you have specified a processing method for the current sequence,
Xcalibur automatically enters the Dil Factor value from the processing
method settings
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Modifying a Sequence
Modifying a
Sequence
Filling Down Columns
The Sequence Setup is equipped with a number of tools and commands to
assist you in compiling a sequence. For instructions on how to use these
tools and commands, see the following procedures provided in this topic.
•
Filling Down Columns
•
Inserting a Row
•
Deleting a Row
•
Going to a Sequence Row
•
Transferring Row Information
•
Printing a Sequence
•
Checking Disk Space
•
Exporting a Sequence
•
Changing the List Separator Character
Use the Fill Down command to copy information from one row or a cell
within a row to any number of rows immediately below it in the Sequence
table.
To fill down sample settings
1. Select the cells in the row to copy.
2. Drag downwards to select the range of columns to be filled (edit the
selection in step 4). You must select at least one row to enable the
command.
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Modifying a Sequence
3. Choose Edit > Fill Down or click the Fill Down button in the toolbar.
The Fill Down dialog box appears (see Figure 50).
Figure 50. Fill Down dialog box
4. Check the extent of the range to be filled. Your selection is shown at the
bottom of the Fill Down dialog box (see Figure 50). Xcalibur identifies
the first selected row as the one to be copied, and all subsequent selected
rows as targets for the Fill Down operation.
Fill Rows Y to Z using Row X
Where:
Row X = the row to be copied
Row Y = the first row of the range to be filled
Row Z = the last row of the range to be filled
If required, type in a new value for Row Z, the last row to be filled with
Row X duplicates. If X is incorrect, click Cancel to close the dialog box
and repeat the procedure from step 1.
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Modifying a Sequence
5. Choose the columns to be copied down by checking the relevant boxes:
•
Click All to select all the column check boxes
•
Click Clear to deselect all the column check boxes
6. Click OK to close the dialog box and execute the Fill Down command.
Xcalibur copies appropriate information from the first row into the
selected range.
Inserting a Row
To insert a row
1. Select the row immediately below where the new row will be inserted.
2. Choose Edit > Insert Row to display the Insert Row dialog box and
click Yes to confirm.
3. The inserted row is a copy of the row immediately prior to the row
selected in step 1.
Deleting a Row
To delete a row
1. Select the row to delete.
2. Choose Edit > Delete Row to display the Delete Rows dialog box.
3. Click Yes to confirm.
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Going to a Sequence Row
Automating Analysis
Modifying a Sequence
To go to a specified row in the current sequence
1. Choose Edit > Go To Row.
2. Type a valid row number in the Go To Line Number dialog box (see
Figure 51).
Figure 51. Go To Line Number dialog box
3. Click OK.
Xcalibur closes the dialog box and highlights the selected row.
Transferring Row
Information
Use Transferring row information to ensure that all occurrences of a
particular Sample ID or Position have the same parameters. Xcalibur copies
the parameters from the first row featuring a Sample ID or Position to all
other rows in the sequence with the same Sample ID or Position.
To transfer row information
1. Choose Change > Transfer Row Information or click the Transfer
Row Info button to display the Transfer Row Information dialog box
(see Figure 52).
Figure 52. Transfer Row Information dialog box
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Modifying a Sequence
2. Do one of the following:
•
Select the Match By Sample ID option to copy the parameters from
the first sequence row with a particular Sample ID to all other
sample rows with the same Sample ID.
•
Select the Match By Position option to copy the parameters from
the first sequence row with a particular Position to all other sample
rows with the same Position.
3. Click OK to close the dialog box.
Xcalibur performs the selected copy operation.
4. To undo the copy operation, immediately choose Edit > Undo or click
the Undo button in the toolbar.
Printing a Sequence
Print a full sequence or a vial list compiled from the active sequence.
To print a sequence list
1. Do one of the following:
•
To preview the appearance of the sequence before printing, choose
File > Print Preview to display the Print Selection dialog box (see
Figure 53). Go to step 2.
•
To print the sequence list without first previewing it, go to step 5.
Figure 53. Print Selection dialog box
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Modifying a Sequence
2. Select one of the following from the Select the Printing Output area:
•
Select the Vial Position List option to review a sequentially
numbered vial position list from the active sequence. The vial
position list summarizes the sequence settings for each vial and is
useful when you are setting up the autosampler tray vial sequence.
•
Select the All Columns option to preview selected rows in the active
sequence.
•
Select the Displayed Columns Only option to review the currently
displayed columns of the active sequence.
3. Click OK.
4. To preview the active list pages, click Next Page, Previous Page, Two
Page, Zoom In or Zoom Out. Click Close to return to the Sequence
Setup view.
5. Choose File > Print or click the Print button on the toolbar to display
the Print Selection dialog box (see Figure 53).
6. Select one of the print output options from the Select the Printing
Output areas, described in step 2 above.
7. Click OK to open the Print dialog box.
8. Complete the printer settings and click OK to print the selected list.
See the Xcalibur online Help for a complete description of all controls
contained in the Print dialog box.
Checking Disk Space
A sequence can generate a large number of raw files. Sequence Setup
provides a utility for you to check the amount of available disk space on
system drive(s).
To check available disk space
1. Choose Actions > Check Disk Space or click the Disk Space button on
the toolbar. The Disk Space dialog box appears (Figure 54). This shows:
•
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The current drive and directory. For example:
C:\Xcalibur\SYSTEM\PROGRAMS
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Modifying a Sequence
•
The number of MB that are available (free) on the current drive and
the percentage of the total capacity of the drive that is available. For
example: 214 MBytes (17.6%) Free
•
A pie-chart showing the available space in the color green and the
used space in the color red.
•
The total capacity of the current drive. For example: 1220 MBytes
Total
Figure 54. Disk Space dialog box
2. Click Directory to check disk space on another disk.
3. Click OK to close the dialog box.
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Exporting a Sequence
Automating Analysis
Modifying a Sequence
Export a sequence as a separator delimited text file with a .csv file extension.
This file format can be read by a text editor, such as Microsoft Notepad, or a
spreadsheet program, such as Microsoft Excel. The exported sequence file
contains the current list separator character (normally a comma) that is set
in the Microsoft Windows dialog box.
To export a sequence
1. Choose File > Export Sequence. The Export Sequence dialog box
appears (see Figure 55).
2. Enter the path and file name of the exported Sequence file in the Export
To File box or click Browse to select a path for the exported Sequence
file. Xcalibur assigns a csv file extension to the exported file.
3. Use the options in the Export Sequence area to select the Sequence
columns to be included in the exported file.
•
Click All to select all the column options.
•
Click Clear to deselect all the column options.
4. Click OK to export the selected columns of the active sequence to the
specified file and location.
Figure 55. Export Sequence dialog box
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Modifying a Sequence
Changing the List
Separator Character
When you export a sequence, Xcalibur creates a text file with a .csv file
extension and inserts a list separator character between each field of each
column of the sequence. This file format can be read by a text editor, such as
Microsoft Notepad, or a spreadsheet program, such as Microsoft Excel.
The list separator can be any alphanumeric character. However, avoid
characters that cannot be distinguished from the characters used in the
sequence text fields, such as alphabetic characters, because they result in
unreadable (invalid) files. The most common list separators are the comma
(,) and the semicolon (;). Each country has a default list separator. For
example, the default list separator for the United States is the comma.
When you import a sequence, the list separator character used in a sequence
file to be imported must be the same as that specified in the Microsoft
Windows XP Professional operating system.
To change the list separator character
1. Click the Start button in the Windows Taskbar and choose Settings >
Control Panel.
2. Double-click the Regional and Language Options icon.
3. Click the Regional Options tab.
4. Click Customize to open the Customize Regional Options dialog box.
5. Enter the new list separator character into the List Separator combo box.
6. Click OK to store the new list separator and close the dialog box.
7. Click OK to close the Customize Regional Options dialog box.
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Running Samples
Automating Analysis
Running Samples
From the Sequence Setup view, run a single sample, a range of samples, or
the full sequence.
To run a sequence containing one or more samples, perform the following
procedures in the order listed:
1. Opening the Run Sequence Dialog Box
2. Setting General Run Options
3. Changing Acquisition Options
4. Selecting a Startup or Shutdown Method
5. Specifying Pre- and Post-Run Acquisition Programs
6. Starting the Run
Opening the Run
Sequence Dialog Box
To open the Run Sequence Dialog Box and run a single sample
1. Select the sample to run by clicking its row number. Xcalibur highlights
the row.
If you do not select a sequence row, Xcalibur runs Sample 1.
2. Choose Actions > Run This Sample or click Run Sample.
Xcalibur displays the Run Sequence Dialog box. Go to the next
procedure: Setting General Run Options.
To open the Run Sequence Dialog Box and run a set of samples
1. Highlight the samples to run. Click the left-most column of the first
sample and drag to the last sample to identify all samples.
2. Choose Actions > Run Sequence or click Run Sequence.
Xcalibur displays the Run Sequence Dialog box. Go to the next
procedure: Setting General Run Options.
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Running Samples
Use the Run Sequence dialog box, shown in Figure 56, to do the following:
•
Identify the range of samples for analysis from the current list
•
Configure instruments to be used in the run
•
Run instrument startup methods before the sequence is initiated
•
Run instrument shutdown methods when the sequence is complete
•
Execute programs before or after each sample acquisition
•
Prioritize the sequence so that it is positioned at the head of the
Acquisition Queue
•
Select processing and reporting options
Figure 56. Run Sequence dialog box
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Setting General Run
Options
Automating Analysis
Running Samples
To set the general run options in the Run Sequence dialog box
1. Check the rows listed in the Run Rows box. If the range is incorrect,
type in the correct range or click Cancel to close the dialog box. Select a
different sample or range of samples and repeat the procedure.
2. Type the name of the operator (up to 10 characters) in the User box.
3. Select the Priority Sequence check box to position the sequence or
sample ahead of all others in the Acquisition Queue. When Xcalibur is
running a quantitation bracket, it queues the priority sequence
immediately after the bracket.
4. Select the Start When Ready check box to perform an autosampler
injection as soon as the system is ready. To initiate an injection using the
Start Analysis command from the Home Page, ensure that the Start
When Ready check box is not selected.
Changing Acquisition
Options
The Acquisition Options window lists the configured modules of your
instrument and the module that triggers the start of a run. The configured
modules are listed in the Instruments column. The module that triggers the
start of the run has a Yes associated with it in the Start Instrument column.
If no instrument is flagged as the start device, Xcalibur expects an unlisted
instrument to provide an appropriate signal to start the acquisition.
Click Change Instruments to open the Change Instruments In Use dialog
box (see Figure 57) and do one of the following:
•
Add or remove an instrument from the list of instruments that are
controlled during the sequence run
•
Have a different instrument start the run
Figure 57. Change Instruments in Use dialog box
To change the status of any instrument in the current configuration, toggle
the In Use field by clicking it.
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Running Samples
To change the Start Instrument assignment, toggle the Start Instrument
fields as appropriate. Only one instrument can be designated as the Start
Instrument.
Selecting a Startup or
Shutdown Method
Specify optional instrument methods to be run before and after the
sequence (for example, for tuning or calibration):
•
Select an existing instrument method to start up the instrument. This
method is run through the instrument before the first sample is queued.
Click Browse to select the drive and directory where the file is located.
•
Select an existing Instrument method to shut down the instrument.
This method is run through the instrument after the last sample has
been analyzed. Click Browse to select the drive and directory where the
file is located.
Data acquisition is not performed during the execution of a start up or shut
down method.
Specifying Pre- and
Post-Run Acquisition
Programs
Specify programs or macros to be run before or after or before and after each
acquisition. These programs or macros might be used, for example, to issue
commands to prepare an instrument not controlled by Xcalibur for
acquisition.
To run a pre-acquisition program
1. To run a pre-acquisition program, click Browse next to the
Pre-Acquisition box. Select the drive and directory where the existing
program is located.
2. In the Run Synchronously box, select Synchronous to wait for a
program to be completed before continuing with its next action. Select
Asynchronous to continue with the next action immediately after
initiating the program.
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Running Samples
To run a post-acquisition program
1. To run a post-acquisition program, click Browse next to the
Post-Acquisition box. Select the drive and directory where the existing
program is located.
2. In the Run Synchronously box, select Synchronous to wait for a
program to be completed before continuing with its next action. Select
Asynchronous to continue with the next action immediately after
initiating the program.
Choosing Processing
Actions
Starting the Run
Choose one or more of the following processing or reporting options:
•
To perform quantitative processing, select the Quan check box.
•
To perform qualitative processing, select the Qual check box.
•
To print the reports that you specified in the processing method, select
the Reports check box.
•
To print the parameters in the Instrument and processing methods used
to acquire and process the samples, select the Print methods check box.
•
To print a summary report for your samples, select the Create Summary
check box.
After you finish making your entries and selections in the Run Sequence
dialog box, click OK to save the settings.
Xcalibur closes the Run Sequence dialog box and places the selected
sample(s) in the run queue or starts processing immediately.
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Reprocessing Samples
Reprocessing
Samples
To reprocess a batch of samples
1. Select the rows to be reprocessed from the current sequence or specify
the row numbers using the Process Rows box in the Batch Reprocess
Setup dialog box – see below. Xcalibur highlights the selected rows.
2. Choose Actions > Batch Reprocess or click Batch Reprocess to display
the Batch Reprocess Setup dialog box (see Figure 58).
Figure 58. Batch Process Setup dialog box
3. Check information in the Process Rows box. If it is incorrect, click
Cancel to close the Batch Reprocess Setup dialog box. Select a different
sample or range of samples and repeat the procedure or type in the
correct range. The format is either [Row] for one sample or [First Row Last Row] for multiple samples.
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Reprocessing Samples
4. Select the Quan check box to reprocess quantitative data. Select the
following quantitative processing options:
•
Select the Peak Detection Integration check box to generate new
peak detection and integration data.
•
Select the Calibration check box to carry out new calibration
calculations using the sequence standards.
•
Select the Quantitation check box to re-calculate the quantitation
data for unknown samples in the sequence.
5. Select the Reports check box to print new reports. Then make the
following selections:
•
Select the Print Sample Reports check box to generate new sample
reports, based on those listed in the processing method.
•
Select the Print Summary Reports check box to generate new
summary reports, based on those listed in the processing method.
6. Select the Programs check box to run the post-processing programs or
macros, based on those listed in the processing method.
7. Select the Print Methods check box to print the Experiment and
processing method used during batch reprocessing.
8. Select the Create Quan Summary Spreadsheet option to generate a
summary spreadsheet for the reprocessed sequence.
9. Select the Advanced Options – Replace Sample Info check box to
replace the sample information generated during data acquisition in the
sample headers with new information generated during reprocessing.
10. Click OK.
Xcalibur initiates batch reprocessing of the selected samples.
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The Acquisition Queue
The Acquisition
Queue
The Acquisition Queue (see Figure 59) shows all the sequences and samples
submitted for analysis. The Explorer style tree view shows two levels of
detail: the sequence names and, within each branch, the raw sample
filenames.
Use the Acquisition Queue to do the following:
•
Delete sequences unless they are currently being run.
•
Delete samples within a sequence unless they have already been
acquired, are currently undergoing acquisition, or are part of the
quantitation bracket currently being acquired.
Figure 59. The Acquisition Queue with the Sample Information window displayed
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The Acquisition Queue
Manipulate entries in the acquisition queue:
•
Right-click the name of the sequence or sample to open a shortcut
menu. Choose Properties to display the Sample Information dialog
box.
•
Double-click a sequence to load it into Sequence Setup.
•
Double-click a sample to open the Sample Information dialog box.
A check box appears alongside each sequence and sample. Select one or
more items for deletion. To delete a sample or sequence from the queue,
select the check box and then press the DELETE key.
Deleted samples are identified by a large cross in the check box. Xcalibur
also appends the word DELETED to the sample or sequence identifier.
Sample Information Dialog
Box
The Sample Information dialog box shows the parameters for all the
sequence fields. See “About Sequences” on page 94 for descriptions of all the
fields.
The Sample Information dialog box closes if you click anywhere outside it.
Click the pin icon to keep it open. Click the close icon to close the dialog
box or unpin the dialog box (by clicking the pin icon again) and click
anywhere outside the dialog box before continuing.
Xcalibur updates a pinned dialog box with the details of any selected
sequence.
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The Acquisition Queue
Managing Tasks
Queue Manager, shown in Figure 60, provides additional functions for
managing queued tasks. It is active whenever samples or sequences are
queued for reprocessing. If it is not visible, it might be minimized to the
Windows toolbar.
Figure 60. Queue Manager window
Use the following procedures to manage the Xcalibur Processing Queue.
Pausing the Processing Queue
To temporarily pause the processing queue, click the Pause button or
choose Queue > Pause.
Resuming the Processing Queue
To resume the processing queue when it is in the Pause mode, click the
Resume button or choose Queue > Resume.
Updating the Display
To update the display with the latest information, choose View > Refresh.
Removing Tasks
To Remove a task from the queue, select the task to be removed. Click the
Remove Job button or choose Analysis > Remove From Queue from
Queue.
To remove all tasks from the queue, choose Queue > Purge Queue.
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The Acquisition Queue
Viewing the Details of a Selected Analysis
To view the details of a selected analysis, select the required analysis in
Queue Manager. Click the Details button in the toolbar or choose Analysis
> Details.
The Details of Selected Analysis dialog box appears (see Figure 61).
Figure 61. Details of Selected Analysis dialog box
This dialog box contains the following readouts:
Thermo Electron Corporation
•
The File readout displays the name of the data file.
•
The Status readout displays the status of the queue.
•
The Submitted readout displays the time and date that the reprocessed
job was submitted.
•
The From readout displays the source of the reprocessing job.
•
The Actions readout displays the tasks required to complete the selected
reprocessing job and their current status.
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Chapter 4
Reviewing Quantitation in
Quan Browser
Xcalibur’s data reviewing component is called Results Review (see
Figure 62). The three core Browsers are:
•
Qual Browser: displays and manipulates chromatograms and spectra,
activate library searches, and view qualitative processing results.
•
Quan Browser: displays and manipulates the peak integration and
calibration curve results of a processing method to be displayed and
manipulated.
•
Library Browser: activates the NIST library utility to match spectra to
library entries.
Figure 62. Results Review section of the Roadmap - Home Page
This chapter describes how to use Quan Browser to analyze processed
quantitation sequences. It explains the properties and uses of each
component within the Quan Browser window. It also describes how to use
Quan Browser to achieve calibration and quantitation reviewing tasks. Qual
Browser and Library browser are described in the Xcalibur Getting
Productive: Qualitative Analysis manual.
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Reviewing Quantitation in Quan Browser
This chapter contains the following sections:
130
•
About Quan Browser
•
The Quan Browser Window
•
The Results Grid
•
Chromatogram View
•
Calibration Companion View
•
Spectrum Companion View
•
Reports
•
Quan Browser Procedures
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About Quan Browser
Reviewing Quantitation in Quan Browser
About Quan Browser
Quan Browser is a powerful and versatile utility for reviewing and
reworking:
•
Component peak identification and integration criteria
•
Standards, QCs, blanks and unknowns
•
Calibration curves for quantitation standards
After making any changes, save the new results with an audit trail describing
the reason for the change.
Quan Browser incorporates a calibration curve display, peak integration,
and results view where you can:
•
Reprocess quantitation sequences
•
Interactively edit processing parameters and audit the changes
•
Create new files that keep track of processing results for individual raw
files and include a copy of the method used to generate the results
Result files changed using Quan Browser do not affect the original
processing method.
This section contains the following topics:
How Quan Browser Works
•
How Quan Browser Works
•
Getting Started in Quan Browser
Quan Browser helps you step through a sequence and review the results for
each component in each file. The Quan Browser helps to quickly review
component peak identification and integration criteria. After making any
changes, save the new results with an audit trail describing the reason for the
change. Result files changed using the Quan Browser do not affect the
original processing method. Only Processing Setup can edit processing
methods and only the Quan Browser can edit result files.
Xcalibur has the following quantitation features:
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•
Calibration Replicates
•
Named Calibration File
•
Non-Bracketed Sequence
•
Open Bracket Sequence
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About Quan Browser
•
Non-Overlapping Bracket Sequence
•
Overlapping Bracket Sequence
Calibration Replicates
Calibration replicates are multiple injections of the calibration mixture at
the same calibration level or amount. These standard samples all contain the
same amount of target compound and therefore correspond to the same
calibration level. Choose which replicates to include or exclude from the
calibration curve by using the Calibration Companion View.
Named Calibration File
After creating a sequence with the Bracket Type set to None, specify a
calibration file name in the Calibration File box. Although, in theory, it is
possible to have a different calibration file name for every sample, in practice
it is usual to have only one name per sequence.
Named calibration files are not available with bracketed sequences.
Non-Bracketed Sequence
Xcalibur processes a non-bracketed sequence using a procedure known as
the continuing calibration method. Each time it processes a non-bracketed
sequence, it creates or updates the calibration file(s) named in the sequence.
Select this process and avoid using of Std Clear to add replicate data
incrementally to a calibration file without discarding the existing replicate
data.
Quan Browser breaks down non-bracketed sequences into logical groups
that are somewhat analogous to brackets. It does this by first ordering the
samples chronologically with respect to acquisition date and time. It then
examines the sequence and starts a new group whenever it encounters a
standard. The group ends at the non-standard sample that immediately
precedes the next standard found.
The first group always starts with the first sample, even if it is not a
standard. The last group always ends with the last sample. Further, a Std
Clear always starts a new group, even if no intervening non-standard sample
has been found following one or more Std Updates.
Note Additional logical groups are formed if different named calibration
files have been specified in the Cal File entries of the sequence. Each cal
file entry causes a new group to be formed. Because using multiple
named calibration files is not typical, their use is not considered any
further in this document, but should be deducible from the discussions
on groups.
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Reviewing Quantitation in Quan Browser
About Quan Browser
As each group is processed, its samples are quantitated against the current
calibration curve. As each standard is encountered, it is processed and either
replaces (sample type set to Std Clear) or adds to (sample type set to Std
Update) the calibration replicate list and a new, calibration curve is
generated.
Quan Browser processing closely emulates that of batch processing (either
that directly after acquisition, or subsequently as a batch re-process
operation). However, it has the additional capability that, if a cal file is
specified but cannot be found nor opened by Quan Browser, the message
Cal File Unavailable - Using Embedded Calibration appears in the
Calibration File edit box and Quan Browser takes replicate data from that
stored in the result file. In most cases this data is identical to that contained
within the original calibration file.
Once Quan Browser has set up the groups, they are independent and are
effectively treated as brackets. In other words, changes in one group do not
affect any other group, unlike in batch processing where subsequent groups
might well be affected.
The following list illustrates the procedure (for a single named
calibration file):
Open Bracket Sequence
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Sample 1
Unknown
Group 1 start
Sample 2
Unknown
Group 1 end
Sample 3
Std Clear
Group 2 start
Sample 4
Unknown
Group 2 end
Sample 5
Std Update
Group 3 start
Sample 6
Std Update
Sample 7
Unknown
Sample 8
Unknown
Sample 9
Blank
Sample 10
QC
Group 3 end
Sample 11
Std Update
Group 4 start/end
Sample 12
Std Clear
Group 5 start
Sample 13
Blank
Group 5 end
Qual Browser creates a replicate list directly from all standard samples in the
sequence without using any calibration data embedded in result files.
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About Quan Browser
Note When you open Quan Browser with a single Result file, it is
treated as a sequence with only one entry and is brought in as an
unknown. To show the calibration curve used to quantitate the sample,
the replicate list is created from the embedded information.
Non-Overlapping Bracket
Sequence
Quan Browser creates a separate replicate list for each bracket. Each replicate
list is created directly from all standard samples in the bracket without
utilizing any calibration data embedded in result files.
Overlapping Bracket Sequence
Quan Browser creates a separate replicate list for each bracket. Each replicate
list is created directly from all standard samples in the bracket without using
any calibration data embedded in result files.
Exceptions occur for shared standard samples between brackets. When a
standard that is shared undergoes a change, that change is reflected in all
brackets that contain that sample. When a shared standard is deleted, it is
deleted in all brackets that contain that sample and the replicate lists for all
brackets are adjusted.
Add a sample to any bracket. When it is added as a standard, it is added to
the replicate list automatically. To add a sample as a shared sample, add it
separately to each bracket.
The exclusion status of the replicates is independent for each bracket. Even
shared samples might be excluded in one bracket but not another. This is
the only exception to a shared sample having identical settings.
Getting Started in Quan
Browser
To start Quan Browser, do one of the following:
•
Click the Quan Browser icon on the Home Page
•
Choose GoTo > Quan Browser.
At startup, Quan Browser displays the Open dialog box allowing you to
select an existing file.
Supported file types are:
•
Sequence files (.sld)
•
Result files (.rst)
•
Quan Browser files (.xqn)
Quan Browser closes if you click Cancel in the Open dialog box.
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About Quan Browser
Quan Browser handles result files as single entry sequences.
When you select a sequence file, Xcalibur checks that all the associated raw
and result files are available. When it encounters a problem with the
sequence file, Xcalibur provides information about the likely cause in a
warning dialog box and asks you to exit the application or select a different
file.
After verifying that the files exist and can be opened, Xcalibur displays the
View Sample Types dialog box shown in Figure 63.
The two options provided in the View Sample Types dialog box determine
how the Result Grid is configured at startup:
•
Select the Show Standard and QC Sample Types option so that the
Result grid displays only Standards and QCs in the Quan Browser Grid
view. Blanks and Unknowns do not display. Select from the following
tabs: Standards and QCs.
•
Select the Show All Sample Types option to display Standards, QCs,
Blanks, and Unknowns in the Quan Browser Grid view. Select from the
following tabs: All, Standards, QCs, Blanks, and Unknowns.
The View Sample Types dialog box includes a Don’t Ask Again check box.
When you select this check box, the dialog box is not displayed when you
start subsequent sessions in Quan Browser and the current selection is used
by default.
Note Make this, and all other Don’t Ask Again type dialog boxes active
by choosing Options > Enable Warnings.
Figure 63. View Sample Types dialog box
Click OK to start the session. Quan Browser now loads the specified
sequence or file and configures the Results Grid using your selected viewing
option.
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The Quan Browser Window
The Quan Browser
Window
Title Bar
The Quan Browser window (see Figure 64) incorporates the following
features, which are described in this section:
•
The Title Bar
•
The Toolbar and Menu Bar
•
Component List
•
Results Grid
•
Chromatogram View
•
Companion View
Menu Bar
Toolbar
Component List
Results Grid
Companion View
Chromatogram View
Figure 64. Quan Browser in action
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The Title Bar
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The Quan Browser Window
The title bar lists:
•
The application name – Quan Browser
•
The active view (Browser or Report)
•
The name of the opened sequence, result or Quan Browser file
•
Additional information: the Bracket in use and the viewing preference.
The bracket information is labeled as Bracket x. The viewing preference
is labeled as View Stds and QCs or View All.
In Figure 64, the title bar display is:
Quan Browser – Browser – steroid.sld (Bracket 1, View All)
The Toolbar and Menu Bar
Toolbar buttons are available on the dockable toolbar. The toolbar can be
customized to display buttons for preferred commands.
The default layout is shown below:
Table 16 lists the name and use of the tools in the Quan view toolbar.
Table 16. Quan view toolbar
Button
Use
Open
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Display the Open dialog box to select a different file.
The software prompts you to save all changes to the
current document. The supported file types are
sequence list (*.SLD), Result (*.RST) and
QuanBrowser (*.XQN) files.
Save
Create an Xcalibur Quan File (*.XQN) from the
current Quan Browser data. This file contains all the
necessary information needed to recreate the
current session.
Calibration Companion View
Set the companion view to display the calibration
curve for the currently selected bracket.
Spectrum Companion View
Set the companion view to display the spectrum
plot. Xcalibur initially displays the spectrum at the
apex of the current peak in the chromatogram view.
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The Quan Browser Window
Table 16. Quan view toolbar
Button
Use
Reports
Opens the Reports dialog box to generate sample or
summary reports for selected samples or the entire
sequence.
Manual Noise Region
Click the Manual Noise Region button and then drag
the cursor horizontally across the region of the
chromatogram to identify as the noise region.
Xcalibur marks the region with a red baseline.
Xcalibur calculates noise based on the data points
you select, using all selected data points as noise
points and calculating noise based on those points.
Select the noise region from an individual trace or
different noise regions from multiple traces.
Note.This button is active after opening a raw file
and selecting a chromatogram.
Delete Manual Noise Region
Click the Delete Manual Noise Region button and
then drag the cursor over the region that was
previously selected as the noise region. Release the
mouse button to delete the noise region.
The next group of buttons are Zoom controls used to adjust the display of
the component chromatogram and companion views. These include the
zoom in and out, for both the vertical and horizontal axes as well as the
‘display all’ button to expand the plot to its limits.
The last button is the application Help button.
Many of Quan Browser’s functions are accessed from shortcut menus from
within the Results Grid, Chromatogram View, or Companion View.
For specific information about Quan Browser’s menu commands or toolbar
buttons, see the Xcalibur online Help.
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The Quan Browser Window
Component List
The Component list displays all the components within the current bracket
sorted by retention time. Click the name of a component to update the
Chromatogram View and the Companion View with data for the selected
component.
Results Grid
The results grid is made up of sequence entries. Each row defines a result file
and associated parameters.
Chromatogram View
The Chromatogram View displays the chromatogram for the currently
selected component from the currently selected result file.
When a filter is stored within the embedded processing method for the
current compound, Xcalibur applies it to the chromatogram. Adjust the
chromatogram plot using the Zoom menu commands or toolbar buttons.
The type of integration used appears in the results grid but can be
overridden. The three types are Method Settings, User Settings, and Manual
Integration. Change the Integration method by using commands on the
shortcut menu within the Chromatogram View.
Companion View
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The Companion View has two display options:
•
Use the Calibration Curve menu option to display the calibration curve
for the current bracket.
•
Use the Spectrum Plot option to display the spectrum at the selected
retention time. Initially the display shows the spectrum at the apex of
the current peak in the chromatogram view.
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The Quan Browser Window
Calibration Companion View
The Calibration Companion View displays a calibration curve for the
current bracket or group.
For a Bracketed Sequence
In a bracketed sequence, Xcalibur calculates the points on the graph from
the embedded calibration information stored in the result file.
For a Non-Bracketed Sequence
For a non-bracketed sequence, the points on the graph consist of the
replicates in the specified calibration file for the current component and any
standards in the first group. Unless Xcalibur encounters a Standard Clear,
each subsequent group includes all the standards from the prior groups as
well as the standards from the calibration file. When Xcalibur encounters a
standard clear, the calibration tables are cleared and only the standards from
the current group are used.
Xcalibur uses all standards within a group to evaluate the calibration curve.
This means that the calibration curve is the same for all samples within a
group. At the time of sample acquisition, non-standard samples are
processed immediately after acquisition using a calibration curve
determined from the standards acquired so far (that is, omitting any
standards following the sample in the sequence).
Spectrum Companion View
140
The spectrum companion view displays a spectrum from the current
chromatogram in the chromatogram view. View spectra from the apex, left
peak edge or right peak edge using commands from the shortcut menu.
When the View is pinned, view scans from any part of the chromatogram by
clicking on the chromatogram.
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The Results Grid
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The Results Grid
The Results Grid (see Figure 65) is made up of sequence entries. Each row
defines a result file and associated parameters. Above the Results Grid,
Xcalibur displays the following: the Bracket/Group In Use combo box and
the Calibration File box.
This section contains the following topics, which describe the features of the
Results grid:
•
Bracket/Group in Use
•
Calibration File
•
Results Grid Columns
Figure 65. The Results Grid
There are two viewing configurations. These are determined by your choice
in the Viewing Sample Types dialog box at startup (see Figure 63 on
page 135) or by your choice in the Options menu.
When the Options menu viewing preference is set to View Stds and QCs,
the results grid has three pages:
Thermo Electron Corporation
•
Use the All tab to view all standard and QC samples.
•
Use the Standards tab to view only Standard samples.
•
Use the QCs tab to view only QC samples.
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The Results Grid
When the Options menu viewing preference is set to View All, the results
grid has two additional pages:
Bracket/Group in Use
•
Use the Blanks tab to view only Blank samples.
•
Use the Unknowns tab to view only Unknown samples.
For bracketed sequences, this combo box lists the available brackets in
sequential order. Xcalibur selects the first bracket in the list when the file is
first loaded into Quan Browser, and displays the samples within this bracket
in the results grid.
When you load a non-bracketed sequence, the samples are broken into
logical groups (see “How Quan Browser Works” on page 131). The combo
box lists the available groups.
Selecting a new bracket or group from the combo list refills the results grid
with the samples from the selected bracket or group. Xcalibur updates all the
other Views and dialog boxes automatically.
Calibration File
This read only box shows the calibration method applied to the current
bracket or group.
When the calibration information for the current bracket was obtained from
the embedded processing method and not from a separate calibration file,
the box displays Embedded Calibration.
For non-bracketed sequences, the box displays the name of the calibration
file associated with the current Group in the sequence. To change the
calibration file, choose File > Replace Calibration. This option is not
available for bracketed sequences.
Results Grid Columns
Xcalibur lists samples under some or all of the headings described in
Table 17.
Table 17. Description of the columns in the Result grid
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Column Heading
Description
File Name
The raw file containing the acquisition data for this run
Sample Type
Standard, QC, Blank or Unknown
Sample Name
Sample name given to this sample when the sequence was
prepared in Sequence Setup
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The Results Grid
Table 17. Description of the columns in the Result grid
Working Directly With The
Grid
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Column Heading
Description
Integration Type
The method applied to integrating the peak. The choices are
Method, User and Manual
Area (or Height)
Integrated area (or height) under the detected peak (count secs or
counts)
ISTD Area
(or ISTD Height)
Integrated area (or height) under the Internal Standard peak (count
secs or counts)
Area Ratio
(or Height Ratio)
The area (or height) ratio between the selected peak and the
Internal standard
Specified Amount
The amount of the component at the Cal or QC level
Calculated Amount
Amount of component as determined by the response ratio and
calibration curve
% Diff
Percentage difference between the calculated amount and the
specified amount
% RSD
Relative Standard Deviation of the difference between the
calculated amount and the specified amount as expressed as a
percentage of the specified value
Peak Status
Low appears if the %Difference is < 0, High appears if
the %Difference is > 0 and Fail appears if the %Difference is
greater than the QC fail percentage test value
Level
The name of the calibration or QC level of the sample
Units
The units defined in the processing method for quantity or
concentration
RT
Retention time in minutes at the peak apex
Sample ID
Unique sample identification string given to this sample when the
sequence was prepared in Sequence Setup
Exclude
Indicates if the sample point is to be included or excluded from the
calibration curve for the current bracket or group
Change the information in any of the following columns by clicking the
appropriate grid cell:
•
Click the Sample Type cell and select Standard, QC, Blank or Unknown.
•
Click the Integration Type cell and select Method Settings, User Settings
or Manual Integration.
•
Click the Levels cell and select another defined level from the list.
•
Click the Exclude check box and select or clear the check box to exclude
or include the sample in the bracket calibration. Selecting excludes the
data and is indicated in the grid by Yes.
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The Results Grid
When a sample is shared between two brackets, you cannot change its
Sample Type. Xcalibur notifies you when a sample is part of two
overlapping brackets if you attempt to change its Integration Type, Level, or
Exclude state.
Results Grid Shortcut Menu
144
Most of the commands for manipulating the Results Grid are available on a
shortcut menu. Display this menu by right-clicking anywhere within the
grid. Use the menu to choose the following commands:
•
Choose Columns to display the Result List Column Hiding dialog box
(see Figure 66) to select the columns to be displayed in the results grid.
•
Choose Delete Selected Samples to remove the currently selected
sample(s) from the Results grid. Select samples by dragging a range
across one or more columns in the rows to be deleted. When you delete
Standards or QCs, the RSD% parameters are recalculated for the
bracket.
•
Choose Add Sample to display the Open Result Data File dialog box to
select a new file. Xcalibur adds this file to the current bracket and
displays it in the Results grid according to the new sort order. When the
sample(s) added are standards, Xcalibur recalibrates the bracket and
recalculates the RSD% parameters. RSD% is also recalculated if the new
sample is a QC sample.
•
Choose Copy Row to duplicate the selected row. When the copied
sample is a standard, Xcalibur recalibrates the bracket and recalculates
the RSD% parameters. RSD% is also recalculated if the copied sample
is a QC sample.
•
Choose Set Sorting Order to open the Quantitation Results Sorting
Order dialog box to set the sorting criteria for samples in the results grid.
•
Choose Send to Qual Browser to launch Qual Browser with the results
file for the currently selected sample.
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Hiding or Displaying Columns
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The Results Grid
Quan Browser’s Results Grid contains many columns. Choose to display
some or all of these columns using the Result List Column Heading dialog
box shown in Figure 66.
Figure 66. Result List Column Hiding dialog box
To open the Result List Column Hiding dialog box
1. Right-click within the Result Grid and choose Columns from the
shortcut menu.
2. Select the check box for a column heading to display it. Clear the check
box to hide the column.
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The Results Grid
Changing the Sort Order
To change the sort order for entries in the results grid, right-click the grid,
and choose Set Sorting Order from the shortcut menu. Xcalibur displays
the Quantitation Results Sorting Order dialog box shown in Figure 67.
Figure 67. Quantitation Results Sorting Order dialog box
Base the first sort order of the Results Grid view on any of the following
column headings or file properties:
•
•
•
•
•
•
•
•
•
•
•
•
•
<none>
%Difference
%RSD
Area/Height
Area/Height Ratio
Exclude
Filename
Integration Type
Level Name
Peak Status
Sample ID
Sample Type or
Acquisition Date
By default, Xcalibur sets the first order sort to the acquisition date of the file.
Select and sort with any of these sort options even if the corresponding
column is not currently displayed. For example, sort by Sample Type even if
you have selected the Sample Name check box in the Result List Column
Hiding dialog box. The second and third sort criteria can be any of the
remaining column headings or file properties, even if the column is
currently hidden.
Click Save As Default to replace the default sorting criteria with your new
selections.
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Chromatogram View
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Chromatogram View
The Chromatogram View displays the chromatogram for the currently
selected component from the currently selected result file. Most of the
commands for manipulating the Chromatogram View are available on a
shortcut menu.
This section contains the following topics
Chromatogram View
Shortcut Menu
Thermo Electron Corporation
•
Chromatogram View Shortcut Menu
•
Viewing Peak Information
•
Qualifier Peak Information
•
Spectrum Candidate Information
•
Setting User Peak Detection Parameters
•
Changing Display Options
Display a shortcut menu by right-clicking anywhere within the
chromatogram view. The menu contains the following commands:
Method Settings
Quan Browser uses the integration parameters
embedded within the processing method for the
selected component. The method for a given bracket
derives from the first result file.
User Settings
Quan Browser uses your settings, provided in the User
Identification Settings dialog box (see “Setting User
Peak Detection Parameters” on page 158).
Manual
Integration
Quan Browser integrates the chromatogram according
to the manually dragged baseline markers.
Show Peak Info
Displays the Peak Information dialog box. This shows
information about the peak or one of the peaks used by
the Spectrum search or Ion Ratio Confirmation
routines in a read only format (see “Viewing Peak
Information” on page 148).
User Peak
Detection
Settings
Displays the User Identification Settings dialog box.
Use this box to adjust the peak identification,
detection and integration parameters for the selected
component.
Display Options
Displays the Display Options dialog box. Use this box
to change chromatogram peak labeling.
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Chromatogram View
Manually Add
Peak
Use to place a baseline on the chromatogram plot.
When you use this option, Quan Browser changes the
Integration Type to Manual Integration. This option is
only available if Xcalibur has failed to detect a peak
using the existing method or user settings. Disable the
existing detection using the Set Peak To Not Found
Status command.
Update Expected This command updates the retention time specified in
Retention Time the processing method with the retention time that
Xcalibur detected.
Viewing Peak Information
Set Peak to Not
Found Status
Use this option to override the identification method
and classify the current component as Not Found.
When used, the chromatogram view re-displays over
the whole acquisition range.
Reset Scaling
Use this option to reset the chromatogram scaling to
show the full peak in a normalized window.
Quan Browser displays information about the currently displayed
component peak, qualifier ion or spectrum candidate in the Peak
Information dialog box. The title bar contains the component name. To
open this dialog box, right-click within the Chromatogram View and
choose Show Peak Info from the shortcut menu.
When the peak is a qualifier ion, the title bar contains the text Qual Ion
Mass xxx.x where the xxx.x represents the mass of the qualifier ion.
When the peak is for a Spectrum candidate, the title bar contains the text
Spectrum Candidate.
For a component peak, the Peak Information dialog box has 5 tabbed pages:
Info
Flags
More Flags
Suitability
Spectrum
Shows statistics about the chromatogram peak
Displays integration and detection flag results
Shows flags for detection, calibration and quantitation
thresholds
Shows system suitability test results
Displays the spectrum at the apex retention time
When the component peak is the leading Spectrum detection candidate, the
dialog box features an additional page labeled More Info. This page shows
information about ion coelution and ion ratio testing.
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Chromatogram View
For a qualifier ion, the dialog box has 7 tabbed pages. These include the
Info, Flags, More Flags, Suitability and Spectrum pages described above.
The two additional pages are:
More Info
Chro
Displays the results of the Ion Coelution and Ion
Ratio tests.
Displays the chromatogram for the qualifier ion.
For a spectrum candidate, the dialog box has three tabbed pages:
Info
Chro
Spectrum
Shows information about the chromatogram peak
and spectrum matching.
Displays the TIC for the spectrum candidate
Displays the spectrum at the apex of the spectrum
candidate chromatogram peak.
When the component peak is not found, then the dialog box consists of a
single tab labeled No Peak. This tab displays the text No Peak Found:
Cannot show Peak Info.
The Peak Information dialog box is read only. If you select other
components or samples, the dialog box is updated with peak information
for the displayed component chromatogram peak.
Info Page
The Peak Info page, shown in Figure 68, displays the following properties:
Left (min)
Apex (min)
Right (min)
Height
Area (cts-secs)
Baseline
Base Peak (m/z)
Signal to Noise
Expected RT
(min)
ISTD Response
Response Ratio
Calculated
Amount
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Left point in minutes of integration baseline
Location of apex in minutes
Right point in minutes of integration baseline
Height at peak apex
Area measured in count seconds
Baseline height directly beneath the apex
Mass to charge ratio of ion with largest response
Measured signal-to-noise
Expected retention time in minutes of peak
Area (or Height) of internal standard peak
Ratio of this peak’s area (or height) to the ISTD peak’s
area (or height)
Amount in sample as calculated with response ratio
and calibration curve
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Chromatogram View
Figure 68. Info Page of Peak Information dialog box
Flags Page
The Flags page, shown in Figure 69, displays information about peak
detection.
Figure 69. Flags page of Peak Information dialog box
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Chromatogram View
The displayed parameters are:
Detected By
A read only edit box which contains the description of
the detection method used. The available types are
Spectrum, Highest Peak and Nearest RT. In the case
where an LC method is used, only the Highest Peak
and Nearest RT are available.
Valid
A flag indicating whether or not the peak was
successfully detected.
Left Edge Type
Displays one of the following, based on the detection
method used during peak detection: Baseline, Valley,
Manual, Stripe, Tail, Tilt or Unknown.
Right Edge Type
Displays one of the following, based on the detection
method used during peak detection: Baseline, Valley,
Manual, Stripe, Tail, Tilt or Unknown.
The page also displays the state of the following calibration flags. If the flag
is true, its box is checked.
Thermo Electron Corporation
Saturated
Indicates that one or more of the scans within the peak
were saturated.
Calculated
Amount
Indicates that a quantitation calculation was
performed.
Valley Detect
Indicates that valley detection was enabled in the
processing method.
QC Failed
Indicates whether or not the sample failed a QC
check.
RT Ref OK
Indicates whether Xcalibur found the Retention Time
Reference component.
Response OK
Indicates that Xcalibur calculated a Response Factor.
Response Low
Indicates if the calculated amount was less than the
lowest level of the component and therefore
determined by extrapolation from the lowest level.
Response High
Indicates if the calculated amount was greater than the
highest level of the component and therefore
determined by extrapolation from the highest level.
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Chromatogram View
More Flags Page
The More Flags page, shown in Figure 70, displays the state of flags for
detection, calibration and quantitation thresholds. If the flag is true, its box
is checked.
Figure 70. More Flags page of Peak Information dialog box
The detection threshold flags (defined in Processing Setup in the Data Flags
dialog box accessed from the method’s Detection page) are:
Area
True if the peak area exceeds the defined absolute peak
area.
Height
True if the peak height exceeds the defined absolute
peak height.
The calibration and quantitation threshold flags (defined in Processing
Setup in the Calibration and Quantitation Flags dialog box accessed from
the method’s Calibration page) are:
152
R-squared
True if the R-squared threshold value (a test of the
goodness of fit of the calibration curve) is greater than
the threshold value, otherwise false.
Detection Limit
True if the quantified component concentration is less
than the Detection Limit threshold, otherwise false.
Linearity Limit
True if the quantified component concentration is less
than the Linearity Limit threshold, otherwise false.
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Suitability Page
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Chromatogram View
Quantitation
Limit
True if the quantified component concentration is less
than the Quantitation Limit threshold, otherwise false.
Carryover Limit
True if the quantified component concentration is less
than the Carryover Limit threshold; otherwise false.
The Suitability page, shown in Figure 71, displays the results of specific tests
that might have been performed (as determined by the System Suitability
parameters in the processing method) on the component peak to determine
its suitability to be considered a valid peak.
Figure 71. Suitability page of Peak Information dialog box
There are three possible responses for each test:
•
Passed
•
Failed
•
Not Tested
The tests reviewed on the Suitability page are:
Thermo Electron Corporation
Symmetrical
Determines if the peak is symmetrical about the apex.
Resolution
Determines if peaks are well resolved into individual
peaks.
Peak Width
Determines if peak width is within specified limits.
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Chromatogram View
Tailing
Determines if tailing has occurred.
Column
Overload
Determines if the column was overloaded during
acquisition.
Baseline Clipping Determines if the baseline is clipped (no noise) outside
the peak.
Spectrum Page
Signal-ToNoise Ratio
Determines if the minimum Signal-to-Noise ratio is
met.
Concave
Determines if the peak exhibits a concave depression
(local minimum) due to noise.
Saturation
Determines if the detector was saturated during
acquisition.
The Spectrum page, shown in Figure 72, displays the current spectrum at
the Apex retention time. No adjustments can be made to the plot.
Figure 72. Spectrum page of Peak Information dialog box
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Qualifier Peak Information
More Info Page
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Chromatogram View
When you select a qualifier ion, the Peak Information dialog box also
features More Info and Chro pages.
The More Info page (see Figure 73) displays the results of Ion Coelution
and Ion Ratio tests. In Processing Setup, these are defined in the Ion Ratio
Confirmation area on the Detection page for the method.
Figure 73. More Info page of Peak Information dialog box for a qualifier ion
Ion Coelution
Test Passed
Indicates whether the selected qualifier ion passes the
Coelution test. To do this, its mass chromatogram
must have a peak apex within the Qualifier
Coelution Window specified on the processing
method’s Detection page (see Chapter 2, “Processing
Setup”).
When the qualifier ion fails the Coelution test, the
Ion Ratio test is not performed and the Ion Ratio
Test area is not displayed.
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Ion Ratio Test
Passed
Indicates whether the selected qualifier ion passes the
Ion Ratio test. The Target ratio% and Absolute
Window% parameters display the results of the test.
Target Ratio
The calculated Target Ratio Percentage. See Chapter
2 for more details.
Absolute
Window
The calculated Absolute window Percentage. See
Chapter 2 for more details.
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Chromatogram View
Chro Page
The Chro page (see Figure 74) displays the qualifier ion mass chromatogram
within the component peak window. No adjustments can be made to the
plot.
Figure 74. Chro page of Peak Information dialog box for a qualifier ion
Spectrum Candidate
Information
After selecting a Spectrum candidate the Peak Information dialog box
features three tabbed pages: Info, Chro and Spectrum.
Spectrum candidates are only displayed if Spectrum detection was specified
in the processing method. Spectrum detection is only available if the GC
chromatography option is selected. See “Peak Detection for GC” on page 50
for more details.
Info Page
The parameters in the Peak Info area (see Figure 75) are the same as those
described for the standard peak and qualifier ion Info page.
See the Xcalibur online Help for a more detailed description of these
parameters and Spectrum detection.
Note If Xcalibur detects the main component peak using Spectrum
detection, Xcalibur displays the standard Info page with the Spectrum
Results area on an additional tabbed page called More Info.
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Chromatogram View
Figure 75. Info page of Peak Information dialog box for a Spectrum candidate
The Spectrum results group parameters are:
Forward
Calculated forward matching factor for the spectrum
candidate and the reference spectrum
Reverse
Calculated reverse matching factor for the spectrum
candidate and the reference spectrum
Match
Calculated probability matching factor for the
spectrum candidate and the reference spectrum
Chro Page
The Chro page displays a TIC plot for the Spectrum candidate. The plot
has the width used by the component peak display.
Spectrum Page
The Spectrum page is effectively the same as that described for a standard
peak. It displays the spectrum corresponding to the apex of the spectrum
candidate chromatogram.
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Chromatogram View
Setting User Peak
Detection Parameters
When you first open a sequence in Quan Browser component
identification, Xcalibur gets peak detection, calibration, and quantitation
information from the Result file.
Within Quan Browser, apply unique peak detection parameters to the
chromatogram using the User Identification Settings dialog box. This box
duplicates the parameters available in the Identification and Detection pages
in the Quan View of Processing Setup, so you can adjust and test the effect
of different values. You can:
•
Save the settings in a Quan Browser file (*.xqn). Choose File > Save or
File > Save As.
•
Export the User Settings as a full processing method using the File >
Export Method menu command.
To open the User Identification Settings dialog box, right-click the
Chromatogram View and choose User Peak Detection Settings from the
shortcut menu.
The User Identification Settings dialog box for the ICIS peak detection
algorithm consists of the following tabbed pages:
Identification
Parameters used by Xcalibur to identify the selected
component in the chromatogram
Detection
Settings used by Xcalibur to confirm peak detection
ICIS Integration
ICIS peak detection algorithm parameters applied to
the component peak
ICIS Advanced
ICIS advanced parameters used by Xcalibur during
peak identification and integration
Flags
Detection flagging thresholds applied to the selected
component to validate detection
The User Identification Settings dialog box for the Genesis peak detection
algorithm consists of the following tabbed pages:
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Identification
Parameters used by Xcalibur to identify the selected
component in the chromatogram
Detection
Settings used by Xcalibur to confirm peak detection
Genesis
Integration
Genesis peak detection algorithm parameters applied
to the component peak
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Chromatogram View
Genesis
Advanced
Genesis advanced parameters used by Xcalibur during
peak identification and integration
Flags
Detection flagging thresholds applied to the selected
component to validate detection
The User Identification Settings dialog box for the Avalon peak detection
algorithm consists of the following tabbed pages:
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Identification
Parameters used by Xcalibur to identify the selected
component in the chromatogram
Detection
Settings used by Xcalibur to confirm peak detection
Avalon
Avalon peak detection algorithm parameters
Integration applied to the component peak
Flags
Detection flagging thresholds applied to the selected
component to validate detection
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Chromatogram View
Identification Page
Xcalibur uses the parameters on the Identification page (see Figure 76) to:
•
Generate a chromatogram from raw data
•
Identify the component within the chromatogram
The parameters are identical to those on the Identification page of Quan
View in Processing Setup. These are described in “Identification” on
page 30.
Figure 76. User Identification Settings - Identification page
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Detection Page
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Chromatogram View
Xcalibur uses the parameters on the Detection page (see Figure 77) to
confirm the identity of the component within the retention time window
defined by the Identification settings. The options available on this page
depend on the Chromatography mode selected in the original processing
method used to generate the raw data (see “Detection” on page 41).
The parameters are identical to those in the Peak Detection area on the
Detection page of Quan View in Processing Setup. These are described in
“Detection” on page 41.
Figure 77. User Identification Settings - Detection page
The controls on the Detection page vary depending upon whether you are
using a GC or LC and also whether the detection method is Spectrum,
Highest Peak, or Nearest RT. For more information, see the Xcalibur online
Help.
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Chromatogram View
Genesis Integration Page
Xcalibur applies the settings on the Integration page (see Figure 78) during
peak integration.
The parameters are identical to those in the Genesis Peak Integration area
on the Detection page of Quan View in Processing Setup. These are
described in “Peak Integration” on page 43.
Figure 78. User Identification Settings - Genesis Integration page
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Genesis Advanced Page
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Chromatogram View
Xcalibur applies the Genesis Advanced page (see Figure 79) parameters
during Genesis peak detection and integration.
The parameters are identical to those in the Advanced Detection Options
dialog box accessed from the Advanced button on the Detection page of
Quan View in Processing Setup.
Figure 79. Genesis Advanced page of User Identification Settings dialog box
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Chromatogram View
Flags Page
Xcalibur applies the parameters on the Flags page (see Figure 80) to test the
validity of detected peaks.
The parameters are identical to those in the Data Flags dialog box accessed
from the Detection page of Quan View in Processing Setup. These are
described in “Data Flags” on page 61.
Figure 80. Flags page of User Identification Settings dialog box
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Changing Display Options
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Chromatogram View
Use the Display Options dialog box (see Figure 81) to change the way Quan
Browser displays the Chromatogram View. To open this dialog box,
right-click on the Chromatogram View and choose Display Options from
the shortcut menu.
Figure 81. Display Options dialog box
For more information about these settings, see the Xcalibur online Help.
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Calibration Companion View
Calibration
Companion View
Calibration Companion
View Shortcut Menu
Excluding a Data Point
166
To display the Calibration Companion View, choose
View > Set Companion View > Show Calibration Curve or use the
shortcut menu from within the Companion View. If the companion View is
currently displaying a spectrum plot, right-click within it and choose Show
Calibration Curve.
Right-click on the calibration curve plot to display the Calibration
Companion View shortcut menu. The following menu commands are
available:
Calibration
Settings
Displays the Calibration Settings dialog box. Use this
box to change ISTDs, apply a new calibration curve,
adjust levels, and change flag thresholds. Normally,
Xcalibur uses the settings from the embedded
processing method.
Save Calibration
File
Displays the Save As dialog box. Use the Save As dialog
box to save the calibration settings in a calibration file
with an .xcal extension.
Exclusion List
Displays the Cal Exclusion List dialog box. Use this
box to exclude levels and all associated samples from
the calibration.
Show Spectrum
Plot
Changes the Companion View to the Spectrum
Companion View.
Reset Scaling
Resets the scaling of the calibration curve.
Copy Graph
Copies the calibration graph to the Microsoft
Windows Clipboard. It can then be pasted into other
applications for presentation purposes.
To exclude a data point from the calibration curve, right-click on it and
choose Exclude from the shortcut menu. If the data point is currently
included in the calibration, Xcalibur:
•
Recalculates the calibration curve without it.
•
Updates the corresponding Peak Status and Exclude field in the Results
Grid and Exclusion List to show that it is excluded.
•
Redraws the excluded data point as an unfilled square.
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Restoring an Excluded Data Point
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Calibration Companion View
To restore a data point that you have previously excluded, right-click on the
data point and choose Include from the shortcut menu. Xcalibur:
•
Incorporates the data point into the calibration and recalculates the
curve.
•
Updates the corresponding Peak Status and Exclude field in the Results
Grid and Exclusion List to show that the point is now included.
•
Redraws the included data point as a filled square.
Include or exclude samples that are shared between brackets. Their status
will be unique to the bracket. For example, excluding a shared sample in
bracket 1 will have no effect on the inclusion status in bracket 2.
Adjusting Calibration
Settings
When you first open a sequence in Quan Browser component
identification, Xcalibur performs peak detection, calibration and
quantitation according to the settings of the associated processing method.
Within Quan Browser, apply unique calibration parameters and level
definitions to the chromatogram using the Calibration Settings dialog box.
This box duplicates most of the parameters available in the Calibration and
Levels pages in the Quan View of Processing Setup, so you can adjust and
test the effect of different calibration and quantitation parameters. You can:
•
Save the settings in a Quan Browser file (*.xqn). Choose File > Save or
File > Save As.
•
Export the Calibration Settings as a full processing method using the
File > Export Method menu command.
To open the Calibration Settings dialog box, right-click on the Calibration
Companion View and select Calibration Settings from the shortcut menu.
The dialog box consists of five tabbed pages:
Thermo Electron Corporation
Type
Use this setting to change the sample type: Target or ISTD.
Curve
Use this setting to change the calibration curve calculation
and plotting methods.
Levels
Use this setting to change level definitions for a target
compound.
Isotope %
Use this setting to adjust the isotope contributions of ISTD
and Target compounds.
Flags
Use this setting to adjust the threshold values for calibration
and quantitation flags.
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Calibration Companion View
Type Page
The Type page (see Figure 82) displays the component type, target
compound or ISTD. For a target compound, change the ISTD to be used
with it. For ISTDs, change the Amount and Units.
The parameters are identical to those in the Component Type and ISTD
areas on the Calibration page of Quan View in Processing Setup. These are
described in “Calibration” on page 64.
Figure 82. Type page of Calibration Settings dialog box
Curve Page
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Use the Curve page (see Figure 83) to change the way Xcalibur calculates
and plots the calibration curve from the data points.
Calibration
Curve
Use this list to change the type of algorithm applied to
fit the data points. The available types are Linear,
Quadratic, Linear Log-Log, Quadratic Log-Log,
Average RF, Point-to-Point, Cubic Spline and Locally
Weighted
Weighting
Use these options to change the weighting applied to
the individual data points. The available types include
Equal, 1/X, 1/X^2, 1/Y, 1/Y^2 and 1/s^2
Response
Use these options to change the component response
used in the calibration curve: area or height.
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Units
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Calibration Companion View
Use this box to change the units label used in the
Calibration Companion View, on the Levels page, and
in reports.
These parameters are identical to those in the Target Compounds area on
the Calibration page of Quan View in Processing Setup. These are described
in more detail in “Calibration” on page 64.
Figure 83. Curve page of Calibration Settings dialog box
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Calibration Companion View
Levels Page
Use the Levels page (see Figure 84) to change Calibration and QC level
names and their associated amounts. It is not available for ISTD
components (the page displays the message This component does not use
levels).
These parameters are identical to those on the Levels page of Quan View in
Processing Setup. These are described in more detail in “Levels” on page 74.
Figure 84. Levels page of Calibration Settings dialog box
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Isotope % Page
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Calibration Companion View
Use the Isotope% page (see Figure 85) to correct data for:
•
An impurity in the internal standard compound that elutes at the same
time as the target compound.
•
An impurity in the target compound that elutes at the same time as the
internal standard.
These parameters are identical to those in the Correction For Isotope
Contribution dialog box, accessed from the Calibration page of Quan View
in Processing Setup. These are described in more detail in “Calibration” on
page 64.
Figure 85. Isotope% page of Calibration Settings dialog box
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Calibration Companion View
Flags Page
Use the Flags page, shown in Figure 86, to change the threshold values for
calibration and quantitation flags for the selected compound. Enter a value
of 0 to force the flag to be false.
These parameters are identical to those in the Data Flags dialog box,
accessed from the Calibration page of Quan View in Processing Setup.
These are described in more detail in “Calibration” on page 64.
Figure 86. Flags page of Calibration Settings dialog box
When you edit any of the values in the Quantitation Flags group, Xcalibur
checks that the relationships between the four fields are maintained. When
an entry in one parameter forces a change to occur in another, Xcalibur
displays the Automatic Adjustment warning dialog box (see Figure 87).
Figure 87. Automatic Adjustment warning dialog box
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Excluding Calibration Levels
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Calibration Companion View
Use the Cal Exclusion List dialog box (see Figure 88) to exclude levels from
the calibration (see “How Quan Browser Works” on page 131 for a
description of the procedures used to generate this list). This is particularly
useful when you cannot use the Include and Exclude commands because of
overlapping points on the calibration curve. If you are using a named
calibration file, levels might not be represented in the Results Grid but will
always be listed in the Cal Exclusion List dialog box.
Figure 88. Cal Exclusion List dialog box
To open the dialog box, right-click on the Calibration Companion View
and choose Exclusion List from the shortcut menu.
The dialog box lists all the replicates used in the current bracket or group
and their exclusion status. Levels are listed under the following headings:
Level
Shows the name for the level
Expected
Displays the expected amount for level
% Diff
Shows the percentage difference between measured
and expected amounts
Exclude
Denotes excluded levels by the word Yes
To exclude a level, click in the Exclude column adjacent to the level to be
excluded.
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Calibration Companion View
Xcalibur then:
•
Recalculates the calibration curve without any samples using the level.
•
Updates the corresponding Peak Status and Exclude fields in the Results
Grid to show that the samples are excluded.
•
Redraws excluded data points as unfilled squares.
To restore an excluded level, click in the Exclude column adjacent to the
level (on the word Yes) to be restored. Xcalibur then:
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•
Incorporates all samples using the level into the calibration and
recalculates the curve.
•
Updates corresponding Peak Status and Exclude fields in the Results
Grid to show that the points are now included.
•
Redraws the included data point as filled square.
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Spectrum Companion
View
Reviewing Quantitation in Quan Browser
Spectrum Companion View
To display the Spectrum Companion View choose View >
Set Companion View > Spectrum Plot.
Or, use the shortcut menu from within the Companion View. If the view is
currently displaying the calibration curve, right-click on it and choose View
Spectrum Plot.
Use the Spectrum Companion View to examine the identity of peaks and
other features (such as the background) in the chromatogram. For further
analysis, including library matching of spectra, export data to Qual Browser
using the Send to Qual Browser option in the Result List shortcut menu.
Initially, Xcalibur displays the spectrum corresponding to the scan at the
current chromatogram’s apex retention time. If no peak was detected,
Xcalibur displays the expected retention time as defined by the processing
method.
Change the spectrum companion view by:
•
Selecting options on the shortcut menu.
•
Pinning the cell and selecting a scan in the chromatogram view.
Access the shortcut menu by a right-click within the Spectrum Companion
View. The menu contains four viewing options:
Spectrum at Peak Apex Displays the spectrum at the current
chromatogram’s apex retention time.
Spectrum at Peak Left Displays the spectrum at the current integration
Edge
baseline’s left edge retention time.
Spectrum at Peak
Displays the spectrum at the current integration
Right Edge
baseline’s right edge retention time.
Show Calibration
Changes the Companion view to display the
Curve
calibration curve.
Reset Scaling
Resets the view to display the full spectrum in a
normalized window.
Click the pin in the spectrum companion view to display spectra from other
regions of the chromatogram. Any click within the Chromatogram View
will then result in the Spectrum Companion View being updated with a
spectrum corresponding to the scan at the clicked retention time.
In an active (pinned) Spectrum View, use the cursor to rescale the plot. The
Zoom menu commands and toolbar buttons are also effective.
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Reports
Reports
To generate reports for the current sequence, either
•
The Reports Dialog Box
Click the Reports button on the toolbar or choose View > Reports
dialog.
The Reports dialog box (see Figure 89) duplicates the Reports view in
Processing Setup. When opened, it displays the reports specified in the
processing method associated with the active sequence. The displayed
parameters might change as you select different brackets.
Figure 89. Reports dialog box
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Reports
For a description of the Sample Reports and Summary Reports tables, see
“Reports” on page 86. The Reports dialog box features the following
additional parameters:
Selecting Samples for
Reports
Include Sample
Reports
Select this check box to include sample reports in
any print run.
Include Summary
Reports
Select this check box to include summary reports
in any print run.
Select Samples
Click Select Samples to open the Select Report
Samples dialog box and choose samples in the
sequence for report generation and printing.
Print Reports
Click Print Reports to initiate report generation
and printing as defined in the dialog box.
Use the Select Samples button in the Reports dialog box to open the Select
Report Samples dialog box (see Figure 90) and choose the samples to be
processed during report generation.
Figure 90. Select Report Samples dialog box
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Reports
To include a sample in report processing
1. Click its name in the Sample Choices list.
2. Click Add.
Select multiple files using the SHIFT and CTRL keys:
•
Hold the SHIFT key down to select a range of samples.
•
Hold the CTRL key down to select multiple samples.
3. Click Add All to add all the samples listed in the Sample Choices list to
the Selected Samples list.
To exclude a sample from report processing
1. Click its name in the Selected Samples list.
2. Click Remove.
3. Click Remove All to remove all samples listed in the Selected Samples
list.
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Quan Browser
Procedures
Editing a Sequence
Reviewing Quantitation in Quan Browser
Quan Browser Procedures
This section describes procedures for the common tasks associated with
reviewing calibration and quantitation results.
•
Editing a Sequence
•
Reviewing Samples
•
Reviewing a Chromatogram
•
Modifying Detection and Identification
•
Integrating Chromatogram Peaks Manually
•
Modifying Calibration Parameters
To review and edit an existing sequence
1. Inspect the sequence. Verify that the correct raw files are listed in the
Results Grid. Make sure that each raw file in the sequence is properly
associated with a calibration level, QC level, blank, or unknown.
2. To remove raw files from the sequence:
a. Select the row(s) in the sequence to delete.
b. Right-click the sequence to display a shortcut menu.
c. Choose Delete Selected Samples to delete the selected row(s) in the
sequence.
3. To add raw files to the sequence:
a. Select the row in the sequence above where the new row (sample)
will be located.
b. Right-click the Results Grid to display a menu.
c. Choose Add Sample to open the Open Rawfile dialog box.
d. Locate the raw file to add to the sequence and click Open to open
the Add Sample dialog box.
e. Specify sample information in the Add Sample dialog box and click
OK.
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Quan Browser Procedures
4. To change the sample type:
a. Click the Sample Type list down-arrow to display a list of sample
type options.
b. Select the new sample type. Quan Browser displays the new sample
type in the Sample Type list.
5. To save the sequence with all current detection and calibration settings,
choose File > Save or File > Save As. The resulting Xcalibur Quan file
(extension .xqn) contains all the necessary information required to
recreate the current Quan Browser session.
Reviewing Samples
To review and rework samples:
1. Select a component from the Component List. Xcalibur automatically
updates the Result List, Chromatogram, and Companion Views.
2. Click the Standards tab to display calibration standards results.
3. Inspect the calibration curve in the Companion View. If it is not
currently displayed, either:
•
Choose View > Set Companion View > Show Calibration Curve,
or right-click in the Companion View and choose Show
Calibration Curve from the shortcut menu.
4. Inspect the calibration curve according to the criteria used in your
laboratory.
5. Select a row in the Results Grid. Each row corresponds to a data file.
6. Check the peak detection and integration fields in the Result Grid for
peak detection and integration problems. Make sure that the selected
data file corresponds to the correct level and sample type.
7. Inspect the plot in the Chromatogram View.
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•
Confirm that Xcalibur found the peak. Xcalibur shades found peaks
gray and marks the starting and ending points with square
integration markers.
•
Confirm that Xcalibur integrated the peak properly. Check that the
shaded area accurately represents the contribution of the component
to the chromatogram.
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Quan Browser Procedures
8. Modify the peak detection and integration settings:
•
Right-click the Chromatogram View and choose User Peak
Detection Settings. Xcalibur opens the User Identification Settings
dialog box.
•
Click the Detection tab to open the Detection page to change the
detection method. Modify the settings.
•
If you have problems with noise in the peak, unresolved peaks, or
peak tailing, click the Integration tab to open the Integration page.
Modify the settings.
•
If baseline noise is interfering with peak identification or
integration, click the Advanced tab to open the Advanced page.
Advanced options should only be used if the standard options do
not provide sufficiently selective detection criteria.
•
Manually integrate the peak. Manually change the starting and
ending points and baseline of the peak by clicking and dragging the
square integration markers to the desired location.
9. Repeat the procedure for the remaining components.
10. Repeat the procedure for all the data files to review.
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Quan Browser Procedures
Reviewing a
Chromatogram
To review a chromatogram
1. Right-click in the Chromatogram View and choose Show Peak Info.
Xcalibur displays the Peak Information dialog box. Review the
chromatogram peak data:
•
Review the properties of the detected peak on the Info page.
•
Review the integration information and flags on the Flags page.
•
Review the System Suitability test results on the Suitability page.
•
Review the spectrum for the peak apex scan on the Spectrum page.
2. Adjust the chromatogram in Chromatogram View:
•
Change detection or integration parameters. See “Modifying
Detection and Identification” on page 183.
•
Manually integrate peaks. See “Integrating Chromatogram Peaks
Manually” on page 184.
•
Change chromatogram peak labeling. To change the labels,
right-click in the Chromatogram View and choose Display
Options to open the Display Options dialog box. Click the Labels
tab to open the Labels page. Select the labels to display.
3. To view spectra for the chromatogram peak, display the Spectrum
Companion View:
•
Choose View > Set Companion View > Show Spectrum Plot from
the Quan Browser menu or right-click the Companion View to
open a shortcut menu and choose Show Spectrum Plot.
4. View spectra across the chromatogram. Pin the Spectrum Plot
Companion View. Click points of interest in the chromatogram to view
the corresponding spectrum.
5. To carry out a detailed qualitative analysis of the chromatogram, export
the results file to Qual Browser. Right-click on the Results Grid and
choose Send to Qual Browser.
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Modifying Detection and
Identification
Reviewing Quantitation in Quan Browser
Quan Browser Procedures
To modify and test component peak detection criteria
1. Review the displayed data for the selected component to determine if
the results are consistent with your expectations:
•
Are there peaks that were not found?
•
Are neighboring peaks resolved?
•
Are tailing peaks detected properly?
2. To modify detection criteria, right-click in the Chromatogram View and
choose User Peak Detection Settings to open the User Identification
Settings dialog box.
3. To change the chromatogram trace or adjust the retention time window,
modify the settings in the Integration tab.
4. To change the detection method, modify the settings in the Detection
tab.
5. If you have identified problems with noise in the peak, unresolved
peaks, or peak tailing, modify parameters on the Integration page.
6. If baseline noise is interfering with peak identification or integration,
modify the settings in the Advanced tab. Use advanced options only if
the standard options do not provide sufficiently selective detection
criteria.
7. Save your settings as a new processing method. Choose File >
Export Method.
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Reviewing Quantitation in Quan Browser
Quan Browser Procedures
Integrating Chromatogram
Peaks Manually
Figure 91.
Integrate peaks manually in either of two ways. The first way is to use the
cursor to drag the baseline endpoints to new positions.
See Figure 91 (a) and (b).
Chromatogram (a) shows a partially integrated peak and chromatogram (b) shows a manual
integration of the peak achieved by dragging the baseline endpoint to a new location.
Use the second way when Xcalibur has not detected the peak of choice:
1. Right-click on the Chromatogram View again and choose Manually
Add Peak. Xcalibur changes the cursor shape to denote the mode.
2. In the chromatogram, manually integrate the peak by clicking on one
side of the peak and, while holding down the mouse button, dragging
the mouse across the peak to define the point on the other side.
Repeat the procedure for other samples and components as required. To
restore automatic peak detection and integration, right-click in the
Chromatogram View and choose Method Settings or User Settings.
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4
Modifying Calibration
Parameters
Reviewing Quantitation in Quan Browser
Quan Browser Procedures
To modify the sequence calibration
1. Select a target component. Xcalibur automatically updates the Result
Grid, Chromatogram and Companion Views.
2. Click the Standards tab to display calibration standards results.
3. Inspect the calibration curve in the Companion View. If it is not
currently displayed, do one of the following:
•
Choose View > Set Companion View > Show Calibration Curve
or right-click in the Chromatogram View and choose
Set Companion View > Show Calibration Curve from the
shortcut menu.
4. Inspect the calibration curve according to the criteria used in your
laboratory. The Calibration Companion View displays the calibration
equation, the goodness of fit parameter, R2, and the weighting, W.
To view calibration and quantitation flags
1. Right-click on the Calibration Companion View and choose
Calibration Settings from the shortcut menu.
2. Select the Flags tab.
3. To exclude a point or sample from the calibration curve, right-click on it
and choose Exclude from the shortcut menu. To include a previously
excluded point, right-click on it and select Include from the shortcut
menu.
4. To exclude a level, right-click the Calibration Companion View and
choose Exclusion List from the shortcut menu to open the Cal
Exclusion List dialog box for the selected component.
•
To exclude a level, click in the Exclude column adjacent to the level
to be excluded.
•
To restore an excluded level, click in the Exclude column adjacent to
the level (on the word Yes) to be restored.
5. To adjust the calibration settings, right-click on the Calibration
Companion View and choose Calibration Settings from the shortcut
menu. Xcalibur opens the Calibration Settings dialog box.
Thermo Electron Corporation
Xcalibur: Getting Productive with Quantitative Analysis
185
4
Reviewing Quantitation in Quan Browser
Quan Browser Procedures
•
To adjust the ISTD associated with the component, select a new
ISTD on the Type page.
•
To adjust the calibration equation, weighting, or units, make new
selections and entries on the Curve page.
•
To view the calibration or QC levels, open the Levels page.
•
To make corrections for isotope contributions to ISTD or Target
components, enter new values on the Isotope% page.
•
To change calibration and quantitation flag thresholds, enter new
values on the Flags page.
•
To apply any changes to the sequence, click Apply.
6. To export the calibration settings with peak integration and detection
parameters as a new method, choose File > Export Method.
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Thermo Electron Corporation
Index
A
Absolute Window text box 155
Acquisition Queue page 91, 124
pausing 126
purging 126
resuming 126
Add Samples command 144
Adding
standards to a sequence 105
Amount (Cal Level) text box 75
Amount (ISTD) text box 66
Amount (QC Level) text box 75
Apply Changes dialog box 18
Area 143
ratio 143
Area Scan Window text box 59
Area Tail Extension text box 59
Area Threshold flag 61, 152
Automating analysis 91
Avalon Event List dialog box 58
Avalon peak detection algorithm 31, 43
User Identification Settings dialog box 159
Average RF 68
B
Baseline 63
and noise window 63
minimum number of scans in 63
noise tolerance 63
Baseline clipping 83
Baseline clipping suitability test 154
Batch Reprocess Setup dialog box 122
Blanks 12, 105
Bracket Type group box 102
Brackets 140
Brackets/Groups In Use combo box 142
Browsers 129
C
Cal Exclusion List dialog box 173
Cal Level text box 75
Thermo Electron Corporation
Calculated amount 143
Calculated Amount flag 151
Calibration
file 107
flag 73
modifying parameters 185
replicates 132
Set Companion View list 140
Calibration Companion view 137, 166
Calibration curve 67, 139
description 3
editing samples 180
excluding a point 166
restoring a point 167
units 68
using external standard (figure) 5
using internal standard (figure) 7
Calibration File text box 142
Calibration Options dialog box 65
Calibration page 64
Calibration settings
Curve page 168
Flags page 172
Levels page 170, 171
Type page 168
Calibration Settings dialog box 166, 166, 167, 168
Calibration standards 11
Carryover Limit flag 153
Carryover Limit text box 73
Cells
states 24
Chro page 156, 157
Chromatogram
cursor actions 24
preview 22
trace 32
Chromatogram view 139
Context menu 147
editing samples 180
reviewing 182
working in 147
Chromatography Options dialog box 42
Column Arrangement dialog box 96
Column overload 82
suitability test 154
Columns command 144, 145
Xcalibur: Getting Productive with Quantitative Analysis 187
Index: D
Columns in sequences
arranging 96
changing user labels 97
Component List text box 139
Components list
Processing Setup 17
Concave suitability test 154
Continuing calibration method 132
Contribution Of ISTD To Target Compound text box 70
Contribution Of Target Compound To ISTD text box 71
Copy Graph command 166
Copy Row command 144
Correction For Isotope Contribution dialog box 69
Cubic spline 68
Cursor actions
active 24
pinning 24
Cursor actions in Processing Setup 24
D
Data Flags dialog box 61
Delete Selected Samples command 144
Details Of Selected Analysis dialog box 127
Detected By flag 151
Detecting components in a chromatogram 41
Detection
limit 4, 73
Detection Limit flag 152
Detection page 41
peak detection 49
User Identification Settings dialog box 161
Diff% 143
Dilution Factor text box 95
Disk Space dialog box 113
Display Options dialog box
Quan Browser 165
E
Editing
a sample 180
a sequence 108, 179
Enable Warnings command 18
Exclude command 143
Exclusion List command 166, 173
Expected text box 35
Export Sequence dialog box 115
Exporting
sequence 115
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Xcalibur: Getting Productive with Quantitative Analysis
External calibration file 132
External standards
considering variables for 5
definition 5
using, for quantitation 5
F
Failure Threshold text box 81
Figures
calibration curve
using external standard 5
using internal standard 7
integrated chromatographic peak 3
File name 142
base 100
File Name text box 95, 106
File Path text box 95, 106
Fill Down dialog box 108
First Peak option button 62
Flags
area threshold 61
baseline clipping 83
carryover limit 73
column overload 82
data 61
detection limit 73
height threshold 61
linearity limit 73
minimum signal-to-noise ratio 83
peak width 81
quantitation limit 73
resolution threshold 79
R-squared 73
symmetry threshold 80
tailing 81
Flags button 61, 72
Flags page 150
Calibration Settings dialog box 172
User Identification Settings dialog box 164
Forward Matching factor 157
G
Genesis
Advanced Detection Options dialog box 58
peak detection algorithm 31, 43
User Identification Settings dialog box 158
Go To Line Number dialog box 111
Groups 132
Thermo Electron Corporation
Index: H
H
Height 143
ratio 143
Height Threshold flag 152
Home Page
Information view 124
Isotope button 69
ISTD 65
area 143
assigning to a target 67
correcting for contribution to target 70
height 143
L
I
ICIS
Advanced page
User Identification Settings dialog box 163
Advanced Parameters dialog box 58, 58
Detection page
peak integration 46
Integration page
User Identification Settings dialog box 162
peak detection 50
peak detection algorithm 31, 43
User Identification Settings dialog box 158
Identification Options dialog box 58, 62
Identification page
Expected text box 35
mass range 34
Retention Time Window text box 35
scan filter 31
trace 32
User Identification Settings dialog box 160
wavelength range 34
Import Sequence dialog box 98
Include command 167
Include Sample Reports check box 177
Include Summary Reports check box 177
INCOS Noise method 59
INCOS Noise option 59
Info page 149, 156
Information view
Acquisition Queue page 124
Injection Volume text box 95, 107
Instrument Method text box 95, 106
Integration Type option box 143
Internal standards (ISTDs)
choosing 7
considering variables for 6
definition 6
using, for quantitation 6
International dialog box 99
Interpreting data 129
Ion Coelution test 155
Ion ratio confirmation 54
Ion Ratio test 155
Thermo Electron Corporation
Labeling peaks 165
LCQUAN
acquiring and processing data with, overview 13
quantitative analysis procedure 13
Left Edge Type flag 151
Level 143
Level text box 95
Levels page
Calibration Settings dialog box 170, 171
Processing Setup 74
Limit
of detection 4
of quantitation 4
Limit Scan Wavelength check box
peak purity 85
Linear 68
Linear log-log 68
Linearity Limit flag 152
Linearity Limit text box 73
List separator character 116
Locally weighted 68
Lower quantitation limit 4
M
Macros 89
Manual Integration command 147
Manually Add Peak command 148
Mass 34
Match by Position option 111
Match by Sample ID option 111
Match Probability factor 157
Max Peak Width text box 81
Method Settings command 147
Min Peak Width text box 81
Minimum
number of scans in baseline 63
signal-to-noise ratio 83
Minimum Peak Height text box 50
Minimum Peak Width text box 59
More Flags page 152
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189
Index: N
More Info page 155
Multiplet Resolution text box 59
N
New Sequence Template dialog box 99
Non-bracketed sequence 132, 140
Non-overlapped, bracket type 134
Number of peak widths for noise detection 83
O
Open, bracket type 133
Opening
files in Quan Browser 137
raw file in Processing Setup 22
sequence in Sequence Setup 94, 94
Origin 68
Overlapped, bracket type 134
P
Path 106
Pausing the acquisition queue 126
Peak
classification parameters 81
detection 49
height
column overload testing 82
peak width testing 81
symmetry threshold 80
integration 46
labeling 147, 165
parameters 59
status 143
width 81
suitability test 153
Peak Coverage text box
peak purity 85
Peak Detection Settings command
User Identification Settings dialog box 158
Peak Height text box
peak tailing 81
Peak Information dialog box 148
Chro page 156, 157
Flags page 150
Info page 149, 156
More Flags page 152
More Info page 155
qualifier ions 155
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Xcalibur: Getting Productive with Quantitative Analysis
spectrum candidates 156
Spectrum page 157
Suitability page 153, 154
Peak purity
Enable check box 85
Limit Scan Wavelength check box 85
PDA chromatograms 85
Peak Coverage text box 85
Scan Threshold text box 85
Wavelength Range text box 85
Peak Purity page 84
Peaks
integrated (figure) 3
Percent Test (QC Level) text box 75
Pinning 25
Point-to-point 68
Previewing processing 22
Previews
active 24
pinning 24
Print
sequence 112
Print Preview dialog box 113
Print Reports button 177
Print Selection dialog box 113
Process Method text box 106
processing actions 121
Processing Method text box 95
Processing Setup 16
Apply Changes dialog box 18
auto open raw file 20
buttons 18
Calibration Options dialog box 65
Calibration page 64
Cancel button 18
Chromatography Options dialog box 42
Correction For Isotope Contribution dialog box 69
customizing setup 20
Data Flags dialog box 61
Identification Options dialog box 62
Load Last Processing Method option button 20
OK button 18
Open Raw File command 22
Programs view 89
Quan view
Calibration Options dialog box 65
Calibration page 64
Correction For Isotope Contribution dialog box 69
Data Flags dialog box 61
Detection page 41
Identification Options dialog box 62
Levels page 74
pages 21
Thermo Electron Corporation
Index: Q
Peak Purity page 84
Standard Dilution dialog box 76
System Suitability page 78
zoom commands 28
Reports view 86
Settings dialog box 20
Spectrum Options dialog box 52
Standard Dilution dialog box 76
view bar 17
Programs 120
Programs view 89
Q
QC Failed flag 151
QC Level table 75
QC Level text boxes 75
Quadratic
log-log 68
Qualifier ions 155
Quality control (QC) samples 11
Quan
quantitative reprocessing option 123
view 22, 23
Quan Browser 129
Cal Exclusion List dialog box 173
Calibration Settings dialog box 167
Chromatogram view 139
component list 139
Display Options dialog box 165
getting started 134
menu bar 137
opening files 134
Peak Information dialog box 148, 148
Quantitation Results Sorting Order dialog box 146
Reports dialog box 176
Result List Column Hiding dialog box 145
results grid 139
Select Report Samples dialog box 177
Set Companion View list 139
title bar format 137
toolbar 137
User Identification Settings dialog box 158
View Sample Types dialog box 135
window features 136
Quan view 21
Calibration page 64
Detection page 41
Levels page 74
Peak Purity page 84
previewing processing 22
System Suitability page 78
Thermo Electron Corporation
using interactively 23
using the toolbar 28
zoom commands 28
Quantitation
flags
carryover limit 73
detection limit 73
linearity limit 73
quantitation limit 73
Quantitation Flags dialog box 172
Quantitation Limit flag 153
Quantitation Limit text box 73
Quantitation limits 4
Quantitation range 4
Quantitation Results Sorting Order dialog box 146
Quantitative analysis
definition 2
discussion 3
reprocessing data 123
sources of error 6
steps of 2
techniques 3
using external standards for 5
using internal standards for 6
Queue manager
Details Of Selected Analysis dialog box 127
updating the display 126
Queue Manager window 126
R
Removing jobs from the acquisition queue 126
Repetitive Noise option 59, 59
Replace Calibration command 142
Replicates 132
Reports
sample 87
Reports command 138, 138
Reports dialog box 176
Select Samples button 177
Reports view 86
Reprocessing 122
Rescaling a preview display 28
Reset Scaling command 148, 166, 175
Resolution
formula 79
Resolution suitability test 153
Resolution threshold 79
Response 69
Response factor 3
Response High flag 151
Xcalibur: Getting Productive with Quantitative Analysis
191
Index: S
Response Low flag 151
Response OK flag 151
Result List Column Hiding dialog box 145
Results grid 139
changing the sort order 146
column headings 142
context menu 144
displaying columns 145
editing a sequence 179
editing samples 180
hiding columns 145
working in 141
Results review 129
Resuming the acquisition queue 126
Retention time
RT reference 35
Window text box 35
Reverse Matching factor 157
Right Edge Type flag 151
Rows
deleting 110
inserting 110
RSD% 143
R-squared 73
R-squared flag 152
RT 143
RT Ref OK flag 151
Run Sequence dialog box 118
Running
programs or macros 89
sample or sequence 117
S
S/N threshold 63
Sample
ID 143
name 142
position 111
type 142
Sample ID text box 95, 106
Sample Information window 125
Sample Type option box 106
Sample types
blanks 12
quality controls (QCs) 11
standards 11
unknowns 11
Samples
editing in Quan Browser 180
ID 106, 111
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Xcalibur: Getting Productive with Quantitative Analysis
name 95
removing from the acquisition queue 126
reports 87
type 106
unknowns 101
volume 95
weight 95
Saturated flag 151
Saturation suitability test 154
Save command 137
Saving
page parameters in Processing Setup 18
sequence in Sequence Setup 94
Scan filters 31
Scan Threshold text box
peak purity 85
Select Report Samples dialog box 177
Select Samples button 177
Send To Qual Browser command 144
Separator character 99
Sequence
See Acquisition Sequence, Processing Sequence.
Sequence Setup 92
Batch Reprocess Setup dialog box 122
Column Arrangement dialog box 96
Disk Space dialog box 113
Export Sequence dialog box 115
Fill Down dialog box 108
Go To Line Number dialog box 111
Import Sequence dialog box 98
International dialog box 99
New Sequence Template dialog box 99
Print Preview dialog box 113
Print Selection dialog box 113
Run Sequence dialog box 118
Transfer Row Information dialog box 111
User Labels dialog box 97
Sequences
brackets 105
columns 94
creating automatically 99
creating manually 106
editing in Quan Browser 179
editing in Sequence Setup 108
exporting 115
importing 98
new 98
New command 99
pausing 126
printing 112
removing from the acquisition queue 126
resuming 126
samples 101
Thermo Electron Corporation
Index: T
standards 105
starting number 100
Set Companion View list 139
Set Peak To Not Found Status command 148
Set Sorting Order command 144, 146
Settings dialog box 20
Show all sample types 135
Show Calibration Curve command 166, 175
Show Peak Info command 147, 148
Show Spectrum View command 166
Show Standards And QC commands 135
Shutdown 120
Signal-to-noise suitability test 154
Smoothing in processing method 46
Sort order 146
Specified amount 143
Spectrum
detection 50
preview 22
cursor actions 24
Spectrum At Peak Apex command 175
Spectrum At Peak Left Edge command 175
Spectrum At Peak Right Edge command 175
Spectrum Companion view 137, 138, 140, 175, 175
Spectrum Options dialog box 52
Spectrum page 154, 157
Spectrum plot 139, 175
Standard
clear 132
update 132
Standard Dilution dialog box 76
Standards 3, 11, 105
Startup 120
Startup Mode group box 20
Status Bar command
Processing Setup 17
Suitability page 153
baseline clipping test 154
column overload test 154
concave test 154
peak width test 153
resolution test 153
saturation test 154
signal-to-noise ratio test 154
symmetrical test 153
tailing test 154
Summary reports 88
Symmetrical suitability test 153
Symmetry threshold 80
System suitability
baseline clipping definition 83
Thermo Electron Corporation
column overload formula 82
peak width defined 81
resolution formula 79
symmetry formula 80
tailing formula 81
System Suitability page 78
T
Tables
Effect of cursor action in an active cell 24
Tailing 81
Tailing suitability test 154
Target 66
correcting for contribution to ISTD 71
Target ratio % 155
Title bar, Quan Browser 137
Toolbar
Processing Setup 17
Quan Browser 137
Trace 32
combinations 32
Transfer Row Information dialog box 111
Type page
Calibration Settings dialog box 168
U
Units 68, 143
ISTD 66
Unknowns 11
Updating queue manager 126
Upper quantitation limit 4
Use as RT reference 35
User Identification Settings dialog box
Detection page 161
Flags page 164
ICIS Advanced page 163
ICIS Integration page 162
Identification page 160
User Labels dialog box 97
User Peak Detection Settings command 147, 158
User Settings command 147
User-defined columns 97, 106
V
Valid flag 151
Valley Detect flag 151
Xcalibur: Getting Productive with Quantitative Analysis
193
Index: W
Value option button 62
Variables, discussion of
quantitation with external standards 5
quantitation with internal standards 6
Vial list 112
Vial number 95
Vial Position text box 95
View all 142
View Sample Types dialog box 135
View Spectrum Plot command 175
View Stds and QCs option 141
Void Time group box 62
W
Warning
automatic adjustment 172
Enable Warning command 18
flags 78
Warning dialog box 135
wavelength 34
Wavelength Range text box
peak purity 85
Weighting 67
Windows
Quan Browser 129
queue manager 126
Sequence Setup 92, 92
Working in the results grid 141
X
Xcal files 132
Z
Zoom commands 138
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Xcalibur: Getting Productive with Quantitative Analysis
Thermo Electron Corporation
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