Xcalibur Getting Productive

Xcalibur Getting Productive
Xcalibur®
Getting Productive:
Qualitative Analysis
XCALI-97101 Revision C
For Research Use Only
Not for use in Diagnostic Procedures
June 2006
© 2006 Thermo Electron Corporation. All rights reserved.
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Contents
Preface ............................................................................................. xi
About This Guide ......................................................................xi
Related Documentation .............................................................xi
Safety and Special Notices.........................................................xii
Contacting Us...........................................................................xii
Assistance ...............................................................................xii
Changes to the Manual and Online Help..............................xiii
Thermo Electron Corporation
Chapter 1
Introduction..........................................................................................1
About Qualitative Analysis..........................................................2
Understanding Spectra ................................................................3
Ionization Modes .....................................................................4
Adduct Formation....................................................................6
Effect of Isotopes......................................................................7
Analysis Modes of Xcalibur .........................................................8
Full Scan ..................................................................................8
Selected Ion Monitoring (SIM)................................................9
Chapter 2
Creating a Processing Method for Qualitative Analysis ...........11
Processing Setup .......................................................................12
The Processing Setup Window..................................................13
The Title Bar .........................................................................14
The Toolbar...........................................................................14
Qual View..............................................................................14
Applying Changes to a Page ...................................................15
Customizing Processing Setup................................................17
Using Qual View Interactively ..................................................18
Previewing Processing ............................................................18
Setting Processing Parameters ................................................20
Cursor Actions .......................................................................20
Using the Toolbar ..................................................................22
Customizing the Previews ......................................................23
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Contents
Identification ............................................................................24
Detector.................................................................................25
Filter ......................................................................................25
Trace Options........................................................................26
Mass (m/z) or Wavelength (nm) ............................................29
Selected Retention Time Window..........................................30
Peak Integration.....................................................................30
Limit Peaks ............................................................................36
Advanced Chromatogram Parameters ....................................37
Identification Options...............................................................38
Spectrum Enhancement ............................................................39
Selecting the Enhancement Method.......................................39
Refine ....................................................................................40
Combine................................................................................42
Threshold...............................................................................46
Library Search Options .............................................................47
Selecting the Type of Library Search ......................................48
Other Search Options ............................................................49
Adding Spectra to a User Library ...........................................51
Selecting Libraries ..................................................................52
Library Search Constraints ........................................................54
Molecular Weight ..................................................................55
Other Databases.....................................................................55
Name Fragment .....................................................................56
Element Constraints...............................................................57
Mass Spectral Peak Constraints ..............................................59
Peak Purity................................................................................61
Enable Peak Purity .................................................................62
Limit Scan Wavelength ..........................................................62
Reports .....................................................................................63
Sample Reports ......................................................................64
Summary Reports ..................................................................65
Programs..................................................................................66
Specifying a Program or Macro ..............................................67
Changing the Program Sample Type......................................68
Example using the Xconvert.exe program...............................68
Method Examples .....................................................................69
Example 1 ..............................................................................69
Example 2 ..............................................................................71
Example 3 ..............................................................................72
Example 4 ..............................................................................74
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Contents
Thermo Electron Corporation
Chapter 3
Automating Analysis in Sequence Setup .....................................77
About Sequence Setup ..............................................................78
The Sequence Setup Window ...................................................79
About Sequences .......................................................................80
Arranging the Columns..........................................................82
Changing User Labels ............................................................83
Creating a New Sequence..........................................................84
Importing a Sequence ............................................................84
Using the New Sequence Template to Create A Sequence......86
Creating a Sequence Manually ...............................................89
Working with a Sequence .........................................................91
Filling Down Columns ..........................................................91
Inserting a Row......................................................................93
Deleting a Row ......................................................................93
Going to a Sequence Row ......................................................94
Transferring Row Information ...............................................95
Printing a Sequence................................................................96
Checking Disk Space .............................................................98
Exporting a Sequence...........................................................100
Running Samples ....................................................................102
Running a Single Sample .....................................................102
Running a Sequence.............................................................103
Setting up the Run...............................................................103
Reprocessing Samples..............................................................108
The Acquisition Queue ..........................................................110
Sample Information Window...............................................111
Managing Tasks ...................................................................112
Chapter 4
Reviewing and Interpreting Data in Qual Browser ..................115
Results Review ........................................................................116
About Qual Browser ...............................................................117
The Toolbar.........................................................................118
The Info Bar ........................................................................121
Windows, Cells, Views and ‘Pinning’...................................122
Getting Started in Qual Browser .............................................124
Opening Single Raw Files ....................................................124
Opening a Sequence.............................................................126
Opening a Result file............................................................128
All About Cells........................................................................130
Creating and Deleting Cells .................................................130
Adjusting Cell Size ...............................................................131
Changing a Cell’s View ........................................................132
Scaling a Plot .......................................................................133
Layouts ................................................................................133
Using the Cell Information Page ..........................................135
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Contents
Using Views Interactively ........................................................137
Using a Chromatogram View..................................................139
Using Chromatogram Plots..................................................140
Chromatogram Ranges.........................................................140
Using the AutoFilter ............................................................148
Setting Chromatogram Options...........................................149
Detecting Peaks....................................................................156
Changing Peak Detection Settings .......................................158
Using a Spectrum View...........................................................160
Using Spectrum Plots...........................................................161
Spectrum Ranges..................................................................162
Setting Spectrum Options....................................................168
Subtracting Background Spectra ..........................................178
Determining the Elemental Composition of a Spectrum......179
Simulating an Isotopic Distribution Spectrum .....................186
Browsing MSn Data.............................................................191
Submitting a Spectrum to a Library Search ..........................198
Customizing a Library Search...............................................200
Exporting a Spectrum to the Library ....................................204
Using a Map View ..................................................................205
Setting the Map Ranges .......................................................205
Setting Map Display Options...............................................206
Using a Spectrum List View ....................................................213
Determining Elemental Composition ..................................214
Setting Spectrum List Ranges ...............................................215
Setting the Spectrum List Options .......................................217
Using a Scan Header View ......................................................223
Setting the Scan Header Range ............................................223
Using a Scan Filter View .........................................................224
Setting the Scan Filter Range ...............................................224
Using a Report View ...............................................................225
Preparing for Presentation.......................................................226
Using Amplify......................................................................227
Adding Text.........................................................................228
Adding Graphics ..................................................................229
Removing Text and Graphics...............................................231
Editing the Heading.............................................................231
Printing Cells .......................................................................233
Copying Cells to the Clipboard............................................234
Tool Menu Utilities ................................................................235
Background Subtract Utility ................................................235
Adding Programs to the Tools Menu ...................................238
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Contents
Chapter 5
Library Browser...............................................................................241
About Library Browser ............................................................242
Exporting Spectra from Xcalibur.............................................243
Manually from Qual Browser...............................................243
Automatically from the Processing Method..........................244
Library Browser Windows.......................................................245
Library Search Window .......................................................245
Other Search Window .........................................................248
Names Window ...................................................................248
Compare Window ...............................................................249
Librarian Window................................................................250
Customizing a Search..............................................................251
Library Search Options Dialog.............................................251
Including a User Library in a Search ....................................254
Limiting a Library Search.....................................................255
Managing User Libraries .........................................................256
Editing a User Library Entry ................................................257
Accessing a User Library Entry.............................................260
Automated Compound Identification Using AMDIS .............261
Index..................................................................................................265
Thermo Electron Corporation
Xcalibur: Getting Productive with Qualitative Analysis
ix
Preface
About This Guide
Welcome to Xcalibur®, the Thermo Electron mass spectrometry data system.
This Xcalibur Getting Productive: Qualitative Analysis manual describes how
to use this Thermo Electron instrument to identify unknown compounds or
carry out trace analysis. It describes how to:
•
Set up a method for automatic qualitative processing
•
Create a sequence or batch of samples for analysis and processing under
full software control
•
Get the best out of the data using Xcalibur’s qualitative reviewing
utilities
•
Submit spectra to library searches
•
Set up personal user libraries of reference spectra
Before reading this manual, read the Getting Started manual for the
instrument and become 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: Quantitative 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 Qualitative Analysis
xi
Preface
Safety and Special Notices
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:
DANGER Highlights laser-related hazards to human beings. It includes
information specific to the class of laser involved. Each DANGER notice
is accompanied by the international laser radiation symbol.
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
[email protected]
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Preface
Contacting Us
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
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
[email protected]
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Chapter 1
Introduction
This chapter provides a basic introduction to mass spectra and explains how
to use Xcalibur®, the Thermo Electron mass spectrometry data system, for
qualitative analysis.
This chapter contains the following sections:
• About Qualitative Analysis
• Understanding Spectra
• Analysis Modes of Xcalibur
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1
Introduction
About Qualitative Analysis
About Qualitative
Analysis
This chapter provides a basic introduction to mass spectra and explains how
to use Xcalibur for qualitative analysis. Qualitative analysis is concerned
with solving two analysis problems:
• The identification of unknown compounds
• Trace analysis and the confirmation of target compounds
This guide describes how to:
• Set up a method for automatic qualitative processing in Processing
Setup.
• Set up a sequence or batch of samples for analysis and processing under
full software control in Sequence Setup.
• Review the data using Xcalibur’s qualitative reviewing utility, Qual
Browser.
• Use the Library Browser to carry out advanced analysis and to set up
personal libraries of reference spectra.
Before reading this manual, read the Getting Started manual for the
instrument to become familiar with the basic features of Xcalibur such as
Home Page and Instrument Setup.
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1 Introduction
Understanding Spectra
Understanding
Spectra
There are many different types of MS detectors but the basic principles are
the same in all cases: the sample is ionized, ions are separated according to
their mass1, and the separated ions are accelerated towards a detector where
they are counted. The data system compiles a spectrum showing the mass
distribution of the ions produced from the sample - a snapshot of ion
intensities plotted against their mass1.
Ionization initially produces molecular ions, but complex secondary
processes can cause the molecular ions to fragment. Together with molecular
ions, these fragment ions make up the mass spectrum. For individual
substances, a mass spectrum can be a characteristic molecular fingerprint.
It is conventional to call the most abundant ion the base peak and give it an
arbitrary abundance or intensity of 100. All the other peaks are reported as a
percentage of the size of the base peak. After this normalization, it is possible
to compare spectra directly.
Figure 1 is an example of a simple library spectrum showing the
fragmentation of acetone C3H6O (molecular weight = 58 u). The most
abundant ions have been labeled with their mass-to-charge ratios. Note that
the molecular ion (58 u) is not the most abundant. The base peak is actually
43 u. This is due to the acetyl ion.
Figure 1.
The 70 eV electron ionization (EI) mass spectrum of acetone
1Strictly,
this should be mass-to-charge ratio (m/z), but in the majority of cases z=1 and the X-axis
becomes equivalent to mass, m.
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1
Introduction
Understanding Spectra
Fragmentation patterns like this can be used to determine molecular
structure. For example, the neutral loss of 15 u from the molecular ion of
acetone indicates the presence of a methyl group in the original molecule. A
subsequent loss of 28 u corresponds to the loss of CO. Commonly observed
neutral losses are listed in Table 1. Assign such losses to help deduce the
structure of an unknown compound. A full structural analysis generally
relies on the presence of a molecular ion and hence the measurement of the
molecular weight of the compound.
Table 1.
Common neutral losses
Loss
Fragment
15
CH3
18
H2O
19
F
29
C2H5 or CHO
35
Cl
46
NO2
59
C3H7O, COOCH3 or CH2COOH
77
C6H5
In some cases, fragmentation is extensive, leaving little or no trace of a
molecular ion. In the absence of a molecular ion, it is difficult to determine
either the molecular weight or the structure.
Ionization Modes
Select an ionization mode that supports the type of instrument used
(LC/MS or GC/MS). This mode can affect the characteristics of a spectrum
of a compound.
This section contains the following topics:
• Ionization Modes for LC/MS Instruments
• Ionization Modes for GC/MS Instruments
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1 Introduction
Understanding Spectra
Ionization Modes for LC/MS
Instruments
LC/MS instruments use a technique called atmospheric pressure ionization
(API). Detectors of this type can be configured to detect positive or negative
ions.
API techniques offer soft ionization. This means that there is usually little or
no fragmentation. An API spectrum typically contains only the protonated
or deprotonated molecular ion. Basic compounds (such as amines) form
protonated molecules. These can be analyzed in positive ion detection
modes, giving a quasi-molecular ion peak at m/z M+1 (where M represents
the molecular weight of the compound).
Acidic compounds (sulphonic acids, for example) form a deprotonated
molecule [M-H]-. These can be analyzed in negative ion modes as
quasi-molecular ion peaks at m/z M-1.
Ionization Modes for GC/MS
Instruments
GC/MS instruments offer two techniques: electron ionization (EI) and
chemical ionization (CI).
EI is very commonly used because it is simple and reproducible. The
fragmentation pattern is effectively determined by the energy of the
impacting electrons alone (electron energy, measured in eV). Virtually
identical spectra can be obtained on very different types of EI mass
spectrometers as long as the electron energy is the same.
This reproducibility has led to the compilation of extensive libraries for
70 eV EI spectra. Xcalibur’s Library Browser can use the optional
NIST/EPA/NIH Mass Spectral Library with over 108000 reference
EI spectra. Use library data to select confirmatory ions for the target
compounds.
Chemical ionization (CI) offers a milder method of forming ions. In CI, a
controlled flow of a reagent gas, commonly ammonia, methane, or
isobutane, is introduced into the area where ionization occurs (the ion
source). Thermal electrons passing across the source ionize the reagent gas.
These ions can then collide with neutral molecules, causing hydrogen
transfer. This process is repeated when the reagent gas ions collide with
analyte molecules.
CI usually produces protonated molecules, generally at a mass one greater
than the molecular mass of the compound. Significantly less fragmentation
occurs than in comparable EI spectra. Depending on the choice of reagent
gas, adduct ions can be formed. For example, M+NH4 is a typical adduct
ion with ammonia as the reagent gas.
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1
Introduction
Understanding Spectra
Under certain conditions, CI produces negative molecular ions formed by
electron capture. The sensitivity of negative ion CI for certain classes of
compounds (those containing double bonds, sulfur, phosphorus, chlorine or
bromine) can be orders of magnitude greater in comparison with positive CI
or EI modes.
For more information about the ionization modes available on the
instrument, refer to the Hardware Manual and Getting Started manual for
the instrument.
Adduct Formation
If ionization takes place in the presence of contaminants or additives such as
ammonium or sodium ions, some compounds are susceptible to adduct
formation. These spectra show other ions in addition to, or instead of, the
molecular ion (see Figure 2). Common adducts are:
[M+18]+ NH4+
[M+23]+ Na+
[M+39]+K+
[M+42]+ ACN+H+
Figure 2.
Mass spectrum showing sodium and acetonitrile adducts
Care must be taken when determining molecular weights to take account of
possible adduct ions.
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1 Introduction
Understanding Spectra
Effect of Isotopes
In some cases, you need to consider the effect of less abundant isotopes and
choose to use an average molecular weight rather than one based on the
most abundant isotopes. When the molecular structure of the target
compound contains large numbers of certain elements, the less abundant
isotopes become significant. This might result in a shift in the mass peaks
from their expected m/z values.
For example, the most abundant isotope of chlorine is Cl35. However, Cl37
occurs with a natural abundance of 24.47%. If a compound contains four
chlorine atoms, its molecular ion would be 2 mass units greater than that
expected from a calculation based solely on Cl35. Using chlorine’s average
atomic weight (35.453), the molecular ion would be correctly identified.
Also you would observe a distribution of molecular ions across 8 mass units
from molecules containing between zero and four Cl37 atoms.
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1
Introduction
Analysis Modes of Xcalibur
Analysis Modes of
Xcalibur
Xcalibur has two analysis modes:
• Full Scan
• Selected Ion Monitoring (SIM)
Full Scan
In full scan operation, the MS detector scans repetitively over a wide mass
range and records successive full spectra throughout the analysis.
Display full scan information in several ways:
• A Total Ion Current (TIC) chromatogram represents the summed
intensities of all the ions in each spectrum plotted against
chromatographic retention time. Each peak in the TIC represents an
eluting compound, which can be identified from the mass scans
recorded during its elution.
• Mass chromatograms show the ion intensities of selected mass-to-charge
ratios (m/z). Xcalibur extracts these from each stored scan and plots
them against retention time. Selectivity can be achieved using this
technique by choosing to display an m/z value characteristic of the
compound of interest but not present in other sample components.
Full scan mode is suited to the identification of unknowns and can also be
used for trace analysis when sensitivity is not important.
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1 Introduction
Analysis Modes of Xcalibur
Selected Ion Monitoring
(SIM)
In SIM mode, configure the MS detector to monitor a limited number of
m/z values, characteristic of a targeted compound or compounds. The mass
analyzer switches between the selected m/z values, each value being
monitored repeatedly for a programmed dwell time before averaging and
moving on to the next.
SIM generates mass chromatograms only of the monitored m/z values, not
complete mass spectra as in full scan operation. Without a complete
spectrum it is not possible to submit a library search to identify an
unknown.
Selected ion monitoring (SIM) offers:
• Improved sensitivity because more time is spent monitoring the ions of
interest rather than scanning across the complete mass range.
• A wide and linear dynamic range (six orders of magnitude without
modification of tuning parameters). This is important in isotope
dilution techniques which use co-eluting labeled standards and also in
the analysis of trace components.
• Better definition of a chromatogram peak profile because more scans
(points) can be recorded across it.
• Reduced file sizes compared to full scan operation because SIM records
only the information of interest.
SIM is ideally suited to trace analysis.
Thermo Electron Corporation
Xcalibur: Getting Productive with Qualitative Analysis
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Chapter 2
Creating a Processing
Method for Qualitative
Analysis
This chapter describes Processing Setup and explains how to use it to create
a Processing Method for the automated analysis of qualitative data. It leads
you through the parameters required for data processing, reporting, and
running additional programs.
This chapter contains the following sections:
• Processing Setup
• The Processing Setup Window
• Using Qual View Interactively
• Identification
• Identification Options
• Spectrum Enhancement
• Library Search Options
• Library Search Constraints
• Peak Purity
• Reports
• Programs
• Method Examples
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2
Creating a Processing Method for Qualitative Analysis
Processing Setup
Processing Setup
Use Processing Setup to create a method for automated batch analysis. It
leads you through the parameters required for data processing, reporting,
and running additional programs (such as file copying procedures).
Xcalibur provides three peak detection algorithms. 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 backwards
compatibility with Xcalibur 1.0 studies. The Avalon peak detection
algorithm has been designed for chromatographic data and supports
detectors other than MS. Specifically, Avalon can detect negative peaks and
shoulders in chromatographic data better than Genesis or ICIS can.
To change the default peak detection algorithms
1. Choose Tools > Configuration in the Roadmap view of the Xcalibur
Home Page.
Xcalibur displays the multi-paged Xcalibur Configuration dialog box.
2. Click on the Peak Detection tab to open the Peak Detection page.
3. Select the appropriate peak detection algorithm for each data type.
Sequence Setup uses the Processing Method to initiate qualitative and
quantitative processing, reporting, and additional programs or macros.
A single Processing Method consists of four views:
Quan
Quantitative processing setup
Qual
Qualitative processing setup
Reports
Reporting setup
Programs
Program and macro selection
This chapter concentrates on the functionality of the Qual, Reports, and
Programs views.
The Xcalibur Getting Productive: Quantitative Analysis manual provides a full
description of the Quan view.
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2
The Processing Setup
Window
Creating a Processing Method for Qualitative Analysis
The Processing Setup Window
The Processing Setup window (see Figure 3) consists of the following:
• A title bar containing a description about the current method
• A menu bar
• A toolbar
• A View bar containing graphical buttons leading to the four views of
Processing Setup: Quan, Qual, Reports, and Programs
• A page or set of pages corresponding to the selected view
• A status bar showing information about activities within
Processing Setup
Title Bar
Toolbar
Qual View
Menu Bar
View Bar
Chromatogram
Plot View
Spectrum
Plot View
Status Bar
Figure 3. Processing Setup window, showing the Identification page of the Qual view
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2
Creating a Processing Method for Qualitative Analysis
The Processing Setup Window
To display or hide the View bar, Toolbar, and Status bar, choose the
appropriate View menu command:
• Choose View > View Bar to display or hide the View bar
• 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 three bars.
The Title Bar
The title bar lists:
• The application name – Processing Setup
• The active view (Quan, Qual, Reports, or Programs)
• The active page (for example, Identification)
• The name of the opened method, or ‘Untitled’ if a new file has not yet
been saved
• The selected type of calibration, internal or external standard
The Toolbar
Qual View
The toolbar contains shortcuts for frequently used menu commands. For
information about the toolbar buttons, refer to the Xcalibur online Help.
The Qual view (see Figure 3) consists of five tabbed pages:
• The Identification page controls the peak detection and integration of
chromatograms
• The Spectrum Enhancement page turns on three spectrum
enhancement options.
• The Library Search Options page consists of the parameters to define a
comparison search of the compound to published compound data.
• The Library Search Constraints page constrains a library search to
increase processing efficiency.
• The Peak Purity page defines scan and wavelength parameters for peak
purity analysis
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2
Creating a Processing Method for Qualitative Analysis
The Processing Setup Window
The Identification page and the Spectrum Enhancement page contain a
Chromatogram Plot view and a Spectrum plot view. Use these views to
preview the results of peak detection and integration in the Chromatogram
preview, and, if enabled, Spectrum Enhancement in the Spectrum preview.
A secondary use of these previews is to set some of the Qual parameters
interactively using an existing raw file.
Note If you select a detector type other than MS or PDA, the Spectrum
Enhancement, Library Search Options and Library Search Constraints
pages are unavailable.
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, otherwise
they are grayed out. When you have changed or edited a parameter:
• 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 file by using the File > Save command from
the open file.
Xcalibur displays the Apply Changes dialog box (see Figure 4) if you
attempt one of the following actions without applying or discarding
changes:
• Switching to another page
• Switching to another component
• Switching to another view
• Changing the chromatography type (Options > Chromatography By)
• Changing the calibration type (Options > Calibration By)
• Clicking on the Close button on the title bar
• Choosing one of the following menu commands:
File > Open
File > <most recently used file list>
File > Save
File > Save As
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Creating a Processing Method for Qualitative Analysis
The Processing Setup Window
File > Exit
File > Import Method
File > New
Apply or undo the page modifications before proceeding with further
actions.
Figure 4.
Apply Changes dialog box
In the Apply Changes dialog box, click:
• Yes to apply changes.
• No to discard any changes and proceed with the selected action.
• Cancel to stop the intended action and return to the current page
without applying or discarding changes.
Select the Don't Tell Me About This Again check box to suppress the
display of the Apply Changes dialog box. In future cases where it would
normally be displayed, Xcalibur treats changes according to the final
selection in the dialog box:
• When you click Yes, Xcalibur applies changes if validation is successful
and continues with the selected action. If validation fails, Xcalibur stops
the intended action and returns to Processing Setup so that changes can
be corrected or discarded.
• When you click No, Xcalibur automatically discards all changes and
continues with the selected action. In such cases, apply changes
explicitly, by clicking on the OK button, before initiating the action.
Choose Options > Enable Warnings to re-enable the warning dialog box.
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Customizing Processing
Setup
Creating a Processing Method for Qualitative Analysis
The Processing Setup Window
By default, Xcalibur loads the most recently used method into Processing
Setup at startup. When a Processing Method is opened, change this option
and configure Xcalibur to open a raw file into the Chromatogram and
Spectrum previews.
To adjust these options choose Options > Settings. Xcalibur displays the
Settings dialog box shown in Figure 5.
Figure 5.
Settings dialog box
Select one of the following options in the Startup Mode area:
• Load Last Processing Method, or
• Create New Processing Method
Select one of the following options in the Auto-Open Raw File area:
• On, or
• Off
Note If you save a Processing Method when a raw file is present,
Xcalibur saves the raw file name in the Processing Method. Xcalibur
automatically opens the associated raw file whenever the Processing
Method is opened if you clicked the Auto-open raw file On option in the
Settings dialog box.
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Creating a Processing Method for Qualitative Analysis
Using Qual View Interactively
Using Qual View
Interactively
Processing Setup displays the Chromatogram and Spectrum previews with
the Identification and Spectrum Enhancement pages. Using a representative
raw file, use these to:
• Preview the results of peak detection and integration in the
Chromatogram preview, and, if enabled, Spectrum Enhancement in the
Spectrum preview.
• Set some of the Identification and Spectrum Enhancement parameters
interactively.
To use the Qual view interactively, choose File > Open Raw File or click
the Open Raw File button on the toolbar. Select a relevant raw file. Then,
click Open.
This section contains the following topics:
• Previewing Processing
• Setting Processing Parameters
• Cursor Actions
• Using the Toolbar
• Customizing the Previews
Previewing Processing
The Chromatogram and Spectrum previews assess processing parameters
for:
• Peak Detection and Integration
• Spectrum Enhancement
Peak Detection and Integration
18
Xcalibur processes the raw file using the parameters of the Identification and
Spectrum Enhancement pages. The Chromatogram preview shades all
detected peaks and indicates the start and end of each peak with a blue
baseline. Initially, the Spectrum preview displays the spectrum
corresponding to the first detected peak in the chromatogram. If no peak
has been detected in the chromatogram, the Spectrum preview shows the
spectrum for the first scan in the raw file.
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Creating a Processing Method for Qualitative Analysis
Using Qual View Interactively
Re-scale the chromatogram or spectrum previews by using:
• Cursor actions (See “Cursor Actions” on page 20.)
• Toolbar buttons (See “Using the Toolbar” on page 22.)
• Zoom menu commands, either from the top-level menu or from the
shortcut menu
To proceed, either:
• Click OK to perform the peak detection processing again using the
current parameters, or
• Click Cancel to discard all changes made to the page
Xcalibur shades all detected peaks and adds the baseline to indicate the peak
start and end positions.
Spectrum Enhancement
In the Spectrum Enhancement page, the Spectrum preview shows:
• The raw spectrum if Spectrum Enhancement is not enabled
• A Refine-enhanced spectrum if Refine is enabled
• A spectrum with the Cutoff threshold applied if Threshold is enabled
• A Combine-enhanced spectrum when you have enabled Combine and
the selected scan lies within the peak start and peak end window of a
detected peak in the Chromatogram preview
If the current spectrum does not lie within a detected peak, Xcalibur shows
no spectrum, displaying the following message: “Combine cannot be
performed. The current spectrum does not lie within a detected chromatographic
peak.”
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Creating a Processing Method for Qualitative Analysis
Using Qual View Interactively
Setting Processing
Parameters
Generate a qualitative Processing Method by typing values for all the
required Qual view parameters. In the Identification and Spectrum
Enhancement pages, use the interactive features of Processing Setup. This
option involves the use of the Chromatogram and Spectrum previews
together with a raw file representative of the analysis requirements.
Use the previews to set:
• The retention time range. See “Selected Retention Time Window” on
page 30.
• The mass range. See “Mass (m/z) or Wavelength (nm)” on page 29.
• The combine range. See “Combine” on page 42.
Cursor Actions
Within the Chromatogram and Spectrum previews, use the cursor in three
ways:
• A click picks a point on the preview
• A line dragged parallel to any axis picks a range
• A line dragged in any diagonal direction selects an area
The effect of these actions depends on the state of the preview:
• Inactive
• Active and unpinned (each preview has a pin icon in its top right corner)
• Active and pinned
Only one of the previews can be active at any one time. The active preview
is highlighted with a gray border. In Figure 3 on page 13, for example, the
Spectrum preview is active, but not pinned.
Pinning fixes the active status of a preview.
To make a preview active
1. Make sure the currently active preview is not pinned. If it is, click the
pin icon to unpin it.
2. Click anywhere within the preview you want to be active. Xcalibur
highlights it with a gray border. Click its pin icon to fix it as the active
preview.
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Creating a Processing Method for Qualitative Analysis
Using Qual View Interactively
Cursor actions in an active preview cause the preview to be scaled according
to the dimensions of the dragged line or area (see Table 2).
Table 2.
Cursor action in active, unpinned, preview
Cursor action in active
preview
Effect
Drag parallel to X-axis
Rescale graph showing selected X range only,
same Y range
Drag parallel to Y-axis
Rescale graph showing selected Y range only,
same X range
Dragged area
Rescale graph showing both the selected X and Y
ranges
The same actions in the unpinned or inactive preview have a very different
effect. In this case, the cursor actions affect the active preview (see Table 3).
Table 3.
Cursor action in inactive or unpinned preview
Qual page(s)
Active preview
Cursor action
Effect
Identification Spectrum Spectrum (must Click in
Enhancement
be pinned)
Chromatogram
preview.
Spectrum preview displays mass scan that occurs
at clicked on retention time.
Identification
Spectrum
Drag across a time
range in
Chromatogram
preview.
Xcalibur enters the time range, indicated by a red
line, into the Retention Time Window, Range
(min) box.
Spectrum Enhancement Spectrum
(with Combine
enabled, and focus on
any of 5 ‘points’ fields)
Drag across a time
range in
Chromatogram
preview.
Xcalibur enters the number of points in the time
range, indicated by a red line, in the Combine
edit field.
Identification
Chromatogram Click single mass
(must be
in Spectrum
pinned)
preview.
Xcalibur enters the mass into the Mass (m/z) box
for any trace combination that includes Mass
Range and Base Peak. The mass is added to any
existing value(s) defined in the mass field.
Important points to note are:
• The cursor action is always applied to the pinned preview.
• Within an active preview, cursor actions rescale the plot.
Note Right-click the active preview to display a shortcut menu with
Display Options and Zoom commands.
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Creating a Processing Method for Qualitative Analysis
Using Qual View Interactively
Using the Toolbar
Use the toolbar buttons to re-scale a chromatogram or spectrum preview.
The toolbar buttons are:
Normalize Intensity
Zoom out Y
Zoom in Y
Auto range
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. This can also be displayed
as a shortcut menu by right-clicking on the appropriate preview. To re-scale
the chromatogram, use the cursor (see Table 2).
The effects of the Reset scaling tool depend on whether you have selected
Auto range or Normalize Intensity. Use Auto range to view negative peaks
rather than Normalize Intensity.
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Customizing the Previews
Creating a Processing Method for Qualitative Analysis
Using Qual View Interactively
To customize the display of a chromatogram or spectrum
1. Click anywhere within the preview to make it active.
2. Choose Options > Display or right-click the appropriate 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.
For detailed information about the Display Options dialog box for the
Chromatogram Plot view, refer to “Setting Chromatogram Options” on
page 149. For detailed information about the Display Options dialog box
for the Spectrum Plot view, refer to “Setting Spectrum Options” on
page 168.
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Identification
Identification
Figure 6 shows the Processing Setup - Qual view - Identification page with
the ICIS peak detection algorithm selected. Xcalibur contains three peak
detection algorithms: ICIS, Genesis, and Avalon. The parameters shown in
the Peak Integration area depend on the Peak Detect selection.
Xcalibur uses the parameters on the Identification page to do the following:
• Generate a chromatogram from a raw file
• Detect and integrate peaks within the chromatogram
• Identify apex scans for each of the detected peaks and submit these to
spectrum enhancement (if enabled) and library searching (if you have
selected MS detection).
This section describes the parameters on the Identification page and
contains the following topics:
• Detector
• Filter
• Trace Options
• Mass (m/z) or Wavelength (nm)
• Selected Retention Time Window
• Peak Integration
• Limit Peaks
• Advanced Chromatogram Parameters
Figure 6. Processing Setup - Qual view - Identification page, showing the ICIS Peak Integration area
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Detector
Creating a Processing Method for Qualitative Analysis
Identification
The Detector area contains three parameters: Type, Delay, and
Peak Detect.
Type
From the Type list, select the type of detector: MS,
Analog, A/D card, PDA, or UV.
Peak Detect
From the Peak Detect list, select a peak detection
algorithm to be used by the Processing Method during
qualitative processing. The available algorithms are
Genesis, ICIS and Avalon.
The Genesis peak detection algorithm has been provided
for backwards compatibility with data from Xcalibur 1.0
studies and, primarily, data of the MS type. The ICIS
peak detection algorithm can be used for all detector
types listed in Xcalibur. ICIS has superior peak detection
efficiency at low MS signal levels. The Avalon peak
detection algorithm supports detectors other than MS.
Specifically, Avalon detects negative peaks and shoulders
in chromatographic data better than Genesis or ICIS
can.
Delay (min)
Filter
Select a non-MS detector type to enable the Delay box.
In this box, specify a delay (in seconds) to synchronize
the data with any acquired MS data. The delay is the
difference in time between the commencement of MS
acquisition and the subsequent start of the non-MS
acquisition.
Select an MS detector type to turn on options in the Filter combo box. 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.
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 if you click OK.
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Identification
For advanced uses, specify your own filter by typing it in the scan filter
format. For example, the scan filter:
c full ms [26.81-251]
uses all scans in a raw file that have centroid data for a Full scan mode with
MS detection and in the mass range from m/z 26.81 to 251.00. See the
online Help for more details about the filter feature.
Trace Options
Use the Trace lists to specify the type of chromatogram you want to use for
qualitative processing. The Trace options depend on the Detector Type
selected:
• 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 three Trace lists to choose:
• A basic chromatogram type, for example, TIC. Then, depending on the
selection:
• A logical operator: + or –. This operator selection turns on:
• A second chromatogram type to add to, or subtract from, the first trace.
For example, Mass Range. The list contains valid trace types which can
be subtracted from, or added to, the trace specified in the first box.
In most cases, use a single trace type such as TIC. A second trace type is
useful for subtracting contributions to a chromatogram from a solvent or
other noise. Table 4 lists the various MS trace types and gives examples of
their use. Table 5 lists trace types for non-MS detectors.
Table 4.
26
MS trace types and combinations
Trace type
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.
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Table 4.
Creating a Processing Method for Qualitative Analysis
Identification
MS trace types and combinations, continued
Trace type
Use
Base Peak
Compiles a chromatogram from the most abundant
ion within the specified mass range.
TIC - Mass Range
Cleans up a TIC by subtracting a range of
background contamination, allowing less abundant
masses to have a more significant effect on the
chromatogram. For example, consider data acquired
from 50 to 1000 with dominant solvent or
contaminant peaks in the range 50 to 150. Use this
trace combination with Mass Range = 50–150.
TIC - Base Peak
Useful in situations where the most intense spectral
peak throughout the run is due to a contaminant.
Subtracting the base peak from the TIC would
remove this. You can also use the TIC-mass range
combination.
Mass Range - Mass Can be used to remove a variety of background,
Range
solvent or contaminant peaks from a chromatogram.
Consider an example where data have been 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 = 150–900; Mass
Range 2 = 500–600.
Mass Range + Mass Similar uses to Mass Range – Mass Range.
Range
Considering the same example as above, identical
results can be obtained using this trace combination
with:
Mass Range 1 = 150–499; Mass Range 2 =
601–900.
Thermo Electron Corporation
Base Peak - Mass
Range
Rarely used. Consider an example in which the most
intense peaks in the spectrum are, say, 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– (125–145) can remove the effect of these
peaks.
Base Peak + Mass
Range
Useful if the Base Peak trace type does not show up
every chromatogram peak of interest. Add the mass
range of interest to enhance the chromatogram.
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Identification
Table 5.
28
Other trace types and combinations
Trace type
Use
Analog x
For monitoring any external detector, such as an
FID detector, that provides an analog signal.
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 1 V). 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), then subtract one from the other,
for example, representing (220 – 500) – (260 –
280) nm.
Analog x + Analog y
As Analog x - Analog y above. You can add two
channels corresponding to the wavelengths of
two compounds of interest (some detectors can
set only single channels rather than ranges).
A/D Card Channel
For monitoring any external detector that
provides a digital signal.
Wavelength Range
PDA detector wavelength range
Wavelength Range +
Wavelength Range
You can add two channels corresponding to the
wavelengths of two compounds of interest (some
detectors can set only single channels rather than
ranges).
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), then subtract one from the
other, for example, (220-500)-(260-280) nm.
Total Scan
PDA detector total scan
Total Scan –
Wavelength Range
Click this option to subtract a single wavelength
or small range (for example, at a contaminants'
specific wavelength) from the total scan.
Spectrum Maximum
PDA spectrum maximum
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Mass (m/z) or Wavelength
(nm)
Creating a Processing Method for Qualitative Analysis
Identification
This parameter is available only if you select an MS or PDA detector type in
the Type list.
For an MS detector type, use the Mass (m/z) box to specify the mass or mass
range for trace combinations featuring Mass Range or Base Peak trace types
(for example, Mass Range, TIC - Base Peak, TIC - Mass Range). If you use
Base Peak ± Mass Range or Mass Range ± Mass Range trace combinations, the
Identification page displays a Mass (m/z) box for each trace type.
For the PDA detector type, use the Wavelength (nm) 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.
If you use a trace combination such as Wavelength Range + Wavelength
Range (refer to Table 5), an additional Wavelength (nm) box appears to
specify the second wavelength range.
To change the range or to add a new range, either
• 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 the following: 123 – 456, or
• Select the range interactively with a representative raw file:
a. Open a representative raw file by choosing File > Open Raw File.
b. Make the Spectrum preview active (pin it, or click within it).
c. Drag the required range on the Spectrum preview or click to select a
single value.
The range is added to the Mass (m/z) or Wavelength (nm) box.
Add up to 50 ranges to the Mass/Wavelength boxes. Separate the ranges
using the list separator setting character, normally a comma. For
instructions on determining the appropriate list separator, refer to
“Changing the List Separator Character” on page 101.
Note You must provide a range for each enabled Mass Range or
Wavelength Range box. If a Mass Range or Wavelength Range box is
blank, you cannot save the parameters or change to another page until
you have provided a range (or switched to a different trace combination
that does not involve Mass/Wavelength Ranges).
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Identification
Selected Retention Time
Window
The Range (min) box describes a time span to limit qualitative processing.
Peak detection occurs over the full time range in the raw file, but Xcalibur
discards detected peaks that lie outside of the Range (min) parameter.
Xcalibur processes a peak only if its apex retention time lies within the
range. The valid, and default, range is 0.0 to 999.0 min.
To specify a time range, either
• Type the range directly in the Range box (for example, 0.3 - 1.6), or
• Select the range interactively with a representative raw file:
a. Make the Spectrum preview active (pin it, or click within it).
b. Drag the cursor horizontally across a range in the Chromatogram
preview. Xcalibur updates the Range (min) box with the dragged
time range.
Peak Integration
Xcalibur contains three peak integration algorithms:
• ICIS Peak Integration
• Genesis Peak Integration
• Avalon Peak Integration
ICIS Peak Integration
The ICIS Peak Integration area shown in Figure 7 contains the following
options for peak integration:
Figure 7.
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Xcalibur: Getting Productive with Qualitative Analysis
ICIS Peak Integration area
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Creating a Processing Method for Qualitative Analysis
Identification
Smoothing Points
Use this box to type the amount of smoothing
that Xcalibur applies before integration. The
valid range is any odd integer from 1 (no
smoothing) through 15 (maximum smoothing).
Baseline Window
When looking look for a local minima, use this
box to type the number of scans to review. 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 this box to specify the noise level multiplier
used to determine the potential peak signal
threshold. The valid multiplier range is 1 to
1000. The default multiplier is 1.
Constrain peak width
Select this 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 this box to specify 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 this box to specify the maximum ratio of the
trailing edge to the leading side of a constrained
peak. The valid range is 0.5 to 9.0.
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
clicked option as a visual reminder of how the option operates on data. For
example, the boxes in the margin above show the large and small values for
the peak integration parameters and illustrate their effects on a simple data
representation, not the actual data.
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Creating a Processing Method for Qualitative Analysis
Identification
Genesis Peak Integration
The Genesis Peak Integration area shown in Figure 8 contains the following
options for peak integration.
Figure 8.
32
Genesis Peak Integration area
Smoothing Points
Use this 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 integer from 1 (no smoothing) through 15
(maximum smoothing).
S/N Threshold
Use this box to set the signal-to-noise threshold for
peak integration. Peaks with signal-to-noise less than
this value are not integrated. Peaks with
signal-to-noise greater than this value are integrated.
The valid range is 0.0 to 999.0.
Enable Valley
Detection
Select this 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.
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Expected Width
(sec)
Creating a Processing Method for Qualitative Analysis
Identification
Use this box to specify 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, Xcalibur ignores any
valley points nearer than the [expected width]/2 to
the top of the peak. If 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 this 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 this box to specify 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 this 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.
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 clicked option as a visual reminder of how the option operates on data.
For example, the boxes in the margin above show the large and small values
for the peak integration parameters and illustrate their effects on a simple
data representation, not the actual data.
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Creating a Processing Method for Qualitative Analysis
Identification
Avalon Peak Integration
The Avalon Peak Integration area is shown in Figure 9.
Figure 9.
Avalon Peak Integration area
To add, delete, or modify Avalon integration events, click Advanced (at the
bottom of the Identification page) to open the Avalon Event List dialog box.
The Auto Calc Initial Events button is active only if a raw file is open.
When you click Auto Calc Initial Events, 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.
The Avalon Peak Integration area contains the following options for peak
integration:
34
Smoothing
Use this 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 integer from 1 (no smoothing) through 15
(maximum smoothing).
Start/End
Threshold
The Start and End Thresholds are directly related to
the RMS noise in the chromatogram. This
integration event is the fundamental control used for
peak detection.
Bunch Factor
The Bunch Factor is the number of points grouped
together during peak detection. It 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.
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Creating a Processing Method for Qualitative Analysis
Identification
Area Threshold
Area Threshold controls the area cutoff. The
software does not detect any peaks with a final area
less than the area threshold. This control is in units
of area for the data.
P-P Resolution
The peak to peak resolution 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. This value is specified as a percent of
peak height overlap.
Negative Peaks
Automatically resets after a negative peak has been
found.
Tension
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. Tension values are set in minutes.
Tangent Skim
Use this event to tangent skim any peak clusters. By
default, it chooses the tallest peak in a cluster as the
parent. You can also identify which peak in the
cluster is the parent. Tangent skim peaks are
detected on either side (or both sides) of the parent
peak. Tangent skim automatically resets at the end of
the peak cluster.
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Identification
Limit Peaks
Use the Limit Peaks area shown in Figure 10 to restrict the number of peaks
submitted to further qualitative processing. The options in this area are
normally not enabled, so Xcalibur processes all the peaks in the specified
Retention Time window range if they meet the detection criteria.
Figure 10. Limit Peaks area
The Limit Peaks options are:
Select Top Peaks
Use the options in this area to limit the processing
of detected chromatographic peaks to a specified
number of the most significant peaks in terms of
area or height:
1. Select the Enable check box.
2. Select one of the options:
• Click the Select By Area option to restrict
processing to the most significant peaks
based on area.
• Click the Select By Height option to
restrict processing to the most significant
peaks based on height.
• In the Num To Select box, type the
maximum number of peaks to be detected.
Xcalibur selects the largest peaks based on
intensity (height) or area.
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Rel Peak Height
Threshold
Creating a Processing Method for Qualitative Analysis
Identification
The options in this area limit processing of detected
chromatogram peaks to those exceeding the
percentage of the most intense peak (in terms of
height) in the chromatogram.
All detected chromatogram peaks meeting these criteria are submitted to
Spectrum Enhancement (if enabled) and then library searching according to
the options set on the Library Search Options and Library Search
Constraints pages.
Advanced Chromatogram
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.
Advanced options are available by clicking Advanced at the bottom of the
Identification page:
Identification Options
Use this dialog box to adjust the parameters
for baseline noise analysis and retention time
correction.
ICIS Advanced
Parameters
This dialog box appears when you use the
ICIS peak detection algorithm.
Genesis Advanced
Detection Options
This dialog box appears when you use the
Genesis peak detection algorithm.
Avalon Event List
This dialog box appears when you use the
Avalon peak detection algorithm.
For more information about ICIS, Genesis, and Avalon advanced detection
options, refer to the Xcalibur online Help.
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.
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Identification Options
Identification Options
Choose Options > Identification to open the Identification Options dialog
box shown in Figure 11. This dialog box contains the parameters used by
Xcalibur to estimate baseline noise and to correct retention time
assignments for void time. For more information about this dialog box,
refer to the Xcalibur online Help.
Figure 11. Identification Options dialog box
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Spectrum
Enhancement
Creating a Processing Method for Qualitative Analysis
Spectrum Enhancement
The Spectrum Enhancement page shown in Figure 12 offers options for:
• Improving the quality of a spectrum using one of two procedures:
Refine or Combine. Both procedures enhance the spectrum from a
peak by estimating and removing the background interference.
• Reducing the number of mass spectral peaks in a spectrum with the
Threshold parameter.
Xcalibur applies Spectrum Enhancement parameters to all peaks detected in
the chromatogram as a result of processing using the parameters on the
Identification page.
Figure 12. Spectrum Enhancement page of the Processing Setup - Qual view with Refine enabled
Selecting the
Enhancement Method
There are three enhancement methods:
• Refine
• Combine
• Threshold
Both Refine and Combine achieve an enhanced spectrum through
background subtraction. Combine also uses spectrum averaging.
You are generally advised to use Refine for spectrum enhancement unless
you fully understand how Combine works and are also sure that it is suited
to the chromatography.
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Spectrum Enhancement
Use the Refine and Combine procedures with care when working with
complex chromatograms featuring large numbers of peaks. Without a
reasonable baseline before and after a peak, neither algorithm can be
expected to estimate the background contribution accurately.
Refine
The Refine algorithm determines which ions in the selected spectrum derive
from a constant chromatography background and removes them to produce
a refined spectrum (see Figure 13).
Window Size (secs)
Abundance %
Apex Scan
Adjusted Intensity
Peak End
Peak Start
Background Noise
Time/Scan Number
Figure 13. Illustration of the Refine procedure
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Creating a Processing Method for Qualitative Analysis
Spectrum Enhancement
With Refine enabled, the Spectrum preview shows a Refine-enhanced
spectrum, permitting a test of the effects of Refine’s two parameters:
Window size (sec)
Start by setting this parameter to a typical peak
width. Refine applies the window to each side of
the peak apex, using it to search for the peak start
and peak end and to estimate the background
noise. In general, a wider window is more effective
than a narrower one.
Noise threshold
Start with a value of zero, increasing the setting
until the procedure eliminates spurious peaks
generated by background noise.
The Refine algorithm examines the mass chromatogram of each ion
contributing to the apex scan:
• It discards those without a peak maximum within ±1 scan of the target
chromatogram peak apex.
• It searches for a minimum within the specified Window Size range
either side of the peak apex. These points define the peak start and end.
• Using scans at and beyond the peak start and peak end, Refine measures
the background noise level in the mass chromatogram.
• Refine uses extrapolation to estimate the contribution of noise to the
scan at the peak apex and adjusts the mass intensity of the apex scan
accordingly.
• Refine uses the value from the Noise Threshold parameter to determine
whether the adjusted intensity is significant in comparison to the
background noise. If:
Adjusted Intensity < Noise Threshold × Background Noise
the mass is discarded from the final spectrum.
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Spectrum Enhancement
Combine
Click the Combine option on the Spectrum Enhancement page (see
Figure 14) to produce a single enhanced spectrum for each target
chromatogram peak by doing the following:
• Averaging all the scans across each peak top
• Subtracting background contributions (averaged from a number of
scans and scaled appropriately) assessed from baseline regions either side
of each peak
Figure 14. Processing Setup – Qual view – Spectrum Enhancement page, showing Combine enabled
Combine requires five parameters to set and test interactively. The
algorithm is applied to all detected chromatogram peaks in the time range
specified in the Range box of the Selected Retention Time Window area
(see Figure 6 on page 24). You might need to examine the peaks in a
reference chromatogram from a representative raw file to confirm that the
Combine settings are appropriate for all the peaks of interest.
For help when setting up the Combine feature, display scan numbers in the
Chromatogram preview. To do this, select the Scan Numbers check box in
the Labels page of the Chromatogram Display Options dialog box (see
“Labels” on page 152).
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Creating a Processing Method for Qualitative Analysis
Spectrum Enhancement
Figure 15. Chromatogram preview, showing scan number labels
The Combine parameters are:
Peak Top Region
Width (points) In this box, type the number of scans for Combine to
average across the apex of the peak. Examine the
chromatogram peak and estimate the number of good
scans across the peak apex.
Background Subtraction Left Region
The parameters in this area define the baseline region analyzed for
background contribution before a peak.
Region width
(points)
In this box, type the number of scans for Combine to
average in the analysis of the background in the left
background subtraction region.
Region end
In this area, select one of the following options to define
the end of the left background subtraction region:
Peak start
Click this option so that Combine uses
the scan at the detected peak start.
Points before
peak top
Click this option to specify the end
point as a specific number of scans
before the peak top.
Background Subtraction Right Region
The parameters in this area define the baseline region analyzed for
background contribution after a peak.
Region width
(points)
Thermo Electron Corporation
In this box, type the number of scans for Combine to
average in the analysis of the background in the right
background subtraction region.
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Creating a Processing Method for Qualitative Analysis
Spectrum Enhancement
Region start
In this area, select one of the following options to define
the start of the right background subtraction region:
Peak end
Click this option to have Combine to
use the scan at the detected peak end.
Points after
peak top
Click this option to specify the start
point as a specific number of scans after
the peak top. Specify the number of
points in the adjacent box.
Figure 16 shows a set of Combine parameters and illustrates their
relationship to a chromatogram peak.
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Creating a Processing Method for Qualitative Analysis
Spectrum Enhancement
Peak top region
Width (points) = 6
Abundance %
Left
Right
Region End
Region Start
8 points
before peak top
9 points after
peak top
Use
previous
5 points
in
background
Use
next
7 points
in
background
Time/Scan Number
Figure 16. Illustration of some optimized Combine parameters and their relationship to the scans forming a
chromatogram peak
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Spectrum Enhancement
Threshold
Click the Threshold option on the Spectrum Enhancement page (see
Figure 17) to limit the number of ions in the final spectrum prior to library
searching.
Figure 17. Processing Setup - Qual view - Spectrum Enhancement page,
showing Threshold option enabled
The single parameter is:
Cutoff threshold
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Xcalibur: Getting Productive with Qualitative Analysis
In this box, type a limiting intensity value as a
percentage of the most intense mass. Xcalibur
discards any ions with an intensity below the
specified threshold.
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Library Search
Options
Creating a Processing Method for Qualitative Analysis
Library Search Options
Use the Library Search Options page (see Figure 18) to specify how the
library search is carried out. Select among the following options:
• Choose between a range of search algorithms
• Limit the search to a maximum number of results.
• Choose the algorithm ‘direction’
• Limit the search by specifying a molecular weight
• Append a spectrum to a specified user library
Figure 18. Processing Setup - Qual view - Library Search Options page
Note Xcalibur uses the NIST Mass Spectral Search Program algorithms
to perform its library searches. The algorithms and related software are
distributed by the Standard Reference Data Program of the National
Institute of Standards and Technology and are the copyright of the US
Secretary of Commerce.
The online Help available in Xcalibur’s Library Browser gives additional
information relating to most of these parameters.
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Library Search Options
Selecting the Type of
Library Search
Select one of the following options from the Search type area shown in
Figure 19:
Identity
Click the Identity option to turn on the Identity
search options. This is the default search type.
Similarity
Click the Similarity option to turn on the Similarity
search options.
The difference between the two search types is primarily in the weightings
of the spectrum as a function of mass.
Figure 19. Search Type area, showing the default settings
Identity
After selecting an Identity search, choose one of the following search types:
Normal
This search algorithm is the default for the Identity
search option and uses the standard pre-screen search
filter.
Quick
Click this option only when the spectrum or compound
exists in the library. The search algorithm uses a fast
pre-screen search filter.
Penalize Rare
Compounds
This option is available only after selecting one or more
of the NIST databases (such as MAINLIB). It has no
effect on spectra in user libraries or other commercial
libraries.
Each reference spectrum in a NIST library contains a
record of other commercial databases containing
information about the compound. A compound is
considered rare if it is present in a limited number of
these databases.
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Similarity
Creating a Processing Method for Qualitative Analysis
Library Search Options
After selecting a Similarity search, choose one of the following search types:
Simple
Click this option to find a large set of spectra to
compare with the submitted spectrum. Because of the
large set, this search is generally slower than an Identity
search. Click this option if:
• The unknown spectrum is not in the library, or
• The spectrum is of poor quality so that a reliable
match is unlikely.
Hybrid
Click this option to choose a search that uses a
combination of the Simple and Neutral Loss searches.
The neutral loss search requires an estimate of the
unknown’s molecular weight. If the unknown
compound contains chemical structures that generate
both characteristic ions and neutral loss patterns, these
structures can be identified from the hit list produced by
this search.
Neutral Loss
Neutral losses in a spectrum are the mass differences
between the molecular ion and other major ions in the
spectrum. For certain classes of compound, neutral
losses can be characteristic spectral features.
If you select this option, Xcalibur examines the
submitted spectrum and identifies the molecular ion.
Xcalibur submits the mass value of the molecular ion to
the search along with the spectrum. The search
algorithm calculates the significant neutral losses and
compares them with library data. Search results are
returned according to matches of the molecular ion and
its neutral losses.
Other Search Options
Thermo Electron Corporation
The Options group shown in Figure 20 contains three ways to customize
the library search. The Reverse Search and Search With MW = options are
normally not active.
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Library Search Options
Figure 20. Options area, showing default settings
Maximum Number
of Hits
The value typed in this box restricts the number of
search results returned by a library search to be
stored in the result file. The valid range is 1 to 25.
The default is 5.
Reverse Search
Select this check box to sort all matching library
spectra by the Reverse Search Factor rather than the
(Forward) Match Factor. This option might prove
useful when the chromatogram has unresolved
peaks. In such cases, there can be interference
between neighboring peaks.
Search With
MW =
Select this check box to restrict the search to library
entries with the specified molecular weight. This
option replaces the standard prescreen with one that
retrieves all compounds with the specified molecular
weight, processing them with the chosen match
algorithm (such as identity or similarity). This
option pre-supposes specific knowledge of the target
compounds. In automatic processing this
knowledge is not normally available.
Note Do not use the Search with MW= option when you have already
clicked the Molecular weight option on the Library Search Constraints
page.
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Adding Spectra to a User
Library
Creating a Processing Method for Qualitative Analysis
Library Search Options
Use the Append to User Library area shown in Figure 21 to add processed
spectra to a specific user library.
Figure 21. Append to User Library area
To set matching criteria to avoid duplicate entries
1. Select the Enable check box.
2. Either select a user library from the combo box list or type a new library
name. The drop down list includes all currently active user libraries.
If you type a new library name, the library is created only if a spectrum
fails the matching criteria. This is why you are advised to enter the
highest possible values in the Threshold boxes. The failed spectrum is
appended to the library.
3. Enter threshold values for Match factor, Reverse match factor, and
Probability%. To confirm that all spectra are appended to the library,
enter 999, 999, and 100 respectively. See “Submitting a Spectrum to a
Library Search” on page 198 for an explanation of these threshold
values.
During processing and after any requested enhancement of a relevant
chromatogram peak apex scan, Xcalibur submits the spectrum to a library
search:
• If the top hit from a library search exceeds (or is equal to) any one of the
threshold values, Xcalibur returns the hit list and the spectrum is not
appended to the specified library.
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Library Search Options
• If the top hit fails to reach any of the threshold values, Xcalibur discards
the hit list (no search results are reported) and appends the searched
spectrum to the specified library. Unless otherwise specified, the library
is stored in the NIST\MSSearch folder.
Selecting Libraries
Click Search List on the Library Search Options page (see Figure 18 on
page 47). The Search List dialog box appears (see Figure 22). This dialog
box lists the names and search order of libraries to be used in library search
processing. The list on the left of the dialog box shows the available libraries.
The list on the right of the dialog box shows the selected libraries in their
search order.
Figure 22. Search List dialog box
Xcalibur generates the Available Libraries list dynamically. Each time you
open the Search List dialog box, it lists the libraries available on the system
at that time.
Libraries listed in the Selected Libraries list, on the other hand, might not
exist on the system - they might have been selected on another system or
deleted or renamed since the method was created.
When you click OK on the Search List dialog box, all libraries in the
Selected Libraries list are checked. If any of them do not exist, a warning
dialog box is displayed.
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Library Search Options
To include a library in the search list
1. Select the library name in the Available Libraries list.
2. Click Add.
The library is added to the Selected Libraries list.
To change the search order of selected libraries
Select the library name in the Selected Libraries list:
• Click Top to move the library to the top of the list.
• Click Up to move the library up one position.
• Click Down to move the library down one position.
• Click Bottom to move the library to the last position.
To exclude a library from the search list
1. Select the library name in the Selected Libraries list.
2. Click Remove.
The library is transferred to the Available Libraries list.
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Library Search Constraints
Library Search
Constraints
For processing efficiency, constrain a library search using some of the
options on the Library Search Constraints page (these are all normally not
enabled). For example, exclude certain high intensity ions that appear in
many compounds or that are present in the spectrum background. You can
target a search to a particular range of molecular weights or to compounds
containing certain elements.
The Library Search Constraints page (see Figure 23) contains five groups of
options to constrain the library search:
• Molecular Weight
• Other Databases
• Name Fragment
• Element Constraints
• Mass Spectral Peak Constraints
Note Using Library Search Constraints requires specific knowledge
about the target compounds in the chromatogram. This knowledge is
not always possible in automated processing.
Figure 23. Processing Setup - Qual view - Library Search Constraints page
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Molecular Weight
Creating a Processing Method for Qualitative Analysis
Library Search Constraints
Select the Enable check box in the Molecular Weight area (see Figure 24) to
constrain the library search to compounds with a specific molecular weight
or molecular weight range. All search results must satisfy the specified
molecular weight range.
Type a molecular weight or molecular weight range in the Range box (for
example, 235, or 200-250). The valid range is 1 to 999999.
Figure 24. Molecular Weight area
Note This constraint is not available if you have already clicked the
Search with MW= option on the Library Search Options page.
Other Databases
Select the Enable check box in the Other Databases area to constrain the
library search to entries in the NIST library that are also featured in other
databases (see Figure 25). NIST library entries contain references to other
commercial databases if they contain information about the compound.
Figure 25. Other Databases area
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Creating a Processing Method for Qualitative Analysis
Library Search Constraints
Select from the following databases:
Fine
Commercially Available Fine Chemical Index
TSCA
Toxic Substances Control Act Inventory
RTECS
Registry of Toxic Effects of Chemical Substances
EPA
EPA Environmental Monitoring Methods Index
USP
US Pharmacopoeia/U.S.A.N.
HODOC
CRC Handbook of Data of Organic Compounds
NIH
NIH-NCI Inventory File
EINECS
European Index of Commercial Chemical Substances
IR
NIST/EPA Gas Phase IR Database
Xcalibur only reports search results if they feature in one or more, but not
necessarily all, of the selected databases.
Name Fragment
Select the Enable check box in the Name Fragment area to constrain the
search to compounds with a specific name or name fragment. The Name
box accepts a text string (up to 39 characters) to represent a fragment of a
compound name; for example, “cyclo”. The library search filters search
results and only returns those containing the specified text in their names.
Note that the entry is not case sensitive; for example “CYCLO” returns
compounds containing the fragments “cyclo”, “Cyclo”, “CYCLO” and any
other case permutations.
Figure 26. Name Fragment area
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Element Constraints
Creating a Processing Method for Qualitative Analysis
Library Search Constraints
Click this option to constrain the library search to compounds with specific
element profiles. There are two methods:
• Individual Element
• Elements in Compound
Individual Element
Use this area to set specific criteria about the elements required in a library
search result (hit). For example, the entries shown in Figure 27 would only
return search results for compounds that contain more than five fluorine
atoms and exactly three chlorine atoms.
You do not need to provide a complete elemental profile. The library search
returns compounds if they satisfy all the specified criteria regardless of any
other elements present. Use this option to specify which elements you do
not want to be in a library hit. For example, C = 0 confirms that no search
results contain carbon.
When a contradiction occurs, for example, C < 2, and C > 4, Xcalibur
displays an error dialog box. Correct the contradiction to proceed.
Figure 27. Element Constraints area
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Library Search Constraints
Elements in Compound
Use this area (see Figure 27) to enter a list of elements that must be present
in returned search results. Separate each element in the list (of up to 30
characters) by the character specified as the list separator character. See
“Changing the List Separator Character” on page 101. If you leave the box
blank, the Elements in Compound Constraint function is not applied. If
you enter an invalid element, Xcalibur reports an error when the edit field
loses focus.
Apply the constraint in one of two ways:
Avoiding Contradictions
58
Some
Click the Some option to have the returned search results to
contain at least one of the specified elements and no elements
other than those listed. For example “C, H, O” would return
CO2, CH4, and HCHO but not CH2Cl2 (Cl not allowed).
All
Click the All option to have the returned search results to
contain all, and only, the listed elements. For example “C, H,
O” would return HCHO but not CO2, CH4, or CH2Cl2.
If there are no contradictions, you can use the two types of elemental
constraints together. For example, if the Individual Element group contains
“C=0” or “C<1" in and the Elements In Compound box contains “C” when
the All option is clicked, Xcalibur displays an error dialog box. Correct the
contradiction to proceed.
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Mass Spectral Peak
Constraints
Creating a Processing Method for Qualitative Analysis
Library Search Constraints
Select the check box in the Mass Spectral Peak Constraints area to build a
profile of ions and their abundances to be matched against library entries
during the search. The search algorithm only returns search results matching
the specified constraints. There are four types of mass spectral peak
constraints:
Normal
Select Normal from the Type list if the constraint
applies to a specific ion represented by its m/z value.
The From and To values represent the ion abundance.
Loss
Select Loss from the Type list to constrain the search to
a neutral loss from a molecular ion. In this case, the
m/z value (limited to 64) represents the mass of the
lost neutral group, for example, for methyl m/z = 15.
For this constraint to be matched, a library spectrum
must contain:
• A fragment ion at an m/z value 15 less than the
molecular ion
• An abundance in the specified From and To range
Rank
Select Rank from the Type list to consider the order of
an ion in the spectrum in terms of relative abundance.
Ions are ranked from the largest (the base ion) to
the 16th. A compound matches a Rank constraint if its
library spectrum contains a mass spectral peak:
• At the specified m/z value
• Ranked between the specified From and To rank
positions
If you specify the same number in both fields, the designated ion must
have that rank in the retrieved spectrum.
Maxmass
Sets a constraint on the m/z value of the most
significant high mass ion. Library search results must
feature:
• An ion at the specified m/z value
• No significantly larger masses at higher m/z values
• An abundance in the specified From and To range
In the case of Normal and Loss type constraints, the
abundance values can be absolute or relative.
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Library Search Constraints
Absolute
Click this option to evaluate all entries as a percentage
of the base (largest) ion in the spectrum. Values must
be between 0 and 100%. For example, if you type 10
and 50 in the From and To fields, the search only
returns search results where the specified ion is present
at an abundance of between 10 and 50%.
Relative
Click this option to consider the second and
subsequent entries relative to the first. This option is
restricted to Normal or Loss types. For example, in the
search algorithm shown in Figure 28, library search
results must contain:
• An ion at m/z 125 with an abundance between 10
and 50% of the base ion
• An ion at m/z 250 with an intensity between 50%
and 999% of the observed intensity of the first ion
in the list
Rank or Maxmass types are not available in Relative
mode.
Note If you switch between Absolute and Relative modes, a warning
dialog box is displayed, and the grid is cleared of all its rows.
Figure 28. Example of a relative mass spectral peak constraint
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Peak Purity
Creating a Processing Method for Qualitative Analysis
Peak Purity
Use the Peak Purity page shown in Figure 29 to specify values for peak
purity parameters to be included in a qualitative Processing Method for the
PDA detector type only. After specifying the Processing Method in a
sequence, apply the parameters to the qualitative PDA analysis when you
acquire data.
Figure 29. Processing Setup – Qual view- Peak Purity page
The Peak Purity page 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
To determine suitable Peak Purity parameters for raw data, process 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 Processing
Setup. To produce a Peak Purity report, use the Reports view of Processing
Setup and include the correlation factor in a Processing Method.
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Peak Purity
Enable Peak Purity
To enable peak purity parameters and calculate peak purity results, select
the Enable check box. Then, type appropriate values in the following boxes:
• Scan Threshold
• Peak Coverage
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 of 40 mAU for scan threshold.
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 the Wavelength Range
box. Select this check box to limit the number of wavelengths to include in
the peak purity computation. Then, type 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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Reports
Creating a Processing Method for Qualitative Analysis
Reports
Xcalibur’s automated reporting creates comprehensive, high quality, printed
documentation. Xcalibur’s XReport reporting package uses Microsoft Word
to create report templates, using a palette of Report Objects for insertion at
any point in a page. To customize reports to suit personal requirements, use
XReport.
Tip To open XReport, double-click on the XReport icon on the desktop or
choose Start > All Programs > Xcalibur > XReport from the
Windows XP taskbar.
The Reports view of Processing Setup shown in Figure 30 lists:
Sample Reports
The reports issued for each sample.
Summary Reports
The summary reports issued after processing of a
sequence quantitation bracket (or non-bracketed
sequence).
Xcalibur is equipped with a number of built-in report templates.
Figure 30. Processing Setup - Reports view
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Creating a Processing Method for Qualitative Analysis
Reports
Sample Reports
The Sample Reports list consists of seven columns:
Enable
Select this check box to enable a report.
Std
Select this check box to produce a report for a Standard
sample type. Yes appears in the cell.
QC
Select this check box to produce a report for a QC sample
type. Yes appears in the cell.
Unk
Select this check box to produce a report for Unknown
sample type. Yes appears in the cell.
Other
Select this check box to produce a report for all other
sample types Yes appears in the cell.
Save As
Select a report export option. Xcalibur saves the exported
file with the sample file name and the appropriate
extension in the Data folder where result files are stored.
Valid export file types are:
Report
Template
Name
None
print only, no exported file
Text
ASCII text file (*.txt), no printed copy
Doc
Word XP file (*.doc), no printed copy
HTML
HTML file, (*.html), no printed copy
Type the full pathname of the template to be used by
Xcalibur in the generation of the 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, right-click the cell, and choose 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. Select the options as
required.
A shortcut menu is available within the grid. Right-click within a row to
access additional commands to:
• Delete the selected row or rows
• Insert a row above the selected row or rows
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Summary Reports
Creating a Processing Method for Qualitative Analysis
Reports
The summary reports list contains three fields:
Enable
Enable report.
Save As
Select a report export option. Xcalibur saves the exported
file with the file name and the appropriate extension in
the Data folder where result files are stored. Valid export
file types are:
Report
Template
Name
None
print only, no exported file
Text
ASCII text file (*.txt), no printed copy
Doc
Word XP file (*.doc), no printed copy
HTML
HTML file, (*.html), no printed copy
Type the full pathname of the template to be used by
Xcalibur in the generation of the report.
Edit cells and rows as described in the topic “Sample Reports” on page 64.
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2
Creating a Processing Method for Qualitative Analysis
Programs
Programs
Use the Programs view of Processing Setup shown in Figure 31 to list
programs or macros to be run by Xcalibur after the analysis of a sample
and the processing of the resulting data.
Figure 31. The Programs view in Processing Setup
Xcalibur runs the programs in the listed order.
Programs are defined by nine headings:
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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.
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Other
Determines whether Xcalibur runs the program after any
other type of sample analysis.
Action
Displays Run Program or Run Excel Macro options.
Program or
Macro Name
Lists the full pathname of the program or Microsoft Excel
macro.
Sync
Determines whether the selected program is to be run
synchronously. If you select Yes, Xcalibur waits for the
program to terminate before starting any other processing
task. If you select No, Xcalibur continues with other
processing tasks without waiting for the program to
terminate.
Parameters
Specifies any command parameters for the selected
program. Use the following macro parameters in the
Parameters column (see Table 6).
Table 6.
Specifying a Program or
Macro
Creating a Processing Method for Qualitative Analysis
Programs
Example Program Parameters
Macro
Parameters
Macro Parameter Replacement
%R
Provides the current raw file
%F
Provides the current result file
%%
Provides a single % character in the run line
%X
Provides the result file name with extension .xls
To specify a Program or Macro, click the Enable field of the appropriate
row. Then:
• Click the Program or Macro Name cell and type the full path name, or
• Double-click the cell to identify the program using a standard Browse
dialog box, or
• Click the cell first, right-click it, and select Browse from the shortcut
menu
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Creating a Processing Method for Qualitative Analysis
Programs
Changing the Program
Sample Type
To change any of the program sample type fields (Std, QC, Unk, or Other),
click the appropriate cell to access a check box: enable the option as
required.
A shortcut menu is available within the grid. Right-click within a row to
access additional commands to:
• 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
Example using the
Xconvert.exe program
To convert a data file for a sample from raw (.raw) file format to ANDI
(.cdf ) file format and copy it to the current default data directory, use the
Xconvert program with the following parameters:
/DA /SL %R,
where DA indicates that the destination file (D) is to be ANDI format (A),
SL indicates that the source file (S) is a raw file (L), and %R is the macro
argument for the current raw file.
To activate this conversion for each Unknown sample in a sequence, set the
Programs parameters as shown in Table 31. For more information about
Xconvert, refer to the Xcalibur online Help.
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Method Examples
Example 1
Creating a Processing Method for Qualitative Analysis
Method Examples
This section describes four typical qualitative Processing Methods and
illustrates how a method might be developed.
The default Processing Method detects peaks in a TIC over the full
retention time range of 0.0 to 999.0 min. Peak Integration options are set at
the default values. The number of peaks to pass to the library search is not
limited (see Figure 32).
Figure 32. Identification page, showing default settings
Spectrum enhancement is unavailable, and no Library Search Options or
Constraints have been applied (see Figure 33 and Figure 35). All identified
peaks are submitted to a Normal Identity library search (see Figure 34).
This method is suited to most common qualitative analysis applications
when little is known about the analytes.
Figure 33. Spectrum Enhancement page, showing default settings
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Creating a Processing Method for Qualitative Analysis
Method Examples
Figure 34. Library Search Options page, showing default settings
Figure 35. Library Search Constraints page, showing default settings
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Example 2
Creating a Processing Method for Qualitative Analysis
Method Examples
The Processing Method is set up to detect peaks in a TIC chromatogram
over a retention time range of 3.0 to 5.0 min. Peak integration defaults are
set at the default values. The library search is limited to peaks with
intensities greater than 5% of the base peak (see Figure 36).
Relative Peak Height
Threshold set to 5% of
Highest Peak
Retention Time Window
Range set
to 3.0 to 5.0 min
Figure 36. Identification page, showing new settings
Spectrum Enhancement is not enabled (see Figure 37).
Figure 37. Spectrum Enhancement page, showing defaults
The Append to Library option is clicked. The software appends spectra to
the Pesticides 1 user library if the Match factor is <500, the Reverse match
factor is <700, and the probability of a hit is less than 10% (see Figure 38).
Figure 38. Library Search Options page, showing the Append to Library option
clicked
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Creating a Processing Method for Qualitative Analysis
Method Examples
No library search constraints are enabled (see Figure 39).
Figure 39. Library Search Constraints page with the default settings
Example 3
The Processing Method is set up to detect peaks in a TIC chromatogram
over a retention time range of 3.0 to 5.0 min. Peak integration defaults are
set at the default values. The library search is limited to peaks with
intensities greater than 5% of the base peak (see Figure 40).
Relative Peak Height
Threshold set to 5% of
Highest Peak
Retention Time Window
Range set
to 3.0 to 5.0 min
Figure 40. Identification page, showing new settings
Spectrum Enhancement is enabled. The Refine option is clicked with the
default settings. The background noise for the peak is determined within a 6
sec. window bracketing the peak apex. Ions are discarded from the enhanced
spectrum unless their intensities are three times the measured background
noise (see Figure 41).
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Creating a Processing Method for Qualitative Analysis
Method Examples
Refine
Enhancement
Option
Figure 41. Spectrum Enhancement page, showing the Refine option clicked
The Append to Library option is clicked.The software appends spectra to
the Pesticides 1 user library if the Match factor is <500, the Reverse match
factor is <700, and the probability of a hit is less than 10% (see Figure 42).
Figure 42. Library Search Options page, showing the Append to Library option
clicked
No library search constraints are enabled (see Figure 43).
Figure 43. Library Search Constraints page with the default settings
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Creating a Processing Method for Qualitative Analysis
Method Examples
Example 4
The Processing Method is set up to detect peaks in a TIC chromatogram
over a retention time range of 3.0 to 5.0 min (the same as examples 2 and
3). Peak integration defaults are set at the default values. The library search
is limited to peaks with intensities greater than 5% of the base peak (the
same as example 3) (see Figure 44).
Relative Peak Height
Threshold set to 5% of
Highest Peak
Retention Time Window
Range set
to 3.0 to 5.0 min
Figure 44. Identification page, showing new settings
Spectrum Enhancement is enabled. The Refine option is clicked with the
default settings of a 6 min window and a 3 noise threshold (see Figure 45).
Refine
Enhancement
Option
Figure 45. Spectrum Enhancement page, showing the Refine option clicked
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Creating a Processing Method for Qualitative Analysis
Method Examples
The Append to Library option is clicked. The software appends spectra to
the Pesticides 1 user library if the Match factor is <500, the Reverse match
factor is <700, and the probability of a hit is less than 10% (see Figure 46).
Figure 46. Library Search Options page, showing the Append to Library option
clicked
The Molecular Weight and the Element Constraints options have been
enabled. The library search is limited to spectra of compounds with a
molecular weight of 200 to 550 that contain Chlorine (see Figure 47).
Molecular Weight
limited to 200 to 550
Compounds must
contain at least one
chlorine atom
Figure 47. Library Search Constraints page with Molecular Weight and Element
Constraints option clicked
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Chapter 3
Automating Analysis in
Sequence Setup
This chapter describes Sequence Setup and explains how to set up and use a
sequence for automated qualitative analysis.
This chapter contains the following sections:
• About Sequence Setup
• The Sequence Setup Window
• About Sequences
• Creating a New Sequence
• Working with a Sequence
• Running Samples
• Reprocessing Samples
• The Acquisition Queue
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Automating Analysis in Sequence Setup
About Sequence Setup
About Sequence
Setup
To automate data acquisition or reprocessing, use Sequence Setup.
Use Sequence Setup to compile a sequential list containing a variety of
sample types. However, for qualitative analysis, the samples would normally
be of type “unknown”. Each row of the list corresponds to one sample
injection. From within Sequence Setup, run a single sample, complete
sequence, or reprocess a batch of previously acquired raw data files.
During acquisition, Xcalibur controls the instrumentation using
information from the sequence. Each time you select processing options 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.
This chapter specifically describes how to set up and use a sequence for
automated qualitative analysis. The companion manual, Xcalibur Getting
Productive: Quantitative Analysis, describes how to set up and use a sequence
solely for automated quantitative analysis. Xcalibur permits both to be
performed simultaneously and, to do this, consult both documents.
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The Sequence Setup
Window
Automating Analysis in Sequence Setup
The Sequence Setup Window
Sequence Setup is one of the view options on the Xcalibur Home Page. To
select Sequence Setup, display the Home Page. Then, do one of the
following:
• Choose View > Sequence Setup View.
• Click the Sequence View button on the View toolbar. If the toolbar is
not displayed, choose View > View Toolbar.
Home Page has four toolbars:
View
Provides options for Home Page view
Road Map
Contains tools for controlling acquisitions and for
accessing Instrument Setup and Processing Setup
Sequence Editor
Contains tools for editing sequences in Sequence
Setup view
Plot
Contains tools for controlling real time plots
during data acquisition
Within Sequence Setup, use the View and Sequence Editor toolbars.
Display or hide a toolbar by choosing the appropriate View menu
command.
For details about Road Map and Plot toolbars and more information about
the use of Home Page, refer to the Getting Started manual for the
instrument.
For specific information about Home Page buttons on the toolbar or menu
commands, refer to the Xcalibur online Help.
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Automating Analysis in Sequence Setup
About Sequences
About Sequences
Each row in the sequence table describes a single sample acquisition. For
qualitative analysis, a sequence is the list of samples to be analyzed (see
Figure 48).
Figure 48. A simple sequence for the qualitative analysis of five samples
Sequence rows can contain many parameters described by the column
headings. Choose which columns are displayed or hidden by using the
method described in “Arranging the Columns” on page 82.
The following list describes those parameters that define a sample
acquisition and that are appropriate to qualitative analysis:
Sample Type
Type of sample, selected from the following:
• Unknown (the ‘normal’ choice for qualitative
analysis; all other types are only normally used for
quantitative analysis)
• Blank
• QC (quality control)
• Standard Clear
• Standard Update
• Start Bracket
• End Bracket
• Standard Bracket
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File Name
Name of the file to contain the sample data.
Sample ID
An identifier unique to the sample. This field can also
be used to import a barcode identifier.
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Automating Analysis in Sequence Setup
About Sequences
Path
The path to the raw file that Xcalibur creates for the
sample data. Xcalibur creates this file with a .raw
extension.
Inst Meth
The path and file name of the Instrument Method to
be used for acquisition.
Proc Meth
The path and file name of the Processing Method to be
used to process the acquired data.
Position
The sample vial number. The format of the entry
depends on the configured autosampler.
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.
ISTD Corr
Amount
A bulk correction factor applied to internal standards.
Study
User-defined topic with a default heading of “Study”.
Client
User-defined topic with a default heading of “Client”.
Laboratory
User-defined topic with a default heading of
“Laboratory”.
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).
To open an existing sequence, click the Open button on the
toolbar or choose File > Open.
To start a new sequence, click the New button on the toolbar or
choose File > New. The New Sequence Template dialog box
appears.
To save the current sequence, click the Save button on the
toolbar or choose File > Save.
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Automating Analysis in Sequence Setup
About Sequences
Arranging the Columns
To change the arrangement of columns in Sequence Setup
Choose Change > Columns or click the Column Arrangement button in
the toolbar to open the Column Arrangement dialog box shown in
Figure 49.
Figure 49. Column Arrangement dialog box
The columns currently displayed are listed in the Displayed Columns pane
in the order that they appear.
To display a column that is currently hidden
1. Select the column heading in the Available Columns list.
2. Click Add. The column title is moved from the Available Columns list
to the Displayed Columns list.
To hide a column that is currently displayed:
1. Select the column heading in the Displayed Columns list.
2. Click Remove. The column title is moved from the displayed columns
list to the Available Columns list.
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Automating Analysis in Sequence Setup
About Sequences
To change the order of the Displayed Columns:
1. Select the column heading you want to move.
2. Click Move Up or Move Down.
3. Repeat the procedure with other columns.
4. Click OK to save the changes and close the dialog box. Xcalibur displays
the columns in Sequence Setup in the new arrangement.
Changing User Labels
To define the caption labels of the five user defined columns, choose User
Labels.
To change a heading caption
1. Choose Change > User Labels or click the User Labels button in the
toolbar to open the User Labels dialog box (see Figure 50).
Figure 50. User Labels dialog box
2. Type the new heading caption in the heading box to replace the current
heading caption. To avoid using a 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 the new captions.
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Automating Analysis in Sequence Setup
Creating a New Sequence
Creating a New
Sequence
There are three ways to create a new sequence:
• Import a sequence from a text file
• Provide Xcalibur with some basic details and allow it to create the
sequence
• Type the sequence manually
Importing a Sequence
Use Xcalibur to import data into some or all of the columns in a sequence.
Xcalibur reads the following:
• Comma separated text files with file extension .csv. This file format can
be created by a text editor, such as Microsoft Notepad, or a spreadsheet
program, such as Microsoft Excel.
• Gilson Unipoint™ files
To import a Sequence
1. Choose File > Import Sequence to open the Import Sequence dialog
box shown in Figure 51.
Figure 51. Import Sequence dialog box
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Automating Analysis in Sequence Setup
Creating a New Sequence
2. Click Browse to select the file for importing, or type the path and file
name directly into the Import from File field.
3. In the Select Columns to Import area, select the sequence columns to be
included in the imported file.
• Click All to select all the column options.
• Click Clear to deselect all the column options.
4. 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 file type, or
• When the separator character is different from the character currently
set in the International dialog box. (See “Changing the List Separator
Character” on page 101.)
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Automating Analysis in Sequence Setup
Creating a New Sequence
Using the New Sequence
Template to Create A
Sequence
To let Xcalibur create a sequence, click the New Sequence button on the
toolbar or choose File > New. The New Sequence Template dialog box
shown in Figure 52 is displayed.
This method is particularly useful when running large numbers of similar
samples.
Figure 52. New Sequence Template dialog box
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Entering General Information
Specifying Samples
Automating Analysis in Sequence Setup
Creating a New Sequence
In the General area of the New Sequence Template dialog box, provide the
following information:
Base File
Name
Type a base file name for the raw files. Xcalibur applies
this name to all of the raw files that it creates using the
new sequence. Xcalibur determines an incremental
numeric suffix for the base name starting at 001. To have
Xcalibur start with a different number, type the number
in the Starting Number box.
Path
Type a path to the directory where you want Xcalibur to
store the raw files or click Browse to locate the drive and
directory.
Instrument
Method
Select an existing Instrument Method. Click Browse to
locate the file.
Processing
Method
Select an existing Processing Method. Click Browse to
locate the file.
Calibration
File
Leave this combo box blank. This is only used for
quantitative processing.
In the Samples area, enter details about the number of samples in the
sequence and select the autosampler tray type.
Note The Tray Type list is not available for all autosamplers that can be
configured in the Xcalibur Instrument Configuration application. For
example, the Tray Type list is not available for the Surveyor
Autosampler.
Enter details about the samples:
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Number of
Samples
In this box, type the number of samples to be analyzed.
Injections
per Sample
In this box, type the number of times each sample is to be
analyzed.
Base Sample
ID
Type the identifier code for the sequence. The Base Sample
ID is an alphanumeric prefix to the Sample ID that
Xcalibur applies to each sample in a new sequence.
Xcalibur adds a suffix to the base sample ID starting
with 001. For example, if you enter a base sample ID of
AB12, Xcalibur numbers the first five samples as follows:
AB12001, AB12002, AB12003, AB12004, AB12005.
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Automating Analysis in Sequence Setup
Creating a New Sequence
Select the autosampler configuration:
Tray Type
Select the autosampler tray type from the drop-down list.
This list is available for only some of the autosamplers
controlled from Xcalibur.
Initial Vial
Position
In this box, type the first vial number in the tray.
Re-Use Vial
Positions
Click this check box to have Xcalibur to use the same vial
number for replicate samples.
Click Select Vials to display the Vial Selection dialog box shown in
Figure 53. 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. To deselect all selected vials (highlighted in blue), click
Cancel Selection in the New Sequence Template dialog box.
Note The Select Vials feature is not available with all autosamplers.
Figure 53. Vial Selection dialog box
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Automating Analysis in Sequence Setup
Creating a New Sequence
Choosing a Bracket Type
In the Bracket Type area, select the None option. The other options are for
quantitative processing only.
Specifying Standards and Blanks
The Calibration area options are for quantitative processing only. Leave all
parameters unchecked.
Specifying QC Samples
The QC area options are for quantitative processing only. Leave all
parameters unchecked.
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 row
in the sequence:
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Sample Type
Click the Sample Type cell and select: Unknown,
Blank, QC, Std Clear, or Std Update from the list.
File Name
Type a file name for storing the sample data.
Sample ID
Type a sample identification number.
Path
Type the directory path of the folder to store the raw
file of the sample, or double-click in the box and
browse to the appropriate directory using the Select
Directory dialog box.
Inst. Meth
Type the path and filename of an Instrument Method
file for data acquisition, or double-click in the box and
browse to the appropriate file using the Select
Directory dialog box. Instrument methods have the file
extension .meth.
Proc Meth
Type the path and filename of the Processing Method
file for data acquisition or file reprocessing, or
double-click in the box and browse to the appropriate
file using the Select Directory dialog box. Processing
methods have the file extension .pmd.
Cal File
Because this file type is only used for quantitative
processing, leave this column blank.
Position
Specify the position of the vial on the autosampler tray.
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Automating Analysis in Sequence Setup
Creating a New Sequence
Inj. Vol.
Type the injection volume (in microliters). Xcalibur
sends this volume to the autosampler. If you do not
enter an injection volume, Xcalibur uses the default
injection volume set in the Instrument Method.
Level
Leave this column blank. This parameter is only used
for quantitative processing.
ISTD Corr Amt
Leave this column blank. This parameter is only used
for quantitative processing.
Dil Factor
Leave this column blank. This parameter is only used
for quantitative processing.
The following columns 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 can be hidden. See “Arranging the Columns” on page 82.
They are not essential for the running of a sample or sequence.
To enter text information under any of these column headings:
1. Click the relevant grid cell.
2. Type the required information.
3. Click any other cell.
Repeat this procedure for all rows in the sequence. To save time in
duplicating column entries use the Fill Down command or button on the
toolbar. See “Filling Down Columns” on page 91.
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Working with a
Sequence
Automating Analysis in Sequence Setup
Working with a Sequence
The Sequence Editor provides a number of tools and commands to help
create a sequence.
This section contains the following topics:
• 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
Filling Down Columns
Use the Fill Down command to copy information from one row to any
number of rows immediately below it in the sequence table. You can copy
information from a single cell or a complete row.
To fill down sample settings
1. Select the cells in the row you want to copy.
2. Drag downwards to select the range of columns to be filled (edit the
selection in step 4). Select at least one row to enable the command.
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Automating Analysis in Sequence Setup
Working with a Sequence
3. Choose Edit > Fill Down or click the Fill Down button in the toolbar.
Xcalibur displays the Fill Down dialog box shown in Figure 54.
Figure 54. Fill Down dialog box
4. Check the selection, shown at the bottom of the Fill Down dialog box.
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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Automating Analysis in Sequence Setup
Working with 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 the appropriate information from the first row into the
selected range.
Inserting a Row
To insert a row
1. Select the row immediately below where you want to insert a row.
2. Choose Edit > Insert Row. A dialog box asks for confirmation of the
action.
3. Click Yes.
4. 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 you want to delete
2. Choose Edit > Delete Row. A dialog box asks for confirmation of the
action.
3. Click Yes.
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Working with a Sequence
Going to a Sequence Row
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 shown
in Figure 55.
Figure 55. Go To Line Number dialog box
3. Click OK.
Xcalibur closes the dialog box and highlights the selected row.
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Transferring Row
Information
Automating Analysis in Sequence Setup
Working with a Sequence
Use the transfer row procedure to confirm 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 on the toolbar.
2. Select from the following options in the Transfer Row Information
dialog box shown in Figure 56.
Figure 56. Transfer Row Information dialog box
Match by Sample
ID
Click this 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.
Match by Position
Click this 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.
To undo the copy operation, immediately choose Edit > Undo or click the
Undo button in the toolbar.
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Working with a Sequence
Printing a Sequence
Print a full sequence or a vial list compiled from the active sequence by
doing one of the following options.
To preview the Sequence before printing
1. Choose File > Print Preview to display the Print Selection dialog box
shown in Figure 57.
Figure 57. Print Selection dialog box
2. Click one of the following:
• Click the Vial Position List option to preview the vial list from the
active sequence
• Click the Full Sequence option to preview the active sequence
• Click the Displayed Columns Only option to preview the displayed
columns
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Working with a Sequence
3. Click OK to open the Print Preview dialog box shown in Figure 58.
Figure 58. Print Preview window
4. Use Next Page, Previous Page, Two Page, Zoom In, or Zoom Out to
preview the active list pages.
5. Click Close to return to Sequence Setup or click Print to print the
displayed list.
To print the Sequence without previewing it
1. Choose File > Print or click the Print button on the toolbar. Xcalibur
displays the Print Selection dialog box shown in Figure 57.
2. Click one of the following:
• To print the vial list from the active sequence, click the Vial Position
List option.
• To print the active sequence, click the All Columns option.
• To print the displayed columns only, click the Displayed Columns
Only option.
3. Click OK to open the Print dialog box.
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Working with a Sequence
4. 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 simple utility for you to check the amount of available disk space
on system drive(s):
1. Choose Actions > Check Disk Space or click the Disk Space button
on the toolbar.
Xcalibur displays the Disk Space dialog box shown in Figure 59.
Figure 59. Disk Space dialog box
This dialog box lists:
• The current drive and directory. For example:
C:\Xcalibur\examples\data.
• 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.
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Working with a Sequence
• A pie-chart in which the available space is shown in the color green
and the used space is shown in the color red.
• The total capacity of the current drive, for example: 1220 MBytes
Total.
2. Click Directory to open the Select Directory dialog box and check disk
space on another disk.
3. Click OK to close the dialog box.
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Working with a Sequence
Exporting a Sequence
To export a sequence as a separator delimited text file with a file
extension .csv, use the File > Export Sequence option. A text editor, such as
Microsoft Notepad, or a spreadsheet program, such as Microsoft Excel, can
read this file format.
The exported sequence file contains the current list separator character
(normally a comma) that is set in the Microsoft Windows dialog box. See
“Changing the List Separator Character” on page 101.
To export a Sequence
1. Choose File > Export Sequence. Xcalibur displays the Export Sequence
dialog box shown in Figure 60.
Figure 60. Export Sequence dialog box
2. Type the path and file name of the exported sequence file in the Export
To File box. The file extension is .csv. Or click the Browse button to
select a path for the exported sequence file. Xcalibur assigns
extension .csv to the exported file.
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Working with a Sequence
3. Use the check boxes in the Select Columns To Export 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.
Changing the List Separator
Character
When you export a sequence, Xcalibur creates a text file with file
extension .csv and inserts a list separator character between each field of
each column of the sequence. A text editor, such as Microsoft Notepad, or a
spreadsheet program, such as Microsoft Excel, can read this file format.
The list separator can be any alphanumeric character. However, characters
that cannot be distinguished from the characters used in the sequence text
fields (such as alphabetic characters) should be avoided 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 operating system.
To change the list separator character
1. In the Windows XP taskbar, click Start. Then, choose Control Panel.
2. Double-click the Regional and Language Options icon to open the
Regional and Language Options dialog box.
3. Click the Regional Options tab.
4. Click Customize to open the Customize Regional Options dialog box.
5. In the List Separator Combo box, type the new list separator character.
6. Click OK to store the new list separator and close the dialog box.
7. Click OK to close the Regional and Language Options dialog box.
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Automating Analysis in Sequence Setup
Running Samples
Running Samples
Running a Single Sample
Run a single sample from a sequence, a range of samples, or the full
sequence. To reprocess raw files, use the Batch Reprocess command.
To run a single sample from the current sequence
1. Select the sample you want to run or process by clicking on its row
number. Xcalibur highlights the row. If you do not select a sequence
row, Sample 1 is the default.
2. Choose Actions > Run This Sample or click the Run Sample button
on the toolbar.
Xcalibur displays the Run Sequence dialog box shown in Figure 61.
3. Proceed to Setting up the Run at the bottom of this page.
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Running a Sequence
Automating Analysis in Sequence Setup
Running Samples
To run a sequence
1. Highlight the samples you want to run or reprocess. Click the left-most
column of the first sample and drag to the last sample in the range.
2. Choose Actions > Run Sequence or click the Run Sequence button on
the toolbar.
Xcalibur displays the Run Sequence dialog box shown in Figure 61.
Figure 61. Run Sequence dialog box
3. Proceed to the next topic: Setting up the Run.
Setting up the Run
Use the Run Sequence dialog box shown in Figure 61 to:
•
•
•
•
•
•
Identify the range of samples for analysis from the current list
Configure instruments to be used in the run
Run instrument start up methods before the sequence is initiated
Run instrument shutdown methods when the sequence is complete
Execute programs before or after each sample acquisition or both
Prioritize the sequence so that it is positioned at the head of the
Acquisition Queue
• Select processing and reporting options
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Running Samples
Setting General Run Options
Set the following run options:
User
Type the name of the operator (up to 10 characters).
Run Rows
Check the Run Rows information. If it is incorrect, either:
• 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.
Choosing Acquisition Options
Priority
Sequence
Click this check box to position the sequence or sample
ahead of all others in the Acquisition Queue. If Xcalibur is
running a quantitation bracket, it queues the priority
sequence immediately after the bracket.
Start When
Ready
Click this check box to have Xcalibur to perform an
autosampler injection as soon as the system is ready. To
initiate autosampler activation using the Start Analysis
command from the Home Page, confirm that the Start
When Ready check box is not clicked.
The Acquisition Options window at the top of the dialog box lists the
Instruments assigned to process the sequence:
Instrument
Names of the instruments assigned to sequence analysis. To
add or remove an instrument, click Change Instruments.
See Changing Instruments.
Start
Instrument
Identifies the instrument used by Xcalibur to start the
acquisition. Xcalibur assumes that the Start Instrument
controls all other active instruments, for example, by way
of contact closure. If no instrument is flagged as the start
device, Xcalibur expects an unlisted instrument to provide
an appropriate signal to start the acquisition.
To change the Start Instrument, click Change Instruments. See
Changing Instruments.
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Changing Instruments
Automating Analysis in Sequence Setup
Running Samples
Click Change Instruments on the Run Sequence dialog box (see Figure 61)
to open the Change Instrument In Use dialog box (see Figure 62).
Figure 62. 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.
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
Use Xcalibur to specify Instrument Methods to be run before and after the
sequence (for example, for tuning or calibration).
Start Up
Select an existing method to start up the instrument.
Xcalibur runs this method before the first sample is
queued. Click Browse to select the drive and directory
where the file is located.
Shut Down
Select an existing method to shut down the instrument.
Xcalibur runs this method after the last sample has been
analyzed. Click Browse to select the drive and directory
where the file is located.
No data are acquired during the execution of a start up or shut down
method.
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Automating Analysis in Sequence Setup
Running Samples
Specifying Pre- and Post-Run
Acquisition Programs
Use Xcalibur to specify programs or macros to be run before or after or
before and after each acquisition. They might be used, for example, to issue
commands to prepare an instrument for acquisition. This feature is of
particular use for instruments that are not controlled directly by Xcalibur.
Pre Acquisition
In this combo box, select an existing program to
run before each acquisition. Click Browse to select
the drive and directory where the file is located.
Post Acquisition
In this combo box, select an existing program to
run after each acquisition. Click Browse to select
the drive and directory where the file is located.
Run Synchronously
Use this area to run Pre Acquisition and Post
Acquisition programs either synchronously (in
series) or asynchronously (in parallel) with data
collection.
If the program(s) are run synchronously, the Run
Manager waits until the program(s) can be run as a
Pre Acquisition or Post Acquisition.
If the program is run asynchronously, the program
is run in parallel with data acquisition. For
example, perform file conversions with
XConvert.exe while taking data. In this case, the
Pre Acquisition or Post Acquisition terminology do
not apply.
Use the Pre-Acquisition check box to run the Pre
Acquisition program displayed in the Pre
Acquisition box either synchronously (in series) or
asynchronously (in parallel) with data collection.
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Running Samples
Check the Pre Acquisition check box to have the
program run synchronously. In this case, the Run
Manager waits until the Pre Acquisition program
can be run prior to data acquisition. For example,
to switch the divert valve before a run, select a
synchronous Pre Acquisition program; or, to
convert data from one data type to another data
type while you are acquiring data, select a Post
Acquisition program.
Uncheck the Pre Acquisition check box to have the
program run asynchronously. For example, use the
XConvert.exe program to perform file conversions
from one data type to another data type during
processing.
Uncheck the Post Acquisition check box to have
the program displayed in the Post Acquisition box
run asynchronously. For example, you can perform
operations that do not involve taking data.
Choosing Processing Actions
Choose from the following processing and reporting options:
Quan
Click this check box to carry out quantitative
processing.
Qual
Click this check box to carry out qualitative
processing.
Reports
Click this check box to print the reports specified
in the Processing Method.
Programs
Click this check box to run the programs and
macros specified in the Processing Method.
Print Methods
Click this check box to print the methods used to
process the sample(s).
Create Quan
Summary
Click this check box to print a quantitative
summary report for the sample(s).
Click OK to save the settings and close the dialog box. Xcalibur 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 raw files
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 shown in Figure 63. Xcalibur highlights the selected
rows.
2. Choose Actions > Batch Reprocess or click the Batch Reprocess
button on the toolbar to display the Batch Reprocess Setup dialog box
shown in Figure 63.
Figure 63. Batch Reprocess Setup dialog box
3. Check the Process Rows information. If it is incorrect, click Cancel to
close the 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. Click the Qual check box to reprocess qualitative data. Click the
following qualitative processing options:
Peak Detection & Click this check box to generate new peak detection
Integration
and integration data.
Spectrum
Enhancement
Click this check box to carry out new Refine,
Combine or Threshold calculations.
Library Search
Click this check box to submit processed spectra to a
new library search.
5. Click the Reports check box to print new reports.
Print Sample
Reports
Click this check box to generate new sample reports,
based on those listed in the Processing Method(s).
Print Summary
Reports
Click this check box to generate new summary
reports, based on those listed in the Processing
Method(s).
6. Click the Programs check box to run the post-processing programs or
macros, based on those listed in the Processing Method(s).
7. Click the Create Quan Summary Spreadsheet option to have Xcalibur
to generate a summary spreadsheet for the reprocessed sequence.
8. Click 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.
9. 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 64) 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.
Figure 64. The Acquisition Queue with the Sample Information window displayed
Use the Acquisition Queue to do the following:
• Rearrange sequences queued for acquisition
• Delete sequences (unless they are currently being run)
• Rearrange samples within a sequence (unless they have already been
acquired, are currently undergoing acquisition, or are part of the
quantitation bracket currently being acquired)
• 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)
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The Acquisition Queue
To manipulate entries in the Acquisition Queue:
1. Right-click the name of the sequence or sample to view the shortcut
menu. This contains a single command: Properties. When selected, this
displays the Sample Information window.
2. Double-click a sequence to load it into Sequence Setup.
3. Double-click a sample to open the Sample Information window.
A check box is displayed alongside each sequence and sample. The check
box provides the option of selecting one or more items for deletion.
To delete a sample or sequence from the queue, select its check box and
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
Window
The Sample Information window shows the parameters for all the sequence
fields. See “About Sequences” on page 80 for descriptions of all the fields.
The Sample Information window 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 on the pin icon again) and click
anywhere outside the dialog box.
A pinned window is updated with the details of any selected sequence.
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The Acquisition Queue
Managing Tasks
The Queue Manager (see Figure 65) provides additional functions for
managing queued tasks. It is active whenever samples or sequences are
queued for acquisition or reprocessing. If it is not visible, it might be
minimized to the Windows toolbar.
Figure 65. Queue Manager window
Use the following procedures to manage the Xcalibur Processing Queue.
To temporarily pause the Processing Queue
Click the Pause button in the toolbar or choose Queue > Pause.
To resume the processing queue when it is in the Pause mode
Click the Resume button in the toolbar or choose Queue > Resume.
To update the display with the latest information
Choose View > Refresh.
To remove a task from the queue
1. Select the task to be removed.
2. Click the Remove Job button in the toolbar, or choose Analysis >
Remove From Queue.
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The Acquisition Queue
To remove all the tasks from the queue
Choose Queue > Purge Queue.
To view the details of a selected analysis
1. Select the required analysis in Queue Manager.
2. Click the Details button in the toolbar or choose Analysis > Details.
Xcalibur displays the Details of Selected Analysis dialog box shown in
Figure 66.
The Details of Selected Analysis dialog box shows:
File
The filename of the sample
Status
The current queue status
Submitted
The time and date the job was submitted
From
The source of the job
Actions
Lists the tasks required to complete the selected job and their
current status
Figure 66. Details of Selected Analysis dialog box
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Chapter 4
Reviewing and Interpreting
Data in Qual Browser
This chapter describes the underlying principles of Qual Browser and
explains how to use it for displaying and manipulating chromatograms
and spectra.
This chapter contains the following sections:
• Results Review
• About Qual Browser
• Getting Started in Qual Browser
• All About Cells
• Using Views Interactively
• Using a Chromatogram View
• Using a Spectrum View
• Using a Map View
• Using a Spectrum List View
• Using a Scan Header View
• Using a Scan Filter View
• Using a Report View
• Preparing for Presentation
• Tool Menu Utilities
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Results Review
Results Review
Xcalibur’s data reviewing component is called Results Review (see
Figure 67). This is organized into three core Browsers.
Figure 67. Results Review section of Xcalibur’s Home Page
The core Browsers are:
Qual Browser
Displays and manipulates chromatograms and spectra,
activates library searches and produces reports.
Quan Browser
Displays a peak list or calibration curve to be
manipulated (described in the Xcalibur Getting
Productive: Quantitative Analysis manual).
Library Browser
Activates the NIST Mass Spectral Search Program to
match spectra to library entries. Also used to generate
user libraries. Chapter 5, Library Browser, describes
Library Browser.
This chapter is primarily concerned with Qual Browser and its use for
analyzing chromatograms and identifying spectra. This chapter describes the
underlying principles of Qual Browser and explains the features of each of
the view types. It does not attempt to describe all of the potential uses for
the browser.
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About Qual Browser
Reviewing and Interpreting Data in Qual Browser
About Qual Browser
Qual Browser is a powerful and versatile utility for viewing chromatograms
and spectra from raw files or qualitative processing results. Use the browser
to view and manipulate data from single or multiple files in any number of
separate data windows (see Figure 68). (The MSn tab is not displayed with
all instruments.) Within each window, do the following:
• Create a grid of cells showing a wide range of data views.
• Save any number of Qual Browser layouts and subsequently apply them
to other raw files.
• Create a variety of reports for raw or result files.
Figure 68. Qual Browser window
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About Qual Browser
Some of the things you might do in Qual Browser are:
• Generate a variety of chromatogram plots and determine suitable peak
detection parameters for subsequent automated analysis using a
Processing Method.
• Optimize a chromatogram peak’s spectrum by averaging scans across its
apex and subtracting other scans averaged from the baseline either side
of the peak.
• Determine the elemental composition of the peaks in the spectrum.
• Simulate the isotopic distribution mass spectrum of a single compound
or mixture of compounds.
• Export a spectrum to the Library Browser to create and maintain user
libraries.
• Submit the spectrum of an unknown compound to a library search (if a
suitable reference library is present).
• Print a report showing data analysis and library results.
To open Qual Browser, click Qual Browser on the Home Page Road Map
view. In other Xcalibur programs, access Qual Browser by choosing the
relevant View menu command.
This section contains the following topics:
• The Toolbar
• The Info Bar
• Windows, Cells, Views and ‘Pinning’
The Toolbar
Qual Browser is equipped with a large number of tools. The display of
toolbars can be turned on or off in the Toolbars dialog box (see Figure 69).
Select View > Toolbars and toggle the tool groups as required. Use this
dialog box also to:
• Toggle the display of ToolTips
• Choose between large or small toolbar buttons
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About Qual Browser
Figure 69. The Toolbars dialog box
The toolbar buttons are divided into two groups:
Main
Tools for loading, saving or printing files, scaling plots,
manipulating cells, peak detection, changing views,
and arranging data windows.
Amplify
Tools for adjusting the normalization in specific
sections of a chromatogram, spectrum or map plot.
By default, these two toolbar groups are positioned along the top of the
Qual Browser window, just beneath the menu bar. They can be dragged
anywhere within the window or docked along any of the other window
edges.
Customizing the Toolbar
To add or remove toolbar buttons to the Main toolbar, use View >
Customize Toolbar. Toolbar buttons are available for most menu
commands.
Choose View > Customize Toolbar to display the Customize Toolbar
dialog box shown in Figure 70.
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About Qual Browser
Figure 70. Customize Toolbar dialog box
To add a toolbar button for a menu command
1. Open the Customize Toolbar dialog box.
2. Select the menu category in the Categories box.
3. Locate and select the menu command in the Commands box using the
scroll bars if necessary.
4. Drag from the Commands box to the appropriate position in the Main
toolbar.
The toolbar button is added to the Main toolbar.
To remove a toolbar button from the Main toolbar
1. Open the Customize Toolbar dialog box.
2. Drag the button from the Main toolbar to the dialog box.
The toolbar button is removed from the toolbar.
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About Qual Browser
To reposition a toolbar button in the Main toolbar
1. Open the Customize Toolbar dialog box.
2. Drag the button in the Main toolbar to its new position.
The button moves to its new position.
Note Use this technique to group buttons together and to put a space
between groups: drag a button to its left to close up a space, or to its right
to open up a space.
The Info Bar
The Info Bar initially occupies the left side of the Qual Browser window.
See Figure 68. Show or hide the Info Bar by clicking on the Info Bar button
on the main toolbar or by choosing View > Info Bar.
The Info Bar has seven tabs. Each tab displays a separate function on a
separate page:
Cell Information
Displays details of the plots contained in the
active cell.
Sequence
Information
Result File
Information
Elemental
Composition
Spectrum Simulation
Shows the raw files available from an open sequence.
Detection Tab
MSn Browser
Information
Thermo Electron Corporation
Shows peak data from a result file.
Calculates the "best matching" chemical formula for
a mass, or a list of masses (from a spectrum).
Creates a simulated isotopic distribution spectrum
of a chemical formula.
Sets peak parameters and advanced noise methods.
The letter in the upper left hand corner of the tab
indicates which algorithm (ICIS, Avalon, or
Genesis) is currently selected.
Displays MSn experimental data for analysis.
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About Qual Browser
Windows, Cells, Views
and ‘Pinning’
Qual Browser’s main window displays raw files, interactive library search
results, and qualitative processing. View raw files in the same or separate
windows. Use the following two Window commands to arrange data
windows within Qual Browser:
Cascade
Arrange windows diagonally so they overlap.
Tile
Arrange windows as non-overlapping tiles.
Each window can be sub-divided into a grid of cells, each displaying a view.
A view can be a chromatogram, spectrum, a mass map, a spectrum list, scan
header, scan filter list, tune method, experiment method, sample
information, status log, or error log. Chromatogram and Spectrum views
can contain up to 8 plots.
The arrangement of cells within a window is termed a layout. Save layouts
to disk for future use.
Various automatic processing options are available for Chromatogram and
Spectrum views. In a Chromatogram view, apply:
• Smoothing to all plots in the cell
• Peak detection to the active plot in the current cell or all plots in the
current cell
In a Spectrum view, apply:
• Smoothing
• Refine enhancement
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About Qual Browser
If no cell has been pinned, the last clicked on cell is active. Shading of its
unpinned icon and a gray border around the cell indicates the active status.
To fix a cell in the active state, click its pin icon:
Pin icon for an unpinned cell
Pin icon for a pinned cell
When you pin a cell, you designate it as the target for operations performed
in other cells. For operations involving the use of menu commands or
toolbar buttons, pinning is not necessary but the target cell must be active.
If no cell is pinned, the active cell is deemed to be the last cell acted on by a
mouse action and is identified by a gray border.
With chromatogram or spectrum views containing more than one plot, any
menu operations target the active plot, indicated by a shaded background.
Select an individual plot in a multi-plot cell by clicking on it.
See Using Views Interactively on page 137 for more information about
cells, views and pinning.
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Getting Started in Qual Browser
Getting Started in
Qual Browser
This section describes how to get started in Qual Browser.
View data by doing the following:
• Opening Single Raw Files. Raw files have a .raw file extension.
• Opening a Sequence. Then select one or more raw files from the
Sequence Information page of the Info Bar. Sequence files have an .sld
file extension.
• Opening a Result file. Result files are the product of reprocessing raw
data files with a Processing Method. Result files have an .rst file
extension.
Opening Single Raw Files
To open a single raw data file
1. Choose File > Open or click the Open button in the toolbar to display
the Open Raw File dialog box shown in Figure 71.
Figure 71. Open Raw File dialog box
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2. Browse to the file.
3. Then, select the options that determine how Qual Browser displays the
raw file.
Replace
Window
If you click this option, Xcalibur replaces all the
plots in the current window (in all cells) with plots
of equivalent type from the selected raw file.
Cell
If you click this option, Xcalibur replaces all plots in
the current cell with plots of equivalent type from
the selected file.
Plot
If you click this option, Xcalibur replaces the current
plot with a plot of equivalent type from the selected
file.
Window
If you click this option, Xcalibur opens the selected
raw file in a new window using the layout of the
currently active window. If no layout is available,
Qual Browser applies the most recently saved layout
file or, if this is invalid, the default layout.
Plot
If you click this option, Xcalibur adds the file as a
plot in the active cell of the current window
(unavailable if the cell already contains the
maximum number (8) of plots).
Add
If the Add Window option is selected, choose the layout to be applied to
the new window.
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Default Layout
If you click this list item, Xcalibur applies the most
recently saved default layout.
Current Layout
If you click this list item, Xcalibur applies the layout
of the currently active window to the new window.
If no layout is available, Qual Browser applies the
default layout.
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Getting Started in Qual Browser
Opening a Sequence
Choose File > Open Sequence. Click Browse to select a sequence file
(extension .sld). The sequence is displayed in the Sequence Information
page of the Info bar (see Figure 72).
Sequence Information
Page
Figure 72. Sequence Information page, showing the shortcut menu
Right-click a sample file to display a shortcut menu. Choose Properties
from the shortcut menu to open the dialog box shown in Figure 73.
Figure 73. Sample Properties dialog box
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Using the Sequence Information
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Reviewing and Interpreting Data in Qual Browser
Getting Started in Qual Browser
Double-clicking any sample file in the sequence opens it in the active
window, replacing the plots in all cells with equivalent spectra,
chromatograms, or maps.
Right-clicking any file within the sequence opens the Sequence
shortcut menu. In the shortcut menu, choose:
Open - Replace >
All in Current Window To open the selected file in the active
window, replacing the plots in all cells with
equivalent spectra or chromatograms (also
achieved by double-clicking on a file)
All in Current Cell
To replace all plots in the active cell with
equivalent plots from the selected file
Current Plot
To replace the current plot in the active
window with an equivalent from the selected
file
Open – Add >
New Window
To open the selected raw file in a new
window
New Plot
To open the selected file as a plot in the
active cell
Open Result File
To open a result file associated with the
selected raw file
Properties
To open the Sample Properties dialog box
shown in Figure 73. The Sample Properties
dialog box shows basic information about
the selected sample including the row,
filename, sample ID, name, sample type and
result file name.
The Sample Properties dialog box closes if you click anywhere outside it.
Click the pin icon to keep the Properties dialog box open. Click the close
box icon to close the dialog box, or ‘unpin’ the dialog box (by clicking the
pin icon again) and click anywhere outside the dialog box.
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Getting Started in Qual Browser
Opening a Result file
A result file contains the list of detected peaks from the chromatogram, and
the qualitative processing results associated with each peak. Qual Browser
displays the result file in a fixed, two-cell arrangement (see Figure 74). This
shows a chromatogram plot in the upper cell, with the detected peaks
highlighted, and the spectrum associated with the currently selected
chromatogram peak, in the lower cell. For more information about using
Chromatogram and Spectrum views, refer to “Using a Chromatogram
View” on page 139 and “Using a Spectrum View” on page 160,
respectively.
To open a result file
1. Click the Open Result File button on the toolbar or choose File >
Open Result File to display the Open Result file dialog box.
2. Use the Browse button to select a result file (extension .rst). Then,
click Open.
Information
Page
Chromatogram
View
Spectrum
View
Figure 74. The Result File view showing the Result File Information page in the Info Bar
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Getting Started in Qual Browser
Library search results for the displayed spectrum are shown in a separate
library search results window. See “Interpreting the Hits List” on page 199.
It is not possible to submit the spectrum from the results display for library
search. If a library search has been carried out during processing (when the
result file was created), search results are stored in the result file and
displayed for each detected peak. To submit a spectrum for library searching
or to export a spectrum to the Library Browser, open the raw file.
Note Many of the features in Qual Browser are not available for use with
a result file because the raw file is not directly available for reprocessing.
The Result File Information Page in the Info Bar shows basic information
about all detected peaks in the result file, including:
• Retention times at the peak start (left), peak apex, and peak end (right)
• The peak area and height
For more detailed information about the peak, including flags, open the
Peak Properties dialog box.
To open the Properties dialog box
1. Right-click the peak identifier in the Peak List.
2. Choose Properties from the shortcut menu.
Xcalibur displays the Peak Properties dialog box shown in Figure 75.
Figure 75. Peak Properties dialog box
The Peak Properties dialog box closes if you click anywhere outside it.
Click the pin icon to keep the Properties dialog box open. Click the Close
box icon to close the dialog box, or ‘unpin’ the dialog box (by clicking the
pin icon again) and click anywhere outside the dialog box.
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All About Cells
All About Cells
Qual Browser displays chromatograms and spectra in a grid of cells. This
section describes the commands used to manipulate cells and contains the
following topics:
• Creating and Deleting Cells
• Adjusting Cell Size
• Changing a Cell’s View
• Scaling a Plot
• Layouts
• Using the Cell Information Page
Creating and Deleting
Cells
When a raw file is open, Qual Browser displays one or more cells according
to the selected layout.
To create a new cell
1. Select the cell adjacent to the cell(s) you want to create.
2. Add additional cells using the appropriate Grid button on the toolbar or
choose the Grid > Insert Cells menu command.
Insert cells above
Insert cells left
Insert cells below
Insert cells right
Note When you add cells, Xcalibur creates duplicate cells that
contain the same view as the active cell. As you add additional cells,
the cell size of all cells becomes smaller. Xcalibur might not be able
to include all header information in views displayed in small cells.
To delete one or more cells
1. Select the cell you want to delete.
2. Delete the cell, row, column, or all other cells using the appropriate
Grid button on the toolbar or choose the Grid > Delete menu
command.
Delete grid row
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Delete grid column
Delete all grid cells (except selected cell)
Note As you delete unwanted cells, the cell size of the remaining
cells becomes larger.
Adjusting Cell Size
To adjust the size of a cell in a multi-cell window, select the cell. Then,
choose Grid > Cell Size or click the Set Cell Size button on the toolbar.
Xcalibur displays the Cell Size dialog box shown in Figure 76.
Figure 76. The Cell Size dialog box
The Cell Size dialog box contains controls for adjusting the column width
and row height. Use this box to make adjustments relative to other columns
and rows. The dialog box also contains a small display area showing the
active cell to preview the effects of different settings before applying them.
Adjust the following:
Column
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Specify the column width within the range 5 to
300%. The Cell Size dialog box displays the current
width below the scroll box.
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All About Cells
Row Height
Specify the row height within a range of 5 to 300%.
The Cell Size dialog box displays the current height
below the scroll box.
Clicking Default Width and Default Height return cell dimensions to
those of the default layout.
Note The Cell Size dialog box is not available if the grid contains a
single cell. The Column control has no effect if the view contains a single
column. Similarly, the Row Height control has no effect in a grid
containing a single row.
There are also a number of toolbar buttons and Grid menu commands for
sizing cells:
Full width (expand active cell to full width of grid)
Full height (expand active cell to full height of window)
Full size (maximize active cell in window)
Reduce active cell size
Grid lines (toggle the display of lines between the cells)
Changing a Cell’s View
To change the view displayed in a cell
• Click the cell where you want to change the view. Then, select the
required view from the main toolbar or choose a view from the View
menu or the shortcut menu (right-click in the active cell). Xcalibur
replaces the view in the active cell with the view that you select.
The available views are:
Chromatogram
Spectrum
Map
Spectrum List
Scan Header
Scan Filter
Tune Method
Instrument Method
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Sample Information
Status Log
Error Log
Scaling a Plot
The Chromatogram, Spectrum, and Map views show plots. Use the Zoom
and Pan menu commands to adjust the display of the active plot:
Zoom in X
Zoom out X (also acts to display all in a data or report view)
Zoom in Y
Zoom out Y
Auto range
Normalize
Zoom reset
Pan graph: Use the Pan Graph button on the toolbar to pan
across a zoomed plot by dragging it to the left or right with
the mouse.
Layouts
Creating a layout
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A layout consists of any arrangement of cells, views and plots within a data
window. Use Xcalibur to create, save, and open layouts. When you open the
Qual Browser window, Xcalibur uses the last layout file to display data from
a raw file in the predefined arrangement and with predetermined option
settings. Open a previously created layout or create a new layout at any time.
You create a layout during the normal processing of a raw file. The basic
composition of a layout is:
Cells
Use the Grid buttons on the toolbar or menu commands to
create the desired arrangement of cells for the data views.
Views
Click the cell to make it active and choose the View Type.
Repeat for each cell to create the desired arrangement of data
views.
Plots
In chromatogram or spectrum cells use the Ranges dialog box
to define the number (up to 8) and characteristics of plots.
Display
Options
Use the Display Options menu command to open the Display
Options dialog box to change style, color, axis, labels and
normalization options.
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All About Cells
Saving a Layout
To save a modified layout with the current name, click the Save Layout
button on the toolbar or choose File > Layout > Save.
Choose File > Layout > Save As to assign a file name and save a new layout.
To save the current layout as the new default layout (default.lyt) click the
Save As Default Layout button or choose File > Layout >
Save As Default.
Applying a Layout to the Active
Window
Click the Apply Layout button or choose File > Layout > Apply to select a
previously saved layout.
To display the current default layout, click the Apply Default Layout
button or choose File > Layout > Apply Default.
Displaying Layout Summary
Information
134
Choose File > Layout > Summary Info to display the File Summary
Information dialog box. This dialog box displays the following information:
User
The user name of the user currently logged in to
Xcalibur and Qual Browser.
Header
Basic details about the layout: the File ID, the date the
layout was created, and the User ID of the originator
of the layout.
Description
Any additional details about the layout such as
modifications.
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Using the Cell Information
Page
Reviewing and Interpreting Data in Qual Browser
All About Cells
The cell information page of the Info Bar displays information about the
active cell (see Figure 77). Its contents depends on whether the plot is a
chromatogram or spectrum.
Figure 77. The Cell Information page of Qual Browser’s Info Bar (with its shortcut menu) showing Spectrum
cell information
Right-click a plot to open the Cell Information page shortcut menu:
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Ranges
Opens the Ranges dialog box (See Chromatogram
Ranges and Spectrum Ranges). This dialog box shows
the properties of all the plots in the active cell. Use this
dialog box to view or modify the time and mass
ranges, change background subtraction and
smoothing parameters.
Delete
Deletes the selected plot from the cell.
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All About Cells
Chromatogram Information
For a chromatogram plot (see Figure 78), the Cell Information page shows:
The plot type and filename
The pathname of the raw file
The scan filter (if applied)
The fixed scale upper limit (if applied)
The chromatogram delay (if applied)
The mass range (for mass range plot type only)
The chromatogram time range or ranges used for background subtraction
(if applied)
Figure 78. Cell Information for a chromatogram plot
Spectrum Information
For a spectrum plot, the Cell Information page (see Figure 79) shows:
The filename
The pathname of the raw file
The scan filter (if applied)
The fixed scale upper limit (if applied)
The chromatogram time range or ranges used for background subtraction
(if applied)
Figure 79. Cell Information for a spectrum plot
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Using Views
Interactively
Reviewing and Interpreting Data in Qual Browser
Using Views Interactively
Qual Browser is interactive, use it to:
• Use a chromatogram to generate a mass spectrum (incorporating single
or averaged scans, with background subtraction if required), or maps
with specific time ranges.
• Use maps to generate a single or averaged spectrum, or mass
chromatograms with specific mass or time ranges.
• Use a spectrum to generate mass chromatograms.
• Apply scan filters to chromatograms using drag and drop.
Within the graphic region of a Chromatogram, Spectrum or Map view, the
cursor becomes a cross hair. The status bar at the bottom of the Qual
Browser window shows the coordinates of the cursor, in appropriate units
for the view. In a Spectrum view, for example, the status bar shows the
cursor position in terms of Mass (m/z) and Intensity.
Use the cross hair cursor in three ways:
• A simple click picks a point on the plot.
• A line dragged parallel to any axis picks a range.
• A line dragged in any diagonal direction selects an area.
The effect of these actions depends on the state of the cell. If it is pinned,
the actions cause the graph to be rescaled according to the dimensions of the
dragged line or area (see Table 7).
Table 7.
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Cursor action in a pinned cell
Cursor action
Effect on view in pinned cell
Click
Makes view ‘active’.
Drag parallel to X-axis
Rescales graph showing selected X range only.
The Y range might rescale depending on the
selected Normalization display options.
Drag parallel to Y-axis
Rescales graph, showing selected Y range only,
same X range.
Dragged area
Rescales graph, showing selected ranges only.
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Using Views Interactively
The same actions in an unpinned cell have a very different effect. In this
case, the action affects the pinned cell (see Table 8). Qual Browser displays
the pinned cell using data appropriate to the selected point, range or ranges.
Table 8.
Cursor action in an unpinned cell
Pinned cell
View actioned by
cursor
Cursor action
Effect on active view in the
pinned cell. No action happens
in the unpinned cell.
Spectrum
Chromatogram
Click retention time (RT) = 1.98 min
in the Chromatogram view.
Cell displays mass scan that occurs
at retention time = 1.98 min.
Status Log
Chromatogram
Click retention time (RT) = 3.16 min
in the Chromatogram view.
Cell displays status log at retention
time 3.16 min.
Scan Filter
Chromatogram
Click retention time (RT) = 1.36 min
in the Chromatogram view
Cell displays the scan filter used
for the scan that occurs at
retention time = 1.36 min.
Spectrum
Chromatogram
Click and drag across a peak of
interest.
Cell displays a spectrum that is the
average of all the scans recorded
across the peak within the selected
range of retention times.
Chromatogram
Spectrum
Click and drag from m/z 198.4
through 299.7.
Cell displays a mass chromatogram
consisting of masses 198.4 through
299.7.
Chromatogram
Map
Click and drag an area enclosing
the ranges 0.5 to 1.0 min and m/z
100 to 200.
Cell displays a mass chromatogram
consisting of masses 100 through
200 with a time range of 0.5 to
1 min.
This table illustrates only a few of the possible effects of Qual Browser’s
interactivity. Important points to note are:
• The target view must be active and in a pinned cell.
• Within a pinned cell, cursor actions rescale the view.
• Use the coordinates in the Status bar to select ranges precisely.
• The Edit > Undo command can be used to correct mistakes.
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Using a
Chromatogram View
Reviewing and Interpreting Data in Qual Browser
Using a Chromatogram View
A Chromatogram view shows the intensities of one or more masses as a
function of time (see Figure 80).
To view a chromatogram in the active cell, do one of the following:
Figure 80. An example of a Chromatogram view with the Chromatogram shortcut menu displayed
• Right-click in the cell and choose View > Chromatogram from the
shortcut menu.
• From the menu bar, choose View > Chromatogram.
• Click the View Chromatogram button on the toolbar.
This section contains the following topics:
• Using Chromatogram Plots
• Chromatogram Ranges
• Using the AutoFilter
• Setting Chromatogram Options
• Changing Peak Detection Settings
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Using a Chromatogram View
Using Chromatogram Plots
Display up to eight plots within a Chromatogram view.
To insert a plot
1. Select the cell containing the view.
2. Right-click the chromatogram above the position where you want to
insert a plot.
3. Choose Plot > Insert from the shortcut menu.
To delete a plot
1. Select the cell containing the view.
2. Right-click the plot to delete.
3. Choose Plot > Delete from the shortcut menu.
Also use the Ranges dialog box to add, delete, or enable plots.
Chromatogram Ranges
Use the Chromatogram Ranges dialog box (Figure 81) to view and edit the
mass range and time range for all the plots in a chromatogram. To display
this view, do one of the following:
• Right-click the Cell Information page (with a chromatogram plot active
or pinned) and choose Ranges from the shortcut menu.
• Right-click a chromatogram plot. Then, choose Ranges from the
shortcut menu.
• From the Qual Browser window (with a Chromatogram view active)
choose Display > Ranges to open the Chromatogram Ranges dialog
box.
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Using a Chromatogram View
Figure 81. The Ranges page of the Chromatogram Ranges dialog box
The Chromatogram Ranges dialog box consists of two tabbed pages:
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Ranges
This page displays the properties of the plots in the
Chromatogram view, including information about the
source raw files, plot types, time and mass ranges.
Automatic
Processing
This page contains parameters for automatic smoothing
and baseline subtraction of all chromatogram plots in
the active cell.
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Using a Chromatogram View
Ranges Page
The settings in the Range area apply to all plots in the selected cell:
Time Range (min)
In this box, type the lower and upper time limits for
the plot in minutes. Separate entries by a dash (no
spaces).
Fixed Scale
Click this check box to set the Y-axis maximum to a
specific value.
Below the Ranges area is a table listing the chromatogram plots
(8 maximum) contained within the selected cell. The table lists the plots
under the following headings: Type, Range, Filter, Delay (min), Scale, Raw
File.
Use the check boxes at the beginning of each line to display or hide plots
within the current cell.
The Plot Properties area displays the properties of the highlighted plot:
Raw File
Shows the path and filename of the raw file used to
generate the selected plot. The list contains all files
active in the current cell. To open an unlisted file, click
Browse, and identify the file in the normal manner.
Scan Filter
Click the down-arrow to display scan filter options
stored in the .raw file.
Plot Type
Use the three Plot Type lists to choose:
• A basic chromatogram type, for example, TIC
• A logical operator: + or –. Select an operator to
turn on:
• A second chromatogram type to add to, or subtract
from, the first trace. For example, Mass Range. The
list features valid plot types.
To change one of the Plot entries, click the
down-arrow to display a list of valid options. Then,
select one of the types.
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Using a Chromatogram View
The list of valid plot types depends on the detector
used to generate the data:
MS
Mass Range, TIC, Base Peak Neutral
Fragment
Analog
Analog 1-4
A/D card A/D Card Ch 1-4
Range(s)
PDA
Wavelength Range, Total
Scan, Spectrum Maximum
UV
Channel A-D
For MS detector types, use this parameter to specify
the mass or mass range for Mass Range or Base Peak
plot types.
For other detector types, use this field to specify the
wavelength or wavelength range for the
chromatogram. If you use a plot combination such as
Wavelength Range + Wavelength Range, an additional
Wavelength (nm) box is displayed for you to specify
the second wavelength range.
To change the range or to add a new range, 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 the
following: 123 – 456.
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Detector
This list shows the type of detector used to generate
the raw file. Valid types are: MS, Analog, A/D card,
PDA, and UV. The type of detector determines the
available Plot types.
Peak Algorithm
Use this list to select the algorithm: ICIS peak
detection algorithm, Genesis peak detection algorithm,
or Avalon peak detection algorithm.
Delay (min)
Use this box to offset the start of a chromatogram
trace. The valid time range is 0.00 to 5.00 min.
Fix Scale To
If you have chosen the Fixed Scale check box in the
Ranges area, this box is enabled for you to specify the
maximum Y-axis value.
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Using a Chromatogram View
Automatic Processing Page
Use the Automatic Processing page shown in Figure 82 to apply smoothing
or baseline subtraction to all the plots in the active Chromatogram view.
This page contains the following areas:
• Smoothing Area
• Baseline Subtraction Area
• Include Peaks Area
• Mass Tolerance Area
• Mass Precision Area
Figure 82. Chromatogram Ranges dialog box - Automatic Processing page
Smoothing Area
Use the Smoothing area settings to smooth all scans defined by the mass
range, time range, and filter settings in the Spectrum Ranges dialog box.
The Smoothing area contains the following parameters:
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Baseline Subtraction Area
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Using a Chromatogram View
Enable
Click the Enable check box to apply chromatogram
smoothing.
Type
Use this list to specify the type of smoothing algorithm you
want to apply to the chromatogram: Boxcar or Gaussian. See
the Xcalibur online Help for more information.
Points
In this box, type the number of points for chromatogram
smoothing. The value must be odd and in the range 3
[minimum smoothing] to 15 [maximum smoothing].
Use the Baseline Subtraction settings to apply baseline subtraction to all
chromatogram plots in the active view. This algorithm fits a smooth curve
through the noise in the chromatogram and subtracts this curve from the
chromatogram, leaving the peaks on a flat baseline.
The Baseline Subtraction area contains the following parameters:
Enable
Click the Enable check box to apply baseline
subtraction.
Polynomial Order Use this box to specify the degrees of freedom allowed
to the fitted curve. With polynomial order set to 0, a
horizontal straight line is fitted. With polynomial
order set to 1, a sloping straight line is fitted. Too high
a value causes the fitted curve to begin to follow the
peak shapes. The valid range for this parameter is 0
to 99.
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Below Curve
Use this parameter to constrain the curve to place the
specified percentage of data points beneath the fitted
background curve. The range for this parameter is
1% to 99%, depending on the abundance and width
of peaks in the chromatogram. For more, or wider
peaks, increase the value.
Tolerance
This value affects the precision of the internal
arithmetic. The valid range is 0.001 (default) to 0.2.
Flatten Edges
Click this check box to confirm that Xcalibur applies
the polynomial so that the beginning and end of the
chromatogram plot is horizontal. This setting is useful
to model abrupt baseline changes that often occur at
the start or end of an analysis program.
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Using a Chromatogram View
Overlay Graph of Click this check box to display the polynomial as a
Fitted Polynomial graphic overlay on the original chromatogram plot.
This option is useful to test the smoothing parameters
and shows whether the intended polynomial correctly
models the baseline. Clear the check box to subtract
the curve and display the smoothed chromatogram.
Include Peaks Area
The Include Peaks area has only one check box. Use this setting to include
or exclude the reference peaks (R) and exception peaks (E) for the mass data
in all the cells in the Qual Browser window.
The Include Peaks area contains the following parameters:
Reference and
Exception Peaks
Use this check box to include or exclude the
reference peaks (R) and exception peaks (E) for the
mass data in a Qual Browser window.
Mass Tolerance Area
Use the Mass Tolerance area to specify a value for mass tolerance. Settings
in this area affect the display of all the mass data in the Qual Browser
window.
The Mass Tolerance area contains the following parameters:
Use User Defined
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Use this check box to specify the values for mass
tolerance and mass units for the mass data in a Qual
Browser window. To enable the parameters and
specify the values, click the Use User Defined check
box. If you deselect the check box, Xcalibur uses the
values for mass tolerance and units that are stored in
the raw file.
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Mass tolerance
Reviewing and Interpreting Data in Qual Browser
Using a Chromatogram View
Xcalibur uses a mass tolerance band to calculate
spectrum averages, to add and subtract spectra, and to
convert profile data to centroid data. The mass
tolerance value controls the number of individual
scans that Xcalibur groups together during data
processing. Xcalibur condenses to a single mass value
all the scans in the range from the lower to the upper
tolerance limits. Individual scans are available for
display if the mass tolerance is small (less than 1
mmu), but can be grouped if the mass tolerance is
larger.
For example, when Xcalibur applies a mass tolerance
of 300 mmu to a mass value, it groups all the
individual scans in the range between -300 and
+300 mmu of the mass value; the result is a single
mass intensity value.
Use the Mass Tolerance box to specify the value for
mass tolerance in the range of 0.1 to 50000.
Set a default value for mass tolerance on the Xcalibur
Configuration dialog box - Mass Options Page (see
Figure 83). Mass tolerance is used commonly with
high-resolution measurements.
Mass Precision Area
Units
mmu
Specifies millimass as the unit of measurement in
which Xcalibur processes the data.
Units
ppm
Specifies parts per million as the unit of measurement
used by Xcalibur to process the data.
Use the settings in the Mass Precision area to apply mass precision to the
mass data in all the cells in the Qual Browser window.
The Mass Precision area contains the following parameters:
Decimals
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Xcalibur applies the number of decimal places to
mass intensity values for data processing calculations.
Specify a number from 0 to 5. If you specify 3, for
example, Xcalibur uses a mass number truncated to 3
decimal places to perform calculations. Set a default
value for mass precision in the Xcalibur
Configuration dialog box - Mass Options page (see
Figure 83). Mass precision is commonly used with
high-resolution measurements.
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Figure 83. Mass Options page for default settings
Using the AutoFilter
Use the Autofilter command you to repopulate a Chromatogram view with:
• A plot showing the chromatogram without any scan filters
• Plots for each scan filter applied to the chromatogram, up to the
maximum of eight chromatogram plots
Choose Actions > AutoFilter to apply the AutoFilter command to a
selected Chromatogram view.
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Setting Chromatogram
Options
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Using a Chromatogram View
To change the style of a Chromatogram view in the Display Options dialog
box, confirm that no other cell is pinned. Then, do one of the following:
• From the menu bar, choose Display > Display Options or right-click
in the cell and choose Display Options.
The Display Options dialog box contains a small display area showing the
active cell. Use this display to preview the effects of different settings before
applying them (see Figure 84).
The Display Options dialog box for the Chromatogram preview consists of
five tabbed pages:
•
•
•
•
•
Style
Style
Color
Labels
Axis
Normalization
The Display Options - Style page for the Chromatogram view is shown in
Figure 84.
Figure 84. Display Options dialog box - Style page for a Chromatogram view
Style options determine the appearance of chromatograms.
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Plotting
Choose between the two plotting options:
Point To Point
Click this option to display point-to-point peak
profiles.
Stick
Click this option to display the chromatogram as
vertical lines.
Arrangement
Choose a 2D or 3D arrangement of plots within the cell:
Stack (2D)
Click this option to stack plots vertically with no
overlap for the active map.
Overlay (3D)
Click this option to overlay plots vertically with an
optional horizontal skew (time offset) for the active
map. When selected, the 3D display options are
available.
3D
The following options are available if you have selected an Overlay (3D)
arrangement:
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Elevation
Use this slide bar to set the elevation angle (from 0 to
60 degrees) for a 3D plot style. Either drag the
Elevation slider or click the Elevation slider left or
right arrow until you reach the desired angle.
Skew
Use this slide bar to set the skew angle for a 3D plot
style. Either drag the Skew slider or click the Skew
slider left or right arrow until you reach the desired
angle (from 0 to 45 degrees).
Draw Backdrop
Click this check box to add a backdrop to 3D plots.
Clear the check box to remove the backdrop.
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Color
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Using a Chromatogram View
The Display Options - Color page for the Chromatogram view is shown
Figure 85. Use the Color page to set the color of chromatogram plots and
the backdrop.
Figure 85. Display Options dialog box - Color page for a Chromatogram view
To select the color of a plot, click the corresponding Plot button. Xcalibur
opens the Color dialog box with a color palette that enables you to select a
preset color or customize a color.
Click Backdrop to select a color for the background of the chromatogram.
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Labels
The Display Options - Labels page for the Chromatogram view is shown in
Figure 86. Use the Labels page to choose the type and style of peak labels.
Figure 86. Display Options dialog box - Labels page for a Chromatogram view
Select the following chromatogram labeling options as required:
Note Signal-to-Noise, Area, and Height are available only when peak
detection occurs and peaks are located.
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Retention
Time
Labels peaks with the time in minutes, into chromatogram
plot. RT prefixes the label.
Decimals
The number of decimal places displayed in the retention
time label.
Name
Labels peaks with the component name.
Scan Number
Labels peaks with number of mass scan at peak maximum.
S# prefixes the label.
Base Peak
Labels the base peak in the mass spectrum of the
chromatogram peak. BP prefixes the label.
Signal-toNoise
Labels peaks with the signal to noise ratio at the peak
maximum. SN prefixes the label.
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Flags
Labels peaks with flags providing supplemental
information about the peak data. For example, if a peak is
saturated, Xcalibur displays an S above the peak.
Area
Labels peaks with the integrated area of the peaks, prefixed
by AA if detected automatically or MA if detected
manually.
Height
Labels peaks with the apex height, prefixed by AH if
detected automatically or MH if detected manually.
Choose from the following label styles:
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Offset
Offset a label from its normal position to avoid conflict
with another label. Type the amount of the offset (in
number of characters) in the Size box.
Rotated
Use vertical labels, rather than horizontal labels.
Boxed
Place a rectangular outline around each peak label.
Label
Threshold
Limit the labeling of peaks to those exceeding the specified
percentage of the base peak.
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Axis
The Display Options - Axis page for the Chromatogram view is shown in
Figure 87. Use the Axis page to set chromatogram axis labels and display
options.
Figure 87. Display Options dialog box - Axis page for a Chromatogram view
The X area displays:
Name
Use this box to type an axis name.
Show Name
Select a display option for the axis label: Never, On
Print, or Always.
Offset
Click this check box to offset the axis label from the
chromatogram.
Split Time Range
Click this check box to split the chromatograms into
two or more separate graphs with equal time ranges.
Divisions
Type the number of split time range graphs
displayed for each chromatogram in the active cell.
This box is enabled if you click the Split Time Range
check box.
The Y area displays:
Separate Labels
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Click this option to apply a label to the left of each
chromatogram displayed on the Axis page.
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Normalization
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Using a Chromatogram View
Plot
Click this option to define which plot “source” and
“units” refers to.
Source
Click the From Detector option to use the label
from the acquired data file. Click the Custom option
to enable the box and type in a label.
Units
Click the Absolute option (intensity) or the Relative
option (relative abundance) for the scaling of the
Y-axis.
Name
Use this box to type an axis name.
Show Name
Click a display option for the axis label: Never, On
Print, or Always.
Offset
Click this check box to offset the axis label from the
chromatogram.
Use the Normalization page to select the normalization (Y-axis scaling)
method used for chromatograms (see Figure 88).
Figure 88. Display Options dialog box - Normalization page for a Chromatogram
view
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The normalization methods are:
Auto Range
Click this option to automatically optimize the
Y-axis for each chromatogram.
Intensity Range
Click this option to set the range manually. Type
the minimum and maximum intensity required for
the Y-axis. The valid range is -200.00% to
+200.00%.
Normalize each plot to:
Largest Peak in
Subsection
Click this option to normalize each split time
range to the largest peak in the division.
Largest Peak in Selected Click this option to normalize the spectrum to the
Time Range
largest peak in the displayed time range.
Largest Peak in All
Times
Detecting Peaks
Click this option to normalize the spectrum to the
largest peak in the entire chromatogram.
Qual Browser provides several ways for detecting chromatogram peaks in a
cell. The following are the three most common:
• Automatic Detection of One Plot
• Automatic Detection of All Plots
• Manual Detection
Automatic Detection of One Plot
To detect and integrate all peaks in a selected chromatogram plot using
the current peak detection and integration settings
1. Click the chromatogram plot in the active cell. Xcalibur shades the
selected plot.
2. Click the Toggle Peak Detection In This Plot button on the toolbar to
detect and integrate all peaks in the selected chromatogram plot using
the current peak detection and integration settings.
3. To undo the peak detection, click the button on the toolbar a
second time or choose Actions > Peak Detection >
Toggle Detection in This Plot from the menu bar.
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Automatic Detection of All Plots
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Using a Chromatogram View
To detect and integrate all peaks in all chromatogram plots in the active cell
using the current peak detection and integration settings:
1. Click the Toggle Peak Detection In All Plots button on the toolbar.
2. Click the button on the toolbar a second time to undo all detected peaks
or choose Actions > Peak Detection > Toggle Detection in All Plots
from the menu bar.
Manual Detection
To manually detect and integrate all peaks in all chromatogram plots in the
active cell, use either the Add Peaks or the Delete Peaks button on the
toolbar.
To add a peak to a chromatogram plot
1. Click the Add Peaks button on the toolbar to detect and integrate any
peak in the selected cell.
Xcalibur changes the cursor to the Add Peaks cursor.
2. Drag the Add Peaks cursor horizontally across the peak to detect and
integrate. Xcalibur marks the added peak with a blue baseline and
integrates the peak. To adjust the positioning of the baseline markers,
click and drag using the cursor.
3. Click the Add Peaks button on the toolbar a second time to restore the
default cursor or choose Actions > Peak Detection > Add Peaks from
the menu bar.
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To delete a peak
Note The delete toolbar buttons and delete menu commands are active
only if peak detection identifies one or more peaks in the active cell.
1. Click the Delete Peaks button on the toolbar.
Xcalibur changes the cursor to the Delete Peaks cursor.
2. Click this cursor within the peak boundary (as indicated by its blue
baseline) of the peak to delete.
3. Click the Delete Peaks button on the toolbar a second time to restore
the default cursor or choose Actions > Peak Detection > Delete Peaks
from the menu bar.
Changing Peak Detection
Settings
You change peak detection settings on the Peak Detection Settings page
shown in Figure 89.
To display the Peak Detection Settings page
• Right-click in an active Chromatogram view and choose Peak
Detection > Settings from the shortcut menu
or
• Choose Actions > Peak Detection > Settings from the menu bar.
For information on the settings in the Peak Detection Settings page, refer to
“Peak Integration” on page 30.
Note The default values in the Peak Detection Settings page are suitable
for most analysis requirements. Change these settings only if standard
chromatogram detection and integration options do not provide the
desired result.
Click the Apply To All Plots check box to apply the current chromatogram
peak identification and integration settings to all displayed plots in the
active view. If the Apply To All Plots check box is not selected, the settings
are only applied to the active plot.
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Peak Detection Setting
Page
Figure 89. Qual Browser - Peak Detection Settings page
In addition to the standard Apply and Help buttons, the Peak Detection
Settings page also features the following buttons:
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Save as Defaults
Click Save as Default to save the current settings as
the default values to be used when you click Load
Default.
Load Defaults
Click Load Default to restore the current default
settings.
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Using a Spectrum
View
A spectrum view shows one or more of the mass scans acquired during an
analysis (see Figure 90).
Figure 90. Example of a Spectrum view with its shortcut menu
To view a spectrum, do one of the following:
• Right-click in the cell and choose View > Spectrum from the shortcut
menu.
• From the menu bar, choose View > Spectrum.
• Click the View Spectrum button on the toolbar.
This section contains the following topics:
• Using Spectrum Plots
• Spectrum Ranges
• Setting Spectrum Options
• Subtracting Background Spectra
• Determining the Elemental Composition of a Spectrum
• Simulating an Isotopic Distribution Spectrum
• Browsing MSn Data
• Submitting a Spectrum to a Library Search
• Customizing a Library Search
• Exporting a Spectrum to the Library
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Using Spectrum Plots
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Using a Spectrum View
Display up to eight plots within a Spectrum view.
To insert a plot
1. Select the cell containing the view.
2. Right-click the spectrum above the position where you want the new
plot.
3. Choose Plot > Insert from the shortcut menu.
To delete a plot
1. Select the cell containing the view.
2. Right-click the plot you want to delete.
3. Choose Plot > Delete from the shortcut menu.
Use also the Ranges dialog box to add, delete, or enable plots.
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Spectrum Ranges
Use the Spectrum Ranges dialog box to view and edit the mass range, time
range, background subtraction, and smoothing parameters for all the plots
in a spectrum cell (see Figure 91).
Figure 91. Spectrum Ranges dialog box
To display the Spectrum Ranges dialog box, do one of the following:
• Right-click a spectrum plot in the Cell Information page of the Info Bar
and choose Ranges from the shortcut menu.
• Right-click an active spectrum plot and choose Ranges from the
shortcut menu.
• From the Qual Browser window (with a spectrum view active), choose
Display > Ranges.
The Spectrum Ranges dialog box consists of two tabbed pages:
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Ranges
This page shows the properties of the plots displayed in the
spectrum cell: the source filenames, the time and mass
ranges of the plots, and background subtraction time ranges.
Automatic
Processing
This page displays any smoothing or refine parameters or
both applied to the data. Use this option to change settings
for mass tolerance, mass precision, and reference/exception
peaks.
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Ranges Page
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The Ranges page contains the following:
Ranges Area
The settings in the Range area apply to all plots in the selected cell.
Mass Range
Type the lower and upper mass limits for the view. Separate
entries by a dash (no spaces).
Averaging
Click this check box to enable spectrum averaging (average
all scans defined by the mass range, time range, and filter
settings).
Fixed Scale
Click this check box to enable and specify the fix scale
setting. To change the value, input the new maximum
Y-axis value in the Fix Scale box.
Spectrum Plot Table
Below the Ranges area is a table listing the spectrum plots (8 maximum)
contained within the selected cell. The table lists plots under the following
headings: Type, Filter, Raw File, Subtract 1, Subtract 2. Use the check
boxes at the beginning of each line to display or remove plots.
Plot Properties Area
The Plot Properties area displays the properties of the selected plot.
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Detector
Shows the type of detector used to generate the raw file. Valid
types are: MS, Analog, A/D card, PDA, and UV. The type of
detector determines the available Plot types.
Time
Shows the time of the mass scan in the host chromatogram.
Type a new value or range as required.
Scan Filter
Lists filters stored in the .raw file. Click a filter from the list,
or type a new one using the scan filter format.
Raw File
Shows the path and filename of the raw file used to generate
the selected plot. The list contains all files active in Qual
Browser. To open an unlisted file, click the Browse button,
and identify the file in the normal manner.
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Background Subtraction Area
The Background Subtraction area shows details of background subtraction
if this has been applied to the spectrum. The background contribution is
determined by averaging the scans from one or two baseline regions:
Time Range 1 First baseline region used for assessing the background.
Time Range 2 Second baseline region used for assessing the background.
Xcalibur enters these settings automatically when you perform a
background subtraction by choosing Actions > Subtract Spectra from the
Qual Browser window, or type time ranges directly into the boxes.
Automatic Processing Page
Use the Automatic Processing page shown in Figure 92 to apply smoothing
or refine window size and noise threshold to all the plots in the active
Chromatogram view.
Figure 92. Spectrum Ranges dialog box - Automatic Processing page
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This page contains the following areas:
Smoothing Area
Use the Smoothing area settings to smooth all scans defined by the mass
range, time range, and filter settings in the Spectrum Ranges dialog box.
The Smoothing area contains the following parameters:
Enable
Click this check box to enable spectrum smoothing.
Type
Specify the type of smoothing algorithm to apply to the
spectrum: Boxcar or Gaussian (refer to the online Help
for more information).
Points
Type the number of points for spectrum smoothing.
The value must be an odd number in the range 3
[minimum smoothing] to 15 [maximum smoothing].
Refine Area
Use this setting to refine all scans defined by the mass range, time range, and
filter settings in the Spectrum Ranges dialog box. The refine spectrum
enhancement feature is described fully in “Spectrum Enhancement” on
page 39.
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Enable
Click this check box to turn on the Refine spectrum
enhancement.
Window Size
Type the number of seconds on either side of the
specified point over which the algorithm uses mass
chromatograms. A reasonable initial value is the peak
width in seconds.
Noise Threshold
Use this parameter to eliminate peaks generated from
baseline noise. Set it to zero (this shows all peaks in the
spectrum) and increase the value until noise peaks are
eliminated. See “Refine” on page 40 for an explanation
of the Refine algorithm.
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Include Peaks Area
The Include Peaks area has only one check box. Use this setting to include
or exclude the reference peaks (R) and exception peaks (E) for the mass data
in all the cells in the Qual Browser window.
The Include Peaks area contains the following parameter:
Reference and
Exception Peaks
Use this check box to include or exclude the reference
peaks (R) and exception peaks (E) for the mass data in a
Qual Browser window.
Mass Tolerance Area
Use the Mass Tolerance area to specify a value for mass tolerance. Settings in
this area affect the display of all the mass data in the Qual Browser window.
The Mass Tolerance area contains the following parameters:
Use User
Defined
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Use this check box to specify the values for mass tolerance
and mass units for the mass data in a Qual Browser
window. To enable the parameters and specify the values,
click the Use User Defined check box. If you clear the check
box, Xcalibur uses the values for mass tolerance and units
that are stored in the raw file.
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Using a Spectrum View
Mass tolerance Xcalibur uses a mass tolerance band to calculate spectrum
averages, to add and subtract spectra, and to convert profile
data to centroid data. The mass tolerance value controls the
number of individual scans that Xcalibur groups together
during data processing. Xcalibur condenses to a single mass
value all the scans in the range from the lower to the upper
tolerance limits. Individual scans are available for display if
the mass tolerance is small (less than 1 mmu), but might be
grouped if the mass tolerance is larger.
For example, when Xcalibur applies a mass tolerance of 300
mmu to a mass value, it groups all the individual scans in
the range between -300 and +300 mmu of the mass value;
the result is a single mass intensity value.
Use the Mass Tolerance box to specify the value for mass
tolerance in the range of 0.1 to 50000.
Set a default value for mass tolerance on the Xcalibur
Configuration dialog box - Mass Options page (see
Figure 83). Mass tolerance is used commonly with
high-resolution measurements.
Units
Use these options to specify the units of measurement for
data processing.
mmu Millimass units
ppm
Parts per million
Mass Precision Area
Use the Mass Precision area settings to apply mass precision to the mass data
in all the cells in the Qual Browser window.
The Mass Precision area contains the following parameters:
Decimals Xcalibur applies the number of decimal places to mass intensity
values for data processing calculations. Specify a number from 0
to 5. If you specify 3, for example, Xcalibur uses a mass number
truncated to 3 decimal places to perform calculations. Set a
default value for mass precision in the Xcalibur Configuration
dialog box - Mass Options page (see Figure 83). Mass precision
is commonly used with high-resolution measurements.
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Setting Spectrum Options
To change the style of a spectrum view in the Display Options dialog box
(see Figure 93), confirm that no other cell is pinned. Then, do one of the
following:
• Right-click in the cell and choose Display Options.
• From the menu bar, choose Display > Display Options.
The Display Options dialog box consists of 6 tabbed pages: Style, Color,
Label, Axis, Normalization, and Composition. It contains a small display
area showing the active cell. Use these options to preview the effects of
different settings before applying them.
Figure 93. Display Options dialog box - Style page for a Spectrum view
Style
Style options determine the appearance of spectra. The Plotting,
Arrangement, and 3D areas offer style selections as follows:
Plotting
Select from the following four viewing styles:
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Automatic
Click this option to have Xcalibur automatically
select the graphic style based upon the data
acquisition method used for the active spectrum.
Point To Point
Click this option to display a point-to-point peak
profile.
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Stick
Click this option to display spectral mass peaks as
vertical lines.
Shade
Click this option to display the spectrum as a shaded
representation of intensity in each amu band for the
active spectrum.
Arrangement
Choose a 2D or 3D arrangement of plots within the cell:
Stack (2D)
Click this option to stack plots vertically with no
overlap for the active map.
Overlay (3D)
Click this option to overlay plots vertically with an
optional horizontal skew (time offset) for the active
map. When selected, the 3D display options are
available (see the following topic).
3D
The following options are available with the Overlay (3D) option:
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Elevation
Use this slider bar to set the elevation angle (from
0 to 60 degrees). Either drag the Elevation slider or
click the Elevation slider left or right arrow until you
reach the desired angle.
Skew
Use this slider bar to set the skew angle. Either drag
the Skew slider or click the Skew slider left or right
arrow until you reach the desired angle (from 0 to 45
degrees).
Draw Backdrop
Click this check box to add a backdrop to 3D plots.
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Color
Use the Color page shown in Figure 94 to customize the colors of spectrum
plots in the various display styles.
Figure 94. Display Options dialog box - Color page for a Spectrum view
Customize the colors of the following spectrum view features:
Regular peaks
Click this button to select the color of normal
(unsaturated) peaks in the stick style.
Saturated peaks
Click this button to select the color of saturated
peaks in the stick style.
Profile
Click this button to select the color of the plot in
peak to peak profile style.
Shade
Click the 0%, 20%, 40%, 60%, 80%, or 100%
button to change the color of map at 0%, 20%,
40%, 60%, 80%, or 100% relative abundance.
Backdrop
Click this button to select the color of the backdrop.
When you click one of these buttons, Xcalibur opens the Color dialog box
with a color palette to select a preset color or customize a color.
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Labels
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Using a Spectrum View
Use the Labels page shown in Figure 95 to choose the type and style of peak
labels.
Figure 95. Display Options dialog box - Labels page for a Spectrum view
The Labels page contains the following spectrum labeling options:
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Mass
Click this check box to label spectrum peaks with
m/z values. Use the Decimals box to specify the
number of decimal places to be used in the label.
Relative to
Xcalibur offsets the m/z label at the top of spectrum
peaks by the amount that you specify in this box.
Flags
Click this check box to label spectrum peaks with
flags. These provide supplemental information about
the peak data. For example, if a peak is saturated,
Xcalibur displays an S above the peak.
Decimals
Use the Decimals box to specify the number of
decimal places to be used in the label.
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Resolution
This check box indicates whether or not Xcalibur
displays Resolution information in the
Spectrum view.
Resolution is a Label Stream parameter and is active
if the raw file has Label Stream data. These values are
written by the instrument.
Resolution can also be active if you have centroided
a profile scan and show a result returned by the
centroider.
Charge
This check box indicates whether or not Xcalibur
displays Charge information in the Spectrum view.
Charge is a Label Stream parameter and is active if
the raw file has Label Stream data. These values are
written by the instrument.
Baseline
This check box indicates whether or not Xcalibur
displays Baseline information in the Spectrum view.
Baseline is a Label Stream parameter and is active if
the raw file has Label Stream data. These values are
written by the instrument.
Noise
This check box indicates whether or not Xcalibur
displays Noise information in the Spectrum view.
Noise is a Label Stream parameter and is active if the
raw file has Label Stream data. These values are
written by the instrument.
Width
This check box indicates whether or not Xcalibur
displays peak width information in the
Spectrum view.
Width is a Label Stream parameter and is active if
the raw file has Label Stream data. These values are
written by the instrument.
Width can also be active if you have centroided a
profile scan and show a result returned by the
centroider.
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Centroid
Click this check box to convert the data from profile
to centroid. This check box in active only if you are
displaying profile data. Click the Centroid check box
to activate the Choose Algorithm button to open the
Choose Centroiding Algorithm dialog box.
Choose Algorithm
Click Choose Algorithm to open the Choose
Centroiding Algorithm dialog box. Click the
Centroid check box to activate the Choose
Algorithm button. The Centroid check box is active
only if you are displaying profile data and the
Centroid check box is selected.
Choose from the following label styles:
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Offset
Click this check box to offset a label from its normal
position to avoid conflict with another label. Choose
the amount of the offset (in number of characters) in
the Size box.
Size
Type the amount that Xcalibur is allowed to offset a
label from its normal position to avoid conflict with
another label. The valid range is 0.0 to 9.0
characters.
Rotated
Click this check box to use vertical labels, rather
than horizontal labels.
Boxed
Click this check box to place a rectangular outline
around each peak label.
Label Threshold
Use this parameter to limit the labeling of peaks to
those exceeding the specified percentage of the base
peak.
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Axis
Use the Axis page shown in Figure 96 to set spectrum axis labels and display
options.
Figure 96. Display Options dialog box - Axis page for a Spectrum view
The X area displays:
Name
Use this box to type an axis name. For example, m/z.
Show Name
Click an axis label display option from the list: Never,
On Print, or Always.
Offset
Click this check box to offset the axis label from the
spectrum.
Split Range
Click this check box to split the spectra into two or
more separate graphs with equal ranges.
Divisions
Type the number of split mass range graphs to be
displayed for each spectrum in the active cell.
The Y area displays:
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Source
Click the Detector option to use the label from the
acquired data file. Click the Custom option to
enable the box and type in a label.
Units
Select the scale to draw the chromatogram by
selecting absolute or relative.
Name
Use this box to type an axis name.
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Show Name
Select a display option for the axis label: Never, On
Print, or Always.
Offset
Select this check box to offset the axis label from the
spectrum.
For the Z-axis:
Normalization
Name
Use this box to type an axis name.
Show Name
Select a display option for the axis label: Never, On
Print, or Always.
Use the Normalization page shown in Figure 97 to select normalization
options for spectrum plots.
Figure 97. Display Options dialog box - Normalization page for a Spectrum view
The Normalize Method area displays:
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Auto Range
Click this option to automatically optimize the
Y-axis.
Intensity Range
Click this option to set the Y-axis range. Use the box
to specify the minimum and maximum intensity.
The valid range is -200.00 to +200.00%.
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The Normalize Spectrum To area displays:
Largest Peak in
Subsection
Click this option to normalize each split mass range
to the largest peak in the division.
Largest Peak in Mass Click this option to normalize the spectrum to the
Range
largest peak in the displayed mass range.
Largest Peak in Scan Click this option to normalize the spectrum to the
largest peak in the entire spectrum.
The Normalize Multiple Scans area displays:
Composition
Individually
Click this option to normalize each mass plot
individually.
All The Same
Click this option to normalize all mass plots equally.
Use the Spectrum Composition page shown in Figure 98 to add chemical
formulas and related labels to the spectrum. Xcalibur determines which
chemical formulas have an m/z value most like that of the experimental
spectrum peaks.
Figure 98. Display Options dialog box - Spectrum Composition page for a
Spectrum view
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Label the mass peaks with:
Elemental
Composition
Xcalibur determines which chemical formulas have a
m/z value most like that of the spectrum peaks. This
check box determines whether or not Xcalibur
displays the chemical formula labels at the top of
spectrum peaks.
Formulae
Specify how many of the most likely chemical
formulas you want Xcalibur to display at the top of
spectrum peaks.
Theoretical Mass
Click the Theoretical Mass check box to display the
theoretical m/z of the chemical formulas that
Xcalibur determines. Xcalibur displays the
theoretical m/z to the right of the formula separated
by an equal sign (=).
Ring and Double
Bond Equivalents
Click the Ring and DB Equiv. check box to display
the value of the ring and double bond equivalents
that Xcalibur calculates for the chemical formulas.
Xcalibur displays the ring and double bond
equivalent value under the chemical formula.
Note Ring and double bond equivalents provides a measure of the
number of unsaturated bonds in a compound. It limits the calculated
formulas to only those that make sense chemically.
Thermo Electron Corporation
Delta
Click the Delta check box to have label the peak with
the difference between the theoretical and
experimental m/z.
Delta Units
Use these options specify the units to use when
calculating the difference between the theoretical and
experimental m/z. The options are amu, mmu, and
ppm.
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Subtracting Background
Spectra
Use a Chromatogram view to subtract background from a Spectrum view,
subtracting background from either one range (either side of the
chromatogram peak of interest) or two ranges (both sides of the
chromatogram peak of interest).
To subtract background spectra
1. Open a Chromatogram view.
2. Open a Spectrum view in another cell (you might need to add a new cell
to the window).
3. Pin the cell containing the Spectrum view.
4. Click and drag the cursor through the chromatogram peak of interest.
This action updates the pinned Spectrum view with an averaged
spectrum using the scans in the indicated range.
5. Select one or two ranges for spectrum subtraction:
a. Choose Actions > Subtract Spectra > 1 (or 2) Range, or
Right-click in the spectrum cell and select Subtract Spectra > 1 (or
2) Range from the shortcut menu.
b. Identify a representative baseline region in the Chromatogram view
close to the peak of interest. Click and drag the new cursor to select
a time range in this region.
c. If you have selected the 2 Range option, choose a further region on
the other side of the peak of interest.
d. Release the mouse button. Xcalibur subtracts an average of the
selected scans and redraws the Spectrum view. The Spectrum view
header shows the number of subtracted scans. For example, SB: 12
indicates that Qual Browser has applied background subtraction to
the spectrum using 12 scans.
6. To see the selected time range(s) of the scans that were subtracted,
choose Display > Ranges to open the Spectrum Ranges dialog box and
review the Time Range 1 box.
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Determining the Elemental
Composition of a
Spectrum
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum View
Use the Elemental Composition page of the Info Bar to set the parameters
that Xcalibur uses to calculate the “best matching” chemical formula for a
mass, or a list of masses (from a spectrum).
To have Xcalibur calculate the best matching chemical formulas for the
peaks in a spectrum
1. Right-click the Spectrum view and choose Elemental Composition
from the shortcut menu.
Xcalibur displays chemical formulas.
2. Click the Elemental Composition tab in the Info Bar to display the
Elemental Composition page (see Figure 99).
Figure 99. Elemental Composition page
Thermo Electron Corporation
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The Elemental Composition area displays:
Mass
Enter a mass or select a mass in the mass list for
Xcalibur to use to calculate probable chemical
formulas. To change the mass value, type a mass
from 0.5 to 100000. To generate a mass list,
right-click a mass spectrum and select Elemental
Composition. To calculate formulas and display
them in the Results area, click the Calculate button.
Maximum Results
Use the Maximum Results box to select the
maximum number of formulas you want Xcalibur to
display.
Calculate
Click Calculate to calculate formulas and display
them in the Results List.
File
Click File to write a formula or a group of formulas
to a file. To select formulas, click an index number in
the Idx column of the Results List, then use
CTRL-click or SHIFT-click to select other results
you want in the file.
List
Click List to display the Spectrum List view. The
Spectrum List tabulates the masses (or wavelengths)
and intensities for each of the selected ions.
Simulate
Click Simulate to simulate the spectrum of a
formula highlighted in the Results List. The
spectrum is based on the full isotope distribution for
that formula.
The Results List displays:
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Idx
The Index (Idx) column displays the number of the
row in the Results List.
Formula
The Formula column displays the formulas
calculated using the values specified in the Elemental
Composition area and the limits you specified in the
Limits area.
RDB
The RDB column displays the ring and
double-bond equivalents calculated for each of the
formulas in the Results List.
Delta [units]
The Delta column displays the difference between
the specified mass and the calculated mass in amu,
mmu, or ppm units for each of the formulas in the
Results List.
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The Limits area displays:
Charge
Use the Charge box to select the charge state used to
calculate probable formulas. To change the charge
state, type a number from -5 to +99.
Nitrogen Rule
Use the Nitrogen Rule list to select whether or not to
use the Nitrogen Rule in the formula calculation.
The choices in the list are as follows: Do Not Use,
Even Electron Ions, and Odd Electron Ions.
For molecular ions of even or odd molecular weight,
specify that formulas contain either an even or an
odd number of nitrogen atoms, respectively, in the
Nitrogen-Rule list. Conversely, for fragment ions of
even or odd molecular weight, specify the reverse;
that is, specify odd or even, respectively, in the
Nitrogen-Rule list.
McLafferty states the Nitrogen Rule as follows: “If a
compound contains no (or an even number of )
nitrogen atoms, its molecular ion is at an even mass
number...[Similarly,] an odd-electron ion is at an
even mass number if it contains an even number of
nitrogen atoms.”
Mass Tolerance
Thermo Electron Corporation
Xcalibur uses a mass tolerance band to calculate
spectrum averages, to add and subtract spectra, and
to convert profile data to centroid data. During data
processing, Xcalibur groups the scans together that
are in the tolerance band, specified in units of amu,
mmu, or ppm. Xcalibur condenses to a single mass
value all the scans in the range from the lower to the
upper tolerance limits. Individual scans are available
for display if the mass tolerance is small (less than 1
mmu), but can be grouped if the mass tolerance is
larger.
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Ring/Double Bond
Equivalents
The Ring/Double Bond Equivalents box displays a
range of values for double bonds and ring
equivalents—a measure of the number of
unsaturated bonds in a compound—and limits the
calculated formulas to only those that make sense
chemically. Specify limits in a range from
-1000.0 to +1000.0.
The value is calculated by the following formula:
imax
D=1+
Σi Ni (Vi - 2)
2
Where D is the value for the RDB, imax is the total
number of different elements in the composition,
Ni is the number of atoms of element i, and Vi is the
valence of atom i.
The calculation results in an exact integer such as
3.0, which indicates an odd-electron ion, or an
integer with a remainder of 0.5, which indicates an
even-electron ion. A value of -0.5 is the minimum
value and corresponds to a protonated, saturated
compound (for example, H3O+).
The Elements in Use List displays:
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Isotope
The Isotope column displays the isotopes to consider
when calculating elemental compositions for a given
mass. To add an isotope, click in an empty area in
the Isotope column to display the Select Isotopes
dialog box (see Figure 100). To remove an isotope,
click an isotope name to bring up a dialog box and
delete the isotope.
Min
The Minimum [Number of Occurrences] column
displays the minimum number of occurrences of a
specified isotope for a determination of formula
composition.
Max
The Maximum [Number of Occurrences] column
displays the maximum number of occurrences of a
specified isotope for a determination of formula
composition.
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DB Eq.
The Double Bond/Ring Equivalents column
displays the values of the lower and upper limits for
double bond and ring equivalents that Xcalibur
calculates for each isotope in the Elements In Use
List. See also Ring / Double Bond Equivalent box.
Mass
The Mass column displays the exact isotopic mass
for each isotope specified in the Elements In
Use List.
The buttons at the bottom of the Elemental Composition page do the
following:
Thermo Electron Corporation
Load
Click Load to display the Open dialog box and
select a file (.lim) that contains a set of isotope limits.
Save As
Click Save As to display the Save As dialog box to
save a list of isotopes to a file with extension .lim.
Apply
Click Apply to apply the Elemental Composition
area settings to the spectrum.
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Use the Select Isotopes dialog box to display the isotopes of each element,
and select one or more isotopes to include in the calculation of chemical
formulas. To display the Select Isotopes dialog box, click an empty cell in
Elements in Use list on the Elemental Composition page.
Figure 100. Select Isotopes dialog box
The Select Isotopes dialog box displays:
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Elements
This list displays the chemical elements. Click an
element in the Periodic Table to add it to the
Elements list. Click the element in the Elements list
to display isotopes. The isotopes appear in the
Isotopes list.
Isotopes
This list displays the name and relative intensities of
the isotopes for the current element selected from
the Elements list. Select an isotope from the list; it
appears in the Selected Isotope text box.
Selected Isotopes
This box displays the isotopes you select from the
Isotopes list.
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Min. Number
This box displays the minimum number of
occurrences of the selected isotope in the formula
that Qual Browser calculates. To change the
minimum number of occurrences, type a number
from 0 to 1000 in the box.
Max. Number
This box displays the maximum number of
occurrences of the selected isotope in the formula
that Qual Browser calculates. To change the
maximum number of occurrences, type a number
from 0 to 1000 in the box.
Add to List
Click Add to List to add the isotopes listed in the
Selected Isotopes list to the Elements in Use list of
the Elemental Composition page.
Periodic Table
Click an element in the Periodic Table to add that
element to the Elements list.
To change the color of the multi-isotopic or
monoisotopic elements, click the Multi Isotopic or
the Mono Isotopic button in the lower left corner of
the periodic table. This action opens the Color
dialog box to select a new color.
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Simulating an Isotopic
Distribution Spectrum
Use the Spectrum Simulation page to create a simulated isotopic
distribution spectrum of a chemical formula.
Click the Spectrum Simulation tab in the Info Bar to display the Spectrum
Simulation page shown in Figure 101.
Figure 101. Spectrum Simulation page
The Isotopic Simulation area displays:
New
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Click New to have Xcalibur place the simulated
spectrum in a new cell.
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Insert
Reviewing and Interpreting Data in Qual Browser
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Click Insert to have Xcalibur place the simulated
spectrum above the selected (highlighted) spectrum
(or add a spectrum if none is present).
Select a spectrum to make the Insert button active.
Replace
Click Replace to have Xcalibur replace the selected
(highlighted) spectrum with the simulated spectrum.
Select a spectrum to make the Replace button active.
Chemical Formula
Use the Chemical Formula combo box to enter or
select the chemical formula for the simulated
spectrum. Then, click Insert or Replace to display
the spectrum.
Type in either upper and lower case letters, however
Xcalibur interprets all lower case input as two-letter
symbols. For example, the parses the string “inau” as
“In Au”. Be more specific in capitalization to force
other interpretations, namely “INAu” or “INaU”.
Xcalibur interprets all upper case input as
single-letter element names. For example, “COSI” is
parsed as “C O S I”. See the Xcalibur online Help for
additional information.
Peptide/Protein
Use the Peptide/Protein combo box to enter or select
the peptide/protein formula for the simulated
spectrum. Then, click the Insert or Replace button
to display the spectrum.
Use either single capital letter abbreviations for
amino acids (for example, CAT) or the standard
three letter abbreviations with the first letter
capitalized. See the Xcalibur online Help for
additional information.
Plus H2O
Click this check box to specify that the simulated
spectrum for a peptide formula includes a water
molecule.
This check box becomes active when you click the
Peptide/Protein option.
Adduct
Thermo Electron Corporation
Click the Adduct check box to specify that the
simulated spectrum is an adduct. Select either H, K,
or Na in the list.
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Mixture
Click the Mixture check box and click the Change
Mixture button to open the Change Mixture for
Simulation dialog box. Use the Change Mixture for
Simulation dialog box to specify the compounds and
amounts you want to include in the mixture.
Change Mixture
Click Change Mixture to open the Specify Mixture
for Simulation dialog box shown in Figure 102. Use
the Specify Mixture for Simulation dialog box to
specify the compounds and amounts to include in
the mixture.
Click the Mixture check box to make the Change
Mixture button active.
Figure 102. Specify Mixture for Simulation dialog box
The Charge Distribution area displays:
Most Abundant
Use the Most Abundant box to select the most
abundant charge of the ion, and Xcalibur calculates
the masses accordingly. The range is -99 to +99, and
the default value is +1.
Half Width
Use the Half Width box to simulate a distribution of
charges. Select a value from 0 to 99.
The Output Style area displays:
Pattern
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Click the Pattern option to plot the exact pattern of
isotopic peaks generated by the simulation.
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Profile
Click the Profile option to plot the pattern spectrum
convolved with a gaussian, cosine, triangular, or
lorentzian broadening function.
Samples/Peak
Use the Samples Per Peak box to specify the number
of data points across the peak. Here, the peak is
defined as either the FWHM or the width to the
10% or 5% intensity values.
Centroid
Click the Centroid option to have Xcalibur apply the
same algorithm used in the firmware to convert from
profile data to centroid. When you click centroid,
both the Samples/Peak box and the Choose
Algorithm button become active.
Choose Algorithm
Click Choose Algorithm to display the Choose
Centroiding Algorithm dialog box (see Figure 103).
Use the Choose Centroiding Algorithm dialog box
to select a centroiding algorithm.
Figure 103. Choose Centroiding Algorithm dialog box
The Resolution area displays:
Thermo Electron Corporation
Daltons
Use the Daltons option and box to specify a value for
simulated peak width in Daltons. When you click
the option, the box becomes active.
PPM
Use the PPM option and box to specify a value for
simulated peak width in parts per million. When you
click the option, the box becomes active.
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Resolving Power
Use the Resolving Power option and box to specify a
quality factor for simulated peak width in units of
resolving power. When you click the option, the box
becomes active.
The Valley area displays:
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FWHM
Click this option to make the peak width at half
maximum equal to the resolution. For example, if
you select a resolution of 1 Dalton, the peak is 1
Dalton wide at half maximum.
10%
When you select this option, Xcalibur adjusts the
peak width such that two equal-height peaks that are
separated by the specified resolution have a valley
height between them equal to 10% of the peak
height. In other words, the width of a single peak
equals the resolution at 5% of the peak height.
5%
When you click this option, Xcalibur adjusts the
peak width such that two equal-height peaks that are
separated by the specified resolution have a valley
height between them equal to 5% of the peak height.
In other words, the width of a single peak equals the
resolution at 2.5% of the peak height.
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Browsing MSn Data
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum View
Use the MSn Browser Information page of the Info Bar to display and
analyze MSn experimental data.
Click the MSn tab in the Info Bar to display the MSn Browser Information
page shown in Figure 104.
Figure 104. MSn Browser Information page
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Use the MSn Browser Information page to set the following parameters:
Time Range
Use this box to specify a chromatogram time range.
The MSn Browser changes the Time axis on the
chromatogram (display by choosing View >
Chromatogram) and limits the spectra available for
display to those taken during the specified time
range.
There are two ways to change the Time Range:
• Type the time range in minutes in the Time
Range box. The valid range is 0.00 to 999.0 min.
The format is [From]-[To]. For example, to view
the chromatogram time range from 0.01 min to
5.10 min, type 0.01-5.10.
• Click the Track check box to enable tracking of
the time range using the Chromatogram view.
Then, click and drag the cursor horizontally
across the Chromatogram view from the
minimum time to the maximum time of interest.
Xcalibur changes the range in the Time Range
box and displays the chromatogram with its
revised range. To return to the original range,
click the zoom reset button.
Track
Click the Track check box to enable tracking of the
time range using the Chromatogram view. Then,
click and drag the cursor horizontally across the
Chromatogram view (display by choosing View >
Chromatogram) from the minimum time to the
maximum time of interest. Xcalibur changes the
range in the Time Range box and displays the
chromatogram with its revised range.
To return to the original range, click the Zoom
Reset button.
Mass Range
Use this box to specify a mass range to view. The
MSn Browser limits the viewable spectra to the
specified mass range.
To change the mass range, type the range in the Mass
Range box. The valid range is m/z 65.00 to 2000.00.
The format is [From]-[To]. For example, to view
spectra having mass range m/z 100 to 500, type
100.00–500.00.
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Mass Tolerance
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum View
This box displays the current mass range over which
spectra are not distinguished (grouped). The valid
range is m/z 0.00 to 10.00. The default value is
m/z 0.50.
• If the mass tolerance is greater than 0.50,
Xcalibur groups scans that meet the range
tolerance so that the number of individual scans
can be reduced.
• If the mass tolerance is less than 0.50, Xcalibur
displays each scan from the specified precursor.
Normalize
Composite
Spectrum
Click this check box to normalize the composite
spectrum displayed in the Spectrum view (display by
choosing View > Spectrum). This action results in a
composite of individual spectra (MS2, MS3, MS4,
and so on) where each spectrum is normalized to the
highest intensity in the MS2 experiment, not using
the relative number of counts. This option supports
easy comparison of MSn experimental data, however
the peak ratio of the MSn data is not maintained.
Deselect this check box if you do not want to
normalize the composite spectrum displayed in the
Spectrum view (display by choosing View >
Spectrum). This action results in a composite of
individual spectra (MS2, MS3, MS4, and so on)
where the MS2 spectrum is normalized to Relative
Abundance 100 and all other spectra are displayed
relative to their actual number of counts. This
option increases the difficulty of comparison of MSn
experimental data, however the peak ratio
information is maintained.
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The MSn Browser Information page displays the following:
MS2
Precursor
The
icon is the top of the tree view displayed in the Info
bar of the MSn Browser feature available in the Qual Browser
window. Use this tree view to select MSn spectra. A typical
starting view is shown below:
The MS2 precursor ion m/z is displayed to the right of the
icon. The MS2 precursor ion is the parent ion mass from the
MS experiment that is used for the MS2 (MS/MS)
experiment.
If you click the plus (+) sign, all of the MS3 precursor icons
appear and the icon for the MS2 average spectrum appears.
See the example below:
To view a spectrum, click the
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icon of interest.
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Composite A spectrum that is the sum of all MS2, MS3, MS4,
Spectrum MS5,…MS10 spectra, as defined by the Instrument Method.
Not Normalized Spectrum
When the Normalize Composite Spectrum check box is not
enabled, the composite spectrum is normalized (NL) to the
highest intensity of the MS2 experiment. This results in the
intensities of consecutive MS experiments being displayed at
lower and lower intensities.
An example of a processing filter for a not normalized
composite spectrum of an MS3 experiment in which four
microscans were averaged follows. The parent mass of the MS2
experiment is m/z 1108.16, and the parent mass of the MS3
experiment is m/z 775.98. The relative collision energy used
for both experiments was 30%:
Msnbrows#38-185 RT:0.98-4.91 AV:4 NL: 2.73E3
T: + c CMP ms3 [email protected] [email protected]
[200.00-1560.00]
The following peaks are labeled: (Parent Mass) PM MS2 and
PM MS3.
Normalized Spectrum
When the Normalize Composite Spectrum check box is
enabled, each MSn spectrum is individually normalized (NL)
so that its highest peak is displayed at Relative Abundance
100. The relative peak heights of this display are therefore not
meaningful. For example, a composite spectrum (CMP) for an
MS3 experiment displays both the MS2 base peak and the
MS3 base peak at Relative Abundance 100 and maintain all
other relative abundances of the other ions in each spectrum.
Use this display normalization option to view multiple spectra
simultaneously, even if the absolute value of the intensities are
significantly different.
An example of a process filter for a normalized composite
spectrum of an MS3 experiment in which four microscans
were averaged follows. The parent mass of the MS experiment
is m/z 1108.16 and the parent mass of the MS2 experiment is
m/z 775.98. The relative collision used was 30%:
Msnbrows#38-185 RT:0.98-4.91 AV:4 NL: 2.73E3
T: + c CMP ms3 1108.16 775.98 [200.00-2000.00]
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Average
Spectrum
A spectrum that is the sum of all of the microscans taken for a
particular MSn experiment. An average spectrum can be
independently displayed for MS2, MS3, MS4, MS5, …MS10
experiments. The average spectrum is displayed normalized
(NL) to the average base peak.
In the event that there is only one spectrum with one scan to
average, Xcalibur displays the term single spectrum instead of
average spectrum. In other words, single spectrum is a special
case of average spectrum.
An example of a processing filter for the average spectrum of
an MS3 experiment in which two microscans were averaged
follows. The parent mass of the MS experiment is m/z
1108.07 and the parent mass of the MS2 experiment is m/z
776.03:
Msnbrows#38-185 RT:1.01-4.91 AV:2 NL: 2.73E3
T: + MS3 [email protected] [email protected] [200.00-1560.00]
Scan
Single scans (individual scans) are available if you right-click
Number At in the MSn Browser Information page and choose Include
RT
Single Scans from the shortcut menu.
The MSn Browser Information page has the following right-click shortcut
menu commands:
Ranges
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Opens the Spectrum Ranges or Chromatogram Ranges dialog
box. These dialog boxes shows the properties of all the plots in
the active cell. Use the dialog boxes to view or modify the
time and mass ranges and change background subtraction and
smoothing parameters.
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Include
Individual
Scans
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum View
If this command is off, the MSn Browser displays the MS2
Average Spectrum and all MSn Average Spectra and
Composite Spectra that are included in the .raw file.
If this command is on, the MSn Browser displays the MS2
Average Spectrum and all of the individual scans that make up
the average spectrum. The number of individual scans is
controlled by the selection of the Mass Tolerance value.
Individual scans are available for display if the Mass Tolerance
is small (m/z less than 0.50), but can be grouped if the Mass
Tolerance is large (m/z greater than 0.50). In addition, the
MSn Browser displays MSn Average Spectra, MSn Composite
Spectra, and all of the MSn individual scans. The
functionality of the Mass Tolerance value is the same for all
MSn scans as that for MS2 scans, as described above.
Individual scans appear in the following format:
[Scan Number] at [Retention Time]
For example, the icons for scan numbers 76, 115, and 154
appear below:
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Using a Spectrum View
Submitting a Spectrum to
a Library Search
To submit a spectrum to a library search, right-click the spectrum and
choose Library > Search from the shortcut menu.
Qual Browser submits the spectrum to a library search and displays
matching library spectra (hits) in a four-pane Library Search Results
window (see Figure 105).
Figure 105. An example of a library search results window
The four panes are (clockwise from top left):
• A list of Search Results
• The molecular structure (with the formula, molecular weight, name,
and library index number)
• A difference spectrum. Peaks above the X-axis are relatively more intense
in the sample than the library entry. Negative peaks are relatively more
intense in the library entry than in the sample spectrum.
• A comparison of the sample spectrum and the selected search results
item
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Interpreting the Hits List
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum View
The Hits list shows the best matches found during the library search. Three
factors describe the accuracy of the match to the unknown spectrum:
SI
A direct matching factor for the unknown and the
library spectrum.
RSI
A reverse search matching factor ignoring any peaks
in the unknown that are not in the library spectrum.
Prob
A probability factor based on the differences between
adjacent search results in an SI ordered list.
With the SI and RSI matching factors, a perfect match results in a value of
1000. As a general guide 900 or greater is an excellent match; 800 to 900 a
good match; 700 to 800 a fair match. A matching factor less than 600 is a
very poor match. Unknown spectra with many peaks tend to yield lower
match factors than otherwise similar spectra with fewer peaks.
The probability factor is a complex parameter based on the SI matching
factor and the difference between adjacent matches. If a hit has an SI match
factor greater than 900 and the next best hit has a match factor of 300, the
probability of the compound being correctly identified is high. Conversely,
if several results are returned with very similar SI matching factors, the
probability of a correct assignment is low.
You should be wary of making positive assignments based solely on the
statistical result of a library search. Xcalibur might identify a compound
with an unusual structure (or more importantly an unusual mass spectrum)
in a definitive way. More usually, the unknown is a member of a class of
compounds with very similar mass spectra. The ability of Qual Browser
(and indeed any search system) to distinguish between them is limited. In
many cases, the best that the search algorithm can do is identify a class of
compounds that have similar mass spectra—and usually similar structure. In
most cases, you should seek confirmation by other analysis techniques.
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Using a Spectrum View
Customizing a Library
Search
Use the Search Properties dialog box (see Figure 106) to select and order the
libraries used during library searching and change the way that the search is
carried out. The dialog box consists of two pages:
• Search List
• Search Parameters
Figure 106. Search Properties dialog box - Search List page
To open this dialog box, choose Actions > Library > Options or right-click
within a Spectrum view and choose Library > Options from the shortcut
menu.
Search List
Use the Search List Page of the Search Properties dialog box (see
Figure 106) to select the libraries and search order for library searches of
spectra from Qual Browser.
The Search List page contains the following parameters:
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Available Libraries
This box lists the libraries that are currently excluded
from searching during processing. Xcalibur
regenerates this list when you open the dialog box.
Selected Libraries
This box lists the libraries that are currently included
in searches during processing. The order of the
libraries defines the search order.
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Using a Spectrum View
To include a library in the search list, select the library name in the available
libraries list. Then, click the Add button. The selected library is appended to
the selected libraries list in the final position.
To exclude a library from the search list, select the library name in the
selected libraries list. Then, click the Remove button. The library is
transferred to the available libraries list.
To change the search order of selected libraries, select the library name in
the selected libraries list. Then:
• Click the Top button to move the library to the top of the list.
• Click the Up button to move the library up one position.
• Click the Down button to move the library down one position.
• Click the Bottom button to move the library to the last position.
Search Parameters
Use the Search Parameters Page of the Search Properties dialog box (see
Figure 107) to select the type of library search, limit the search by a
molecular weight constraint, and determine how the results of the search are
returned.
Figure 107. Search Properties dialog box - Search Parameters page
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The Search Parameters page contains the following parameters:
Search Type area:
Identity
An Identity search is suited to a spectrum that is
known to have a match in one or more of the
selected search libraries.
Similarity
Select a Similarity search if you are sure the spectrum
does not have a match in the selected search libraries.
The algorithm searches for library spectra similar to
the submitted spectrum.
If you select Identity, select one of the following options:
Normal
This is the default option. Select a Normal Identity
search if the spectrum is low quality or unusual.
Quick
Click this option if the spectrum is of good quality.
Penalize Rare
Compounds
This option is effective only when you have selected
one or more of the NIST databases (such as
MAINLIB). It has no effect on spectra in user
libraries or other commercial libraries.
Each reference spectrum in a NIST library contains a
record of other commercial databases containing
information about the compound. A compound is
considered rare if it is present in a limited number of
these databases. If you click the Penalize Rare
Compounds option, hit compounds present in few,
or no other databases other than the NIST libraries,
have their match factors reduced (the maximum
penalty is 50 out of 1000). This, in effect, leads to a
relative increase in the match factors of common
compounds, placing them higher in the Hits list than
exotic isomers with near identical spectra. This
roughly adjusts for the so-called “a priori
probabilities” of finding a compound in an analysis.
If you select Similarity, select one of the following options:
Simple
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This is the default Similarity search option. Click
this option so that the algorithm finds a large set of
spectra to compare with the submitted spectrum.
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Using a Spectrum View
Hybrid
Click this option to use a combination of the Simple
and Neutral Loss search strategies. As for the neutral
loss search, an estimate of the unknown’s molecular
weight is required. If the unknown compound
contains chemical structures that generate both
characteristic ions and neutral loss patterns, these
structures can be identified from the Hits list
produced by this search.
Neutral Loss
Click this option to apply a Neutral Loss Similarity
search algorithm for library matching of spectra.
In a Neutral Loss search, Xcalibur examines the
submitted spectrum and identifies the molecular ion.
Xcalibur submits the mass value of the molecular ion
to the search along with the spectrum. The search
algorithm calculates the significant neutral losses and
compares them with library data. Hits are returned
according to matches of the molecular ion and its
neutral losses.
Options area:
Use the Options parameters to customize the search:
Search With MW=
Click this check box to restrict the search to library
entries with a particular molecular weight. Use the
associated box to specify the molecular weight.
Reverse Search
Click this check box to sort the search results by the
Reverse Search Match Factor. By default, Xcalibur
sorts results by the Forward Match Factor.
Mass Defect area:
Use the Mass Defect parameters to correct for the differences between the
actual masses and the nominal integer masses of the atoms in a molecule:
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Enable
Click this check box to include mass defect values for
library searches in a Processing Method.
Defect
Use these boxes to specify values (in millimass units)
for mass defect. Specify a smaller value for lower
mass ranges in the first box and specify a larger value
for higher mass ranges in the second box.
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Using a Spectrum View
At Mass
Exporting a Spectrum to
the Library
Use these boxes to specify the masses at which
Xcalibur applies specified mass defect values to
calculations of mass. Specify a smaller mass value in
the first box, and specify a larger mass value in the
second box.
Right-click the spectrum and choose Library > Export to Library Browser
from the shortcut menu.
The Library Browser opens with the exported file listed in the Clipboard
window. Export further spectra from Qual Browser to either overwrite or
append spectra already present in the Spec List.
Chapter 5, “Library Browser,” introduces the Library Browser.
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Using a Map View
Reviewing and Interpreting Data in Qual Browser
Using a Map View
A map is a 2D or 3D representation of an analysis showing all the
mass/wavelength scans acquired during an analysis (see Figure 108).
To view a map
• Right-click in the cell and choose View > Map from the shortcut menu.
• Or, from the menu bar, choose View > Map.
• Or, click the View Map button on the toolbar.
Figure 108. An example of a Map view, showing the Map view shortcut menu
This section contains the following topics:
• Setting the Map Ranges
• Setting Map Display Options
Setting the Map Ranges
To set the ranges for a map view in the Map Ranges dialog box (see
Figure 109), confirm that no other cell is pinned, and either:
• Choose Display > Ranges, or
• Right-click in the cell and choose Ranges from the shortcut menu.
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Using a Map View
Figure 109. Map Ranges dialog box
Set the following:
Setting Map Display
Options
Detector
Use this list to select a detector if the raw file
contains data from multiple detectors.
Mass/Wavelength
Use this box to specify the mass range for the map
plot for MS data or the wavelength range for PDA
data.
Time
Use this box to specify the time range, in the format
[lower time limit] - [Upper time limit] (no spaces).
Scan Filter
[Only used with MS data.] Select a scan filter, if
required, from the list of scan filters stored in
the .raw file, or type a new one adhering to the scan
filter format.
To change the style of a map view, confirm that no other cell is pinned, and
either:
• Right-click in the cell and choose Display Options, or
• From the menu bar choose Display > Display Options
The Display Options dialog box consists of 4 tabbed pages: Style, Color,
Axis, and Normalization (see Figure 110). It contains a small display area
showing the active cell. Use this set of options to preview the effects of
different settings before applying them.
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Figure 110. Display Options dialog box - Style page for a Map view
Style
Style page options determine the appearance of the map. The Style page is
shown in Figure 110 above.
Stack
Click this option to stack 2D plots vertically with no
overlap.
Overlay (3D)
Click this option to overlay plots vertically with
optional horizontal skew (time offset) and elevation.
Density map
Click this option to display a density map, showing
shaded intensities.
If you choose 3D overlay, select from the following 3D effects:
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Elevation
Use this parameter to set the elevation angle of the
map view between 0 and 60 degrees.
Skew
Use this parameter to set the skew angle of the map
view between 0 and 45 degrees.
Fill
Choose a fill option for the map: None, Solid Color,
Intensity shaded or Shaded With Frame.
Draw Backdrop
Click this check box to add a backdrop to 3D plots.
Clear the check box to remove the backdrop.
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Color
Use the Color page (see Figure 111) to set the color of map plots in the
various display styles.
Figure 111. Display Options dialog box - Color page for a Map view
To customize the colors of each display style component, choose any of the
following:
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Line
Click to set the color of lines used for the X-Y grid,
and for plots in an Overlay style using a None, Solid
Color or Shaded with frame fill.
Fill Solid
Click to set the color representing Overlay peaks in a
Solid Color Fill.
Backdrop
Click this button to change the color of the
backdrop (background) of a map view. The current
plot color is displayed to the right of the Backdrop
button.
Grayscale
Selecting this check box discards all color choices and
displays the map as a gray scale.
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Shade
Reviewing and Interpreting Data in Qual Browser
Using a Map View
Click any of these buttons to set the range of colors
used to represent peaks in the Density Map and
Intensity shaded Overlay styles. Xcalibur uses the
colors to represent data within the following
intensity ranges:
0%
20%
40%
60%
80%
100%
no signal
0 to 20%
20 to 40%
40 to 60%
60 to 80%
80 to 100%
When you click any of these buttons, Xcalibur opens
the Color dialog box with a color palette enabling
you to select a preset color or customize a color.
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Log Scale
Select this check box to display the color of the map
in a logarithmic scale. The factor width that you set
in the Factor box determines the scaling between
color bands.
Factor
The Factor determines the scaling between color
bands. The allowable values are 1.1 to 20. Xcalibur
makes the Factor box active when you click the Log
Scale check box.
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Using a Map View
Axis
Use the Axis page (see Figure 112) to set map axis labels and display
options.
Figure 112. Display Options dialog box - Axis page for a Map view
For the X, Y and Z-axes:
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Name
Use this box to specify the axis name: for example,
Name: X: Time, Y: Relative Abundance, Z: m/z.
Show Name
Choose one of the following axis label display
options: Never, On Print, or Always.
Offset
Click this check box to offset the axis label from the
map.
Gridlines
This check box determines whether or not to display
lines from major tic marks on the axis scale.
Split Time Range
Click this check box to split the map into two or
more separate graphs with equal time ranges.
Divisions
Use this box to specify the number of split mass
range graphs displayed for each map in the active
cell. This parameter is enabled if you click Split Time
Range.
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Reviewing and Interpreting Data in Qual Browser
Using a Map View
For the Y axis units:
Source
Use these options to specify that Xcalibur apply
either a custom (user-defined) label or a label from
the detector to the Y-axis of a map plot.
When you specify a custom label in Qual Browser,
Xcalibur retrieves the parameters from a layout (.lyt)
file. If no .lyt file exists, Xcalibur retrieves the
parameters from the default values specified on the
Xcalibur Configuration dialog box - Labeling and
Scaling page.
Units
Normalization
Use these options to apply either absolute or relative
scaling to the Y-axis of a map plot.
Use the Normalization page (see Figure 113) to select normalization (Y-axis
scaling) options for maps in the cell.
Figure 113. Display Options dialog box - Normalization page for a Map view
The Mass Grouping area contains:
Base Peak
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Click this option to have Xcalibur use the largest peak
within each band (mass range) to determine the intensity
of the band.
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Using a Map View
Sum
Click this option to have Xcalibur use the sum of the
intensities within each band (mass range) to determine
the intensity of the band.
Normalize to
Entire File
Click this check box to have Xcalibur normalize the map
to the largest peak in the raw file.
Fix Scale
Click this check box to have Xcalibur normalize the map
to a fixed intensity value. Type an intensity value
between 0.01 and 1e+20 in the Fix Scale box.
The Normalize Method area contains:
Auto Range
Click this option to have Xcalibur optimize the Y-axis
automatically for each chromatogram.
Intensity Range
Use this box to specify the minimum and maximum
intensity required for the Y-axis. The valid range is
-200.00% to +200.00%.
The Normalize Each Mass To area contains:
Largest Peak
in Subsection
Click this option to normalize to the largest peak in each
split range.
Largest Peak
in Time Range
Click this option to normalize to the largest peak in the
displayed time range.
Largest Peak
in All Times
Click this option to normalize to the largest peak in the
entire spectrum.
The Normalize Mass Plots area contains:
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Individually
Click this option to normalize each map individually.
All The Same
Click this option to normalize all maps equally.
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Using a Spectrum List
View
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum List View
A spectrum list tabulates the masses (or wavelengths) and intensities of
peaks in a spectrum (see Figure 114).
Do one of the following to view a spectrum list:
• Right-click in the cell and choose View > Spectrum list from the
shortcut menu.
• From the menu bar, choose View > Spectrum list.
• Click the View Spectrum List button on the toolbar.
Figure 114. Example of a Spectrum List view with its shortcut menu
This section contains the following topics:
• Determining Elemental Composition
• Setting Spectrum List Ranges
• Setting the Spectrum List Options
• Setting the Scan Header Range
• Setting the Scan Filter Range
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Using a Spectrum List View
Determining Elemental
Composition
To have Xcalibur calculate the “best matching” chemical formulas for the
peaks in a spectrum list, right-click the Spectrum List view and select
Elemental Composition from the shortcut menu. A spectrum list with
elemental composition is shown in Figure 115.
Use the Elemental Composition page of the Info Bar to set the parameters
that Xcalibur uses to calculate the best matching chemical formulas for the
spectrum list.
Click
in the Info Bar to display the Elemental Composition page
See Figure 99 on page 179.
Figure 115. Example of a Spectrum List view with elemental composition
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Setting Spectrum List
Ranges
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum List View
To set ranges for a Spectrum List view, confirm that no other cell is pinned,
and either:
• Choose Display > Ranges, or
• Right-click in the cell and choose Ranges from the shortcut menu.
Xcalibur displays the Spectrum List Ranges dialog box shown
in Figure 116.
Figure 116. Spectrum List Ranges dialog box
Set the following:
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Mass
Use this box to specify the mass range for the spectrum list
view.
Detector
Use this list to select a detector if the raw file contains data
from multiple detectors.
Time
Use this box to specify the time range, in the format [lower
time limit] - [Upper time limit] (no spaces).
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Using a Spectrum List View
Scan Filter
Choose a scan filter, if required, from the list of scan filters
stored in the .raw file or type your own, adhering to the
scan filter format.
The Smoothing parameters are as follows:
Enable
Click this check box to enable spectrum smoothing.
Type
Use this list to specify the type of smoothing algorithm:
Boxcar or Gaussian.
Points
Use this box to specify the number of points for spectrum
smoothing. This must be an odd number in the range 3
[minimum smoothing] to 15 [maximum smoothing].
The Refine spectrum enhancement algorithm is described in “Refine” on
page 40. The Refine parameters are as follows:
Enable
Click this check box to enable Refine enhancement.
Window Size
Use this box to specify the number of seconds either
side of the specified point over which the algorithm
uses mass chromatograms. A reasonable initial value
is the peak width in seconds.
Noise Threshold
Use this parameter to eliminate peaks generated
from baseline noise. For a first attempt, set Noise
Threshold to zero to show all peaks in the spectrum.
Increase the value until Refine eliminates all noise
peaks. For a full description of the Refine algorithm,
refer to “Refine” on page 40.
The Background Subtraction parameters are as follows:
Time Range 1
Click this check box to specify the first background
subtraction region. Type a time range in the box.
Time Range 2
Click this check box to specify a second background
subtraction region. Type a time range in the box.
The Mass Tolerance parameters are as follows:
Use User Defined
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Use this check box to specify the values for mass
tolerance and mass units for the MS data in a Qual
Browser window. To make the parameters available
and specify values, click the Use User Defined check
box. If you clear the check box, Xcalibur uses the
values for mass tolerance and units that are stored in
the raw file.
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Reviewing and Interpreting Data in Qual Browser
Using a Spectrum List View
Mass Tolerance
Use this box to specify the value for mass tolerance.
Type a value in the range from 0.1 to 50000 and
select units to apply to the value. Xcalibur uses the
tolerance value to create the limits of a range of
masses.
Units
Use these options to specify the default units that are
used in processing MS data in the Qual Browser
window.
mmu Millimass units
ppm
Parts per million
The Include Peaks parameter is as follows:
Reference and
Exception Peaks
Use this check box to include or exclude the
reference peaks (R) and exception peaks (E) for the
mass data in a Qual Browser window.
Use the Mass Precision settings to apply mass precision to the mass data in
all the cells in the Qual Browser window.
Decimals
Setting the Spectrum List
Options
Xcalibur applies the number of decimal places after
the decimal point to mass intensity values for data
processing calculations. Specify a number from
0 to 5. If you specify 3, for example, Xcalibur uses a
mass number truncated to 3 decimal places to
perform calculations. Set a default value for mass
precision in the Xcalibur Configuration dialog box Mass Options page (see Figure 83 on page 148).
Mass precision is commonly used with
high-resolution measurements.
To change the style of a Spectrum List view, confirm that no other cell is
pinned, and either:
• Right-click in the view and choose Display Options, or
• From the menu bar choose Display > Display Options.
The Display Options dialog box consists of 2 tabbed pages: Style and
Normalization (see Figure 117). It contains a small display area showing the
active cell. Use these options to preview the effects of different settings
before applying them.
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Using a Spectrum List View
Style
Use the Style page (see Figure 117) to select display style options for
spectrum lists.
Figure 117. Display Options dialog box - Style page for a Spectrum List view
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All peaks
Click this check box to display m/z, intensity, and
relative intensity for all spectrum peaks in the range
specified in the Spectrum List Ranges dialog box or
as specified in the active scan filter.
Top
Use this box to specify the maximum number of
peaks in the spectrum list. Clear the All Peaks option
and specify a maximum number of peaks in the Top
box.
Flags
Click this check box to indicate whether Xcalibur
displays letters above spectrum peaks to provide
supplemental information about the peak data. For
example, if a peak is saturated, Xcalibur displays an S
above the peak.
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Resolution
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum List View
This check box indicates whether or not Xcalibur
displays resolution information in the spectrum list.
Resolution is a Label Stream parameter and is active
if the raw file has Label Stream data. These values are
written by the instrument.
Resolution can also be active if you have centroided a
profile scan and show a result returned by the
centroider.
Charge
This check box indicates whether or not Xcalibur
displays charge information in the spectrum list.
Charge is a Label Stream parameter and is active if
the raw file has Label Stream data. These values are
written by the instrument.
Baseline
This check box indicates whether or not Xcalibur
displays baseline information in the spectrum list.
Baseline is a label stream parameter and is active if
the raw file has Label Stream data. These values are
written by the instrument.
Noise
This check box indicates whether or not Xcalibur
displays noise information in the spectrum list.
Noise is a label stream parameter and is active if the
raw file has label stream data. These values are
written by the instrument.
Centroid
Click this option to have Xcalibur apply the same
algorithm used in the firmware to convert from
profile data to centroid. When you click centroid,
both the Resolution check box and the Choose
Algorithm button become active.
Centroid and Choose Algorithm are active when you
have a profile scan.
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Choose Algorithm
Click the Choose Algorithm button to open the
Choose Centroiding Algorithm dialog box. Click the
Centroid check box to activate the Choose
Algorithm button.
Mass
Click this option to order the list by mass (m/z in
ascending order).
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Normalization
Intensity
Click this option to order the list by intensity (in
descending order).
Decimals
Use the Decimals box to specify the number of
decimal places to be used in the label.
Use the Normalization page (see Figure 118) to select normalization (Y-axis
scaling) options for spectrum lists.
Figure 118. Display Options dialog box - Normalization page for a Spectrum List
view
Intensity Range
Use this box to specify the minimum and maximum
intensity. The valid range is -200% to +200%.
Normalize each list to:
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Largest Peak
in Subsection
Click this option to normalize each split mass or
wavelength range to the largest peak in the division.
Largest Peak
in Range
Click this option to normalize to the largest peak in
the selected mass or wavelength range.
Largest Peak
in Scan
Click this option to normalize to the largest peak in
the entire scan.
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Composition
Reviewing and Interpreting Data in Qual Browser
Using a Spectrum List View
Use the Composition page of the Display Options dialog box to calculate
elemental compositions and to add columns containing the results to the
spectrum list. Xcalibur determines which chemical formulas have a m/z
value most like that of the experimental spectrum peaks. Xcalibur displays
the results of the current settings in the graphic on the right side of the page.
Figure 119. Display Options dialog box - Spectrum Composition page for a
Spectrum List
Label the mass peaks with:
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Elemental
Composition
Xcalibur determines which chemical formulas have a
m/z value most like that of the spectrum peaks. This
check box determines whether or not Xcalibur
displays the chemical formula labels at the top of
spectrum peaks.
Formulae
Enter in this box how many of the most likely
chemical formulas you want Xcalibur to display at
the top of spectrum peaks.
Theoretical Mass
Click the Theoretical Mass check box to display
the theoretical m/z of the chemical formulas that
Xcalibur determines. Xcalibur displays the
theoretical m/z to the right of the formula separated
by =.
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Using a Spectrum List View
Ring and Double
Bond Equivalents
Click the Ring and DB Equiv. check box to display
the value of the ring and double bond equivalents
that Xcalibur calculates for the chemical formulas.
Xcalibur displays the ring and double bond
equivalent value under the chemical formula.
Ring and double bond equivalents is a measure of
the number of unsaturated bonds in a
compound—and limits the calculated formulas to
only those that make sense chemically.
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Delta
Click the Delta check box to have Xcalibur label the
peak with the difference between the theoretical and
experimental m/z.
Delta Units
These options specify the units to use when
calculating the difference between the theoretical
and experimental m/z. The options are amu, mmu,
and ppm.
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Using a Scan Header
View
Reviewing and Interpreting Data in Qual Browser
Using a Scan Header View
A scan header contains the following basic information about a scan: total
ion current, base peak intensity, base peak mass, and scan mode (see
Figure 120).
To view a scan header
• Right-click in the cell and choose View > Scan Header from the
shortcut menu, or
• From the menu bar, choose View > Scan Header, or
• Click the View Scan Header button on the toolbar.
Figure 120. Example of a Scan Header view
Setting the Scan Header
Range
To set the range for a Scan Header view, confirm that no other cell is
pinned, and either:
• Choose Display > Ranges, or
• Right-click in the cell and choose Ranges from the shortcut menu.
In the Ranges dialog box, set the time of the scan.
To set the time of the displayed scan header interactively, click in a
Chromatogram view at the required time or scan number.
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Using a Scan Filter View
Using a Scan Filter
View
A Scan Filter view lists the filters applied to a chromatogram over a specified
time range (see Figure 121).
To view a scan filter list
• Right-click in the cell and choose View > Scan Filters from the shortcut
menu, or
• From the menu bar, choose View > Scan Filters, or
• Click the View Scan Filter button on the toolbar.
Figure 121. Example of a Scan Filter view
Setting the Scan Filter
Range
To set the range for a Scan Filter view, confirm that no other cell is pinned,
and either:
• Choose Display > Ranges, or
• Right-click in the cell and choose Ranges from the shortcut menu.
In the Ranges dialog box, set the time or time range for which you require
filter information.
To list the filters applying to a chromatogram, click in a Chromatogram
view at the required time or scan number.
To examine the filters applying to a time range, drag a line across the
appropriate chromatogram time range.
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Using a Report View
Reviewing and Interpreting Data in Qual Browser
Using a Report View
A number of Report views are available:
Tune Method
Instrument Method
Sample Information (see Figure 122)
Status Log
Error Log
To view a report
• Right-click in the cell and choose View > Report from the
shortcut menu, or
• From the menu bar, choose View > Report.
Then, choose the required report from the list, or click the appropriate
report view button in the main toolbar.
Figure 122. Example of a Sample Information view
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Preparing for Presentation
Preparing for
Presentation
Use Xcalibur to enhance plots (see Figure 123) by:
• Amplifying low intensity regions
• Adding text for labels
• Adding graphics (such as arrows or chemical symbols)
Output the plots by:
• Printing
• Copying to the clipboard for pasting into other applications
Figure 123. Section of a Chromatogram plot showing the use of Amplify, and
text and graphic annotation
This section contains the following topics:
•
•
•
•
•
•
•
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Using Amplify
Adding Text
Adding Graphics
Removing Text and Graphics
Editing the Heading
Printing Cells
Copying Cells to the Clipboard
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Using Amplify
Amplifying a Region of a Plot
Reviewing and Interpreting Data in Qual Browser
Preparing for Presentation
Use the Amplify commands and toolbar functions to optimize the plot
display for presentation purposes.
Use Qual Browser’s Amplify command to adjust the normalization in
specific sections of a chromatogram, spectrum or map plot. Use the
following procedure to amplify regions of a graph:
1. Select the amplification factor required from the list in the Amplify
toolbar. Qual Browser features x2, x5, x10 and x50 amplification
factors. To enter any other value (between 1.1 and 1000):
• Type directly into the Amplification Factor box on the toolbar, or
• Choose Display > Amplify > Other Factor. Enter the new factor.
2. Click the Amplify button on the toolbar.
3. Select the region to amplify. Click and drag the cursor horizontally over
the region to amplify.
Xcalibur amplifies the region and places a label above it showing the factor
that was used.
Removing Amplification
To remove all or specific amplified regions, use Xcalibur toolbar buttons or
menu commands.
To remove a specific amplified region
1. Click the Cancel Amplified Region button in the toolbar. Xcalibur
changes the cursor as shown.
2. Drag the cursor across the amplified region(s) to cancel. Xcalibur
removes the amplification, removes the amplification labels and displays
the original cursor.
To turn off the cursor before using it, click the Cancel Amplified
Region button in the toolbar.
To remove all amplified regions
Choose Display > Amplify > Clear All.
Xcalibur removes all amplification regions, removes the amplification labels
and displays the original cursor.
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Preparing for Presentation
Adding Text
To add text to a Chromatogram, Spectrum, or Map view, use Add Text.
Text orientation options are horizontal or vertical. Multiple lines of text can
be aligned to the left, center, or right. Use the cursor to place an annotation
anywhere on the view.
To add text to a view
1. Choose Display > Annotate > Add Text or click the Add Text button
on the toolbar. Xcalibur displays the Add Text dialog box (see
Figure 124).
2. Type one or more lines in the Annotation box. Use the ENTER key to
enter multiple lines.
Figure 124. Add Text dialog box
3. Choose from the following options:
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Marked Position Is
Select Left, Center or Right alignment of text
relative to the cursor marked position.
Multiple Lines
Aligned
Select Left, Center or Right justification.
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Height Drawn
This area contains options to align the text when it
appears on the view:
Just Above Graph
Click this option to display the text slightly above
the nearest peak.
Above Marked
Position
Click this option to display the text just above the
point where you position the cursor on the plot.
Below Marked
Position
Click this option to display the text just below the
point where you position the cursor on the plot.
4. Click OK.
5. Place the cursor at the position of the view where you want the
annotation text to appear and click the plot.
Xcalibur adds the text and changes the cursor back to the default arrow.
Note You cannot move the text. To reposition the annotation,
immediately use the Edit > Undo command or click the Undo button in
the toolbar to remove the text. Then, repeat the procedure. The Add Text
dialog box contains the previous text and settings.
Adding Graphics
To add simple graphics to a view, use Add Graphics. Graphics include
horizontal lines, vertical lines, diagonal lines, boxes, and filled boxes. Use
this option also to select the color of all added lines and fills. Filled boxes
can appear either behind a view or in front of a view.
To add graphics to a view
1. Choose Display > Annotate > Add Graphics or click the Add
Graphics button on the toolbar.
2. Select the options as required in the Add Graphics dialog box (see
Figure 125).
a. Select a graphic style:
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Horizontal line
Click this option to draw a horizontal line.
Vertical Line
Click this option to draw a vertical line.
Diagonal Line
Click this option to draw a diagonal line.
Box
Click this option to draw an unfilled box.
Filled Box
Click this option to draw a filled box.
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b. Select the display colors for the graphic:
Line
Click this button to change line color.
Fill
Click this button to change fill color.
When you click one of these buttons, Xcalibur opens the Color
dialog box with a color palette where you can select a preset color or
customize a color.
c. Select the position for the graphic:
Behind Graph
Click this check box to position the graphic
behind the plot. Clear the check box to
position the graphic on top of the plot.
3. Draw the graphic:
• To draw a line, click and drag the cursor on the graph.
• To draw a box, click to start at any corner of the box and drag the
cursor to the opposite corner.
Note You cannot move a graphic. To reposition it, immediately choose
Edit > Undo or click the Undo button in the toolbar. Then, repeat the
positioning step.
Figure 125. Add Graphics dialog box
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Removing Text and
Graphics
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Preparing for Presentation
To remove a single text or graphic item
1. Choose Display > Annotate > Clear or click the Clear Annotation in
Range button on the toolbar.
2. Click and drag the cursor horizontally above or below the item.
To remove all text and graphic items
Choose Display > Annotate > Clear All or click the Clear All Annotation
button on the toolbar.
To reverse the action, choose Edit > Undo or click the Undo button in the
toolbar.
Editing the Heading
Edit the heading of a raw data graphical view by using the Heading Editor
dialog box shown in Figure 126.
Figure 126. Heading Editor dialog box
The Heading Editor dialog box contains three pairs of columns (Label1,
Value1; Label2, Value2; and Label3, Value3). Each column can have up to
30 rows. Add information to any Value cell by clicking in that cell and
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selecting the desired information from the drop-down list that appears.
Manually add text to any of the cells in any Label column. The program can
automatically add text to any Label cell that has a value in the corresponding
Value cell, and this text describes the information in the Value cell.
The first character of the text or information in a particular column in the
Heading Editor appears in the same user-defined horizontal position in the
heading. Select the color to be used for the text or information in the
heading. However, all text in the Label columns is a single, user-selected
color and all information in the Value columns is also a single, user-selected
color.
To add text to a raw data graphical view heading
1. Confirm that raw data is being displayed in the graphical mode in Qual
Browser and that one cell is above another cell. Right-click anywhere in
the upper cell to display a shortcut menu. Choose Heading Editor to
display the Heading Editor dialog box.
2. Enter the desired information in cells in columns Value1, Value2, and
Value3 as follows: Click in a desired cell to open a drop down list. Select
the desired information from the list. For a detailed explanation of each
item in the Value columns, refer to the Xcalibur online Help.
3. Enter the desired text in cells in columns Label1, Label2, and Label3.
Manually type any text in any Label cell. If a Value cell contains
information, type an asterisk in the corresponding Label cell and the
program automatically enters text that describes the information in the
corresponding Value cell.
4. Click Set Label Color to open the Set Label Color dialog box. Select
the desired color for text in the Label columns. Click OK to close the
Select Label Color dialog box.
5. Click Set Value Color to open the Set Value Color dialog box. Select
the desired color for text in the Value columns. Click OK to close the
Select Value Color dialog box.
6. Set the horizontal position of text in the Label columns of the heading
of the raw data graphical display as follows: Type a number in the
appropriate box in the Column Position Editor area. The first character
of the text in cells in a particular column appears in the same horizontal
position in the heading.
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7. The program can automatically set the horizontal position of
information in the Value columns of the heading of the raw data
graphical display as follows: Check the Auto Value Position check box.
Manually set the horizontal position of information in the Value
columns of the heading of the raw data graphical display as follows:
Clear the Auto Value Position check box. Type a number in the
appropriate box in the Column Position Editor area. In this case, the
first character of the information in cells in a particular column appears
in the same horizontal position in the heading.
8. Click OK in the Heading Editor dialog box. The text and information
are inserted into the heading of the raw data graphical display and the
Heading Editor dialog box closes. Any previously inserted heading text
is replaced.
Printing Cells
Choose File > Print or click the Print button on the toolbar to display the
Print dialog box shown in Figure 127.
Choose File > Print Preview to preview the output before printing.
Figure 127. The Print dialog box
The Print and Print Preview commands open the Print dialog box. Specify
what to print and how to print it:
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Print What
All cells in the selected window or the selected
cell only.
Print How
One page, or each cell on a separate page.
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Copying Cells to the
Clipboard
Copy the active cell to the clipboard using two Edit menu commands:
Copy Cell
The Copy Cell command copies the active cell to the
clipboard as it appears on screen.
Copy Special
Use the Copy Special command to scale the output
size of the clipboard image from the current cell or
window using the Copy To Clipboard dialog box
(see Figure 128).
Figure 128. The Copy To Clipboard dialog box
The Copy To Clipboard dialog box has the following parameters:
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Copy Area
Use these settings to choose whether a single
cell or the entire grid is copied to the clipboard.
Current
Cell
Click this option to copy the currently active
cell.
Grid
Click this option to copy the active window.
Output Size Area
Use these settings to choose the dimensions of
the copied cell or grid.
Width
Width of clipboard image (in mm or in.)
Height
Height of clipboard image (in mm or in.)
Millimeters
Click to scale image in mm.
Inches
Click to scale image in inches.
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Tool Menu Utilities
Reviewing and Interpreting Data in Qual Browser
Tool Menu Utilities
Qual Browser is equipped with a Background Subtract utility to enhance
chromatograms and spectra. Also use the Add Tools option to add other
utilities to the Tools menu.
This section contains the following topics:
• Background Subtract Utility
• Adding Programs to the Tools Menu
Background Subtract
Utility
Use the Subtract Background dialog box to subtract a raw file, or a single
scan from a raw file, from any other specified raw file (see Figure 129). Use
this utility to subtract background effects or deconvolute merged or
superimposed component peaks.
Figure 129. Subtract Background dialog box
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Tool Menu Utilities
To open the Subtract Background dialog box, choose Tools > Background
Subtract.
The dialog box is divided into four areas: Input, Background, Scope, and
Output. The Input settings identify the raw file to be enhanced by the
utility.
The Input area contains the following parameters:
File
Type the pathname of the input raw file. Change the
source of the input file in one of the following ways:
• Click the Browse button adjacent to the box and
browse to the required file.
• Type the full path and filename of the required
file into the box.
Scan Filter
Click the scan filter to be applied to the input file.
Click the arrow on the Filter combo box to display
filter options that are stored in the .raw file, or use
the scan filter format to type a scan filter.
All Detectors
Click this check box to use all detector data sources
to produce the input chromatogram. The check box
is not selected for single source raw files.
Single Detector
Use this list to select a single data source for the
input chromatogram from the chosen raw file. The
box lists the detector sources recorded in the raw file.
The Background and Scope settings identify the raw file containing
background data to be subtracted from the Input file and define the
subtraction method.
The Background area contains the following parameters:
File
Type the pathname of the raw file to be subtracted
from the input file. Change the source of the
background file in one of the following ways:
• Click the Browse button adjacent to the box and
browse to the required file.
• Type the full path and filename of the required
file into the box.
Subtract Whole File Click this option for the whole of the background
file to be subtracted from the input file.
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Tool Menu Utilities
Subtract Single Scan Click this option to specify that a single scan from
(RT)
the background file is to be subtracted from each
scan of the input file. Type the number of the scan in
the adjacent box.
Alignment Offset
(RT)
Type a value in this box to offset the background
subtraction. Type the time, in seconds, that the
subtraction is to be offset.
Scaling Factor
Type a value in this box to scale the subtract
background file operation. Type the factor you want
to apply to the background file prior to its
subtraction from the input file.
The Output settings determine the name and folder to be used for storage
of the raw file resulting from the background subtraction procedure.
The Output area contains the following parameters:
Name
This read only box displays the filename for the
output file resulting from the subtraction of the
background file from the input file. Xcalibur uses the
input filename with the prefix BG_.
Folder
This box displays the folder where the output file is
stored after the subtract background file operation.
Change the folder in one of the following ways:
• Click the Folder button adjacent to the box and
browse to the required folder.
• Type the full path of the folder into the box.
The Subtract Background dialog box features the following buttons:
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Proceed
Click this button to start the Subtract Background
file operation using the settings in the dialog box.
Exit
Click this button to exit the dialog box and stop the
Subtract Background file operation.
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Tool Menu Utilities
Adding Programs to the
Tools Menu
To add application programs to the Qual Browser Tool menu:
1. Choose Tools > Add Tools.
2. Click Add in the Add Programs to Tool Menu dialog box to open the
Add Programs to Tool Menu dialog box shown in Figure 130.
Figure 130. Add Programs To Tool Menu dialog box
3. Click Browse to select the path and file name of the tool, or type the
path and file name of the tool you want to add in the Program box.
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Tool Menu Utilities
4. Click OK to store the path and file name of the tool and close the Add
Tool dialog box.
5. Edit the tool menu entries back in the Add Programs to Tool Menu
dialog box. The dialog box displays the file name in Menu Contents and
Menu Text, the path and file name in the Programs box, and the
directory path in the Initial Directory box. Change:
• The command name of a tool listed in the Menu Contents box.
• The Sequence tool in the Tool menu. Select the tool in the Menu
Contents box and click the Move Up or Move Down button.
To remove tool menu entries, select the tool in the Menu Contents box and
click Remove.
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Chapter 5
Library Browser
This chapter gives a brief overview of Library Browser, describes how to use
data exported from Qual Browser or Processing Setup, and describes how to
build and maintain a User library.
The sections in this chapter are as follows:
• About Library Browser
• Exporting Spectra from Xcaliburr
• Library Browser Windows
• Customizing a Search
• Managing User Libraries
• Automated Compound Identification Using AMDIS
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Library Browser
About Library Browser
About Library
Browser
Xcalibur uses the NIST (National Institute of Standards and Technology)
Mass Spectral Search Program and Library System. This system contains:
• The 2000 version of the NIST/EPA/NIH Mass Spectral Library
containing over 147000 compounds (optional)
• The NIST/EPA/NIH Mass Spectral Selected Replicates Library of
27750 spectra (optional)
• A default User library, NISTDEMO
Create User libraries using spectra:
• Exported from Qual Browser
• Exported automatically by a Processing Method
• From other sources including text files
The NIST libraries give access to:
• An extensive collection of chemical names
• Chemical Abstracts Service (CAS) registry numbers
• Molecular formulas and weights
• Chemical structures
The program permits searches in many different ways:
• Finding reference spectra most closely matching a submitted spectrum
• Locating spectra or compounds having certain specified characteristics
(for example, the abundance of certain peaks)
• Displaying the mass spectra of selected molecules
This chapter gives a brief overview of the Library Browser, and describes
how to:
• Use data exported from Qual Browser
• Build and use a User library
It is beyond the scope of this manual to describe all the features and
functions of the Library Browser. For detailed information, consult the
Library Browser online Help.
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5 Library Browser
Exporting Spectra from Xcalibur
Exporting Spectra
from Xcalibur
To export spectra from Xcalibur to the Library Browser, use either of the
following methods:
• Manually from Qual Browser
• Automatically from the Processing Method (refer to “Adding Spectra to
a User Library” on page 51 for more information)
Manually from Qual
Browser
If you use Qual Browser’s Library > Export to Library Browser command,
Xcalibur creates:
• A copy of the exported spectrum in the Xcalibur\Libspecs folder. The
file is given an .msd extension with the same filename as the original
data file, although this is truncated to eight characters (8.3 filename
format).
• An index file containing details about all exported spectra.
If you select the Export To Library Browser command, Xcalibur opens the
Library Browser automatically. Open the Library Browser from the Home
Page or by choosing GoTo > Library Browser.
Xcalibur places the exported spectrum into the Spec List (Spectrum List),
and the Library Browser immediately searches the selected libraries for
matching spectra if the Automation option in the Library Search Property
dialog is checked. To display the Library Search Property dialog, choose
Tools > Search Options. To display the Automation option check box,
choose either the Search tab or the Automation tab. See “Library Search
Options Dialog” on page 251.
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Library Browser
Exporting Spectra from Xcalibur
Automatically from the
Processing Method
The Processing Method contains the Append to User Library option to
export a spectrum directly into a User library. If the specified library does
not exist, Xcalibur creates it.
When a spectrum is added to a User library, it contains the following basic
information:
• The complete mass/intensity list
• A name, in the format:
<filename>#<scannumber> RT: <rt> AV: <av> NL: <nl>,
for example: steroids02#1 RT: 0.01 AV: 1 NL: 2.14E3
• A comment showing the Scan Filter, such as:
T: + c Full ms2 363.30 [150.00 - 375.00]
The Mol Weight and CAS Number fields are set to zero.
You might want to modify these entries and provide:
• A chemical formula
• A list of synonyms
• The molecular weight
• The CAS number
• A chemical structure (in MOL, SDF or MDL file format)
To edit a library entry, refer to “Editing a User Library Entry” on page 257.
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Library Browser Windows
Library Browser
Windows
The Library Browser contains five main windows:
• Library Search Window
• Other Search Window
• Names Window
• Compare Window
• Librarian Window
A description of each of these windows follows. Further information can be
found in the Library Browser online Help.
To display a specific window, click the appropriate tab at the bottom of
the screen.
Library Search Window
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The Library Search window of the Library Browser (see Figure 131) features
eight panes:
Spec List
A “scratch pad” for storage of imported spectra and a
general repository
Histogram
A graphical display of the number of matches as a
function of the match factor. Clicking on a bar
changes the information displayed in the other
windows
Hit List
The set of data found by the search
Plot of Search
Spectrum
The plot of the unknown spectrum being
investigated
Text of Search
Spectrum
Text about the unknown spectrum being investigated
Compare Result
A graphical display of peaks from a first spectrum, a
second spectrum, and the difference between the two
Plot of Hit
A graphical display of the spectrum of a hit
Hit Text
Information
Text concerning an item from the search results
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Library Browser
Library Browser Windows
Figure 131. Library Browser - Library Search window
Using the Spec List
The Library Browser stores spectra in the Spec List pane of the Library
Search window when you do either of the following:
• Export spectra from Qual Browser
• Import spectra from other sources using the File > Open command
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Library Browser Windows
The Browser identifies the origin of each Spec List entry with an initial
letter:
(T)
Spectra that came directly from a text file such as that exported
by Qual Browser
(E)
Spectra created with the Edit or New buttons in the User Library
Manager window
(M)
Spectra from the NIST/EPA/NIH Main library
(R)
Spectra from the NIST/EPA/NIH Replicates library
(U)
Spectra from a User library
To submit a Spec List spectrum to a Library search
1. Set the Library Search options. See “Customizing a Search” on
page 251.
2. Double-click the spectrum title.
The Browser displays all library matches in the Hit List pane (see below).
Comparing a Spec List Spectrum
in the Compare Pane
To transfer a Spec List entry to the Compare pane in the Library Search
window, click the entry.
To transfer a Spec List entry to the Compare window, double-click the
entry. See “Displaying Spectra in the Compare Window” on page 249.
Using the Hit List
The Hit List displays in the Library Search window. Each search result is
listed with its order number, forward and reverse matching factors, and a
library identifier:
• (M) for the Main NIST library
• (R) for the Replicates library
• (U) for a User library
Click a name in the Hit List to display its spectrum and structure in the Plot
pane and to display text describing the hit in the Text pane. The spectrum
also appears in the Compare pane.
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Library Browser
Library Browser Windows
The Plot and Text Panes
The Plot panes display the following:
• The full spectrum or expanded views of a selected m/z range
• The compound structure (if available)
The Text panes display the following:
• Compound Information – name, formula, molecular weight, CAS
number, NIST number, ID number, and search library
• The ten largest peaks in the mass spectrum
• Synonyms or alternate names used in the library
Other Search Window
The Other Search window of the Library Browser can carry out searches
based on the following:
• Formula
• ID number
• Molecular weight
• Any peaks
• CAS Registry number
• Sequential method
• NIST Library number
Launch these searches by selecting the appropriate option from the Search
Type list in the top left corner of the window or use the appropriate
command from the Search menu. Consult the Library Browser online Help
for more information.
Names Window
Use the Names window to search a NIST library for a compound name or a
text fragment. Type a search name into the box in the top left corner of the
window. The Browser lists potential matches to the text as you type.
The default name search mode accepts both alphabetic (a–z) and numeric
(0–9) characters. Punctuation and spacing are ignored (for example,
1-butene is equivalent to 1butene). Greek characters must be spelled out (for
example, alpha).
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Library Browser Windows
With the a to z only, no prefix option enabled, the Browser accepts only
alphabetic characters and ignores common prefixes (for example, cis, trans,
dl, di, tri,), numbers and Greek characters. In this mode, entering butene
finds 1-butene and cis 2-butene.
Displaying the Spectrum from a
Name Search
To display the details of a name search
1. Carry out the search as described above. Xcalibur displays spectrum,
structure, and text information in panes in the Name Search window.
2. Right-click an entry. Choose Library Search from the shortcut menu.
The Browser loads details of the compound into the Compare window and
the Plot, Text, Compare, and Hit List panes of the Library Search window.
Compare Window
The Compare window is used for the comparison of spectra only—it has no
searching capabilities. It supports comparison of any set of spectra and,
unlike the Library Search Window, it can show multiple spectra. There are
three panes in the Compare window. The top pane is a plot of the spectral
display. The middle pane is a standard Difference Display pane. The
bottom pane is a pane for the display of multiple spectra from the Hit List.
The Compare Window uses the standard Difference Display options:
Difference, Head to Tail, Side by Side, and Subtraction. The Difference
option shows peaks from the first (often upper) spectrum and second (often
lower) spectrum along with the difference. The Head to Tail option shows
only the two spectra being compared. The first spectrum points upward and
the second spectrum points downward from a common axis. The Side by
Side option shows only the two spectra being compared. Both spectra point
upwards from a common axis. The Subtraction option creates a spectrum
derived by subtracting the second spectrum from the first.
Displaying Spectra in the
Compare Window
To add spectra to the top pane
• Double-click an entry in the Spec List of the Library Search window.
or
• Select an entry in either the Spec List or the Hit List of the Library
Search window. Right-click the entry and choose Copy from the
shortcut menu. Go to the Compare window and use the Paste
command on the shortcut menu to paste the desired spectra in the top
pane.
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5
Library Browser
Library Browser Windows
Formatting the Compare Window
Change the number of spectra displayed in the lowest pane by right-clicking
in the pane and choosing Properties from the shortcut menu. This displays
the Compare Tool Properties dialog. Choose the Comp. List tab and specify
the desired number of spectra in the Spectra Per Page box.
Librarian Window
The Librarian window is the Library Browser User Library manager. Use it
to create User Libraries and to add spectrum files and compound
information to them. Also use it to create, edit, and save mass spectra and
compound identification information as text files.
The Librarian window is divided into three panes:
• A Spec List showing the names of all files currently being considered
• A Plot pane showing the spectrum of the file selected in the Spec List
• A Text pane displaying text concerning the file selected in the Spec List
Buttons available in the Librarian toolbar include:
Delete
Removes a spectrum from the list and its associated
library (if it has one) without saving it anywhere.
Add
Adds the highlighted spectrum on the list to a
designated User library.
Move
Removes the highlighted spectrum from the list and
puts it into a designated User library.
DelLib
Deletes an entire User library.
Clicking on the Add, Move, and DelLib buttons opens a dialog listing the
available user libraries. Select the library required for the command. When
adding or moving a spectrum, type a new User library name, if required.
The dialog also features buttons for managing individual library entries:
250
New
Create a new library entry.
Edit
Change a current library entry in the Spectrum
Information dialog.
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5 Library Browser
Customizing a Search
Customizing a Search
The Library Browser automatically loads all exported spectra into the Spec
List window.
Initiate a library search by double-clicking on the appropriate entry. The
search uses the settings defined in the Library Search Options dialog (see
Figure 132).
Figure 132. Library Search Options dialog
Library Search Options
Dialog
The Library Search Property dialog contains five pages that allow you to
define search and library parameters. See the Library Browser online Help to
find more detailed information on each of the pages.
To display the Library Search Options dialog
Choose Tools > Search Options.
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Library Browser
Customizing a Search
Search Page
Libraries Page
Automation Page
Use the Search page to define search parameters. The Identity and Similarity
algorithms and other search options have already been described in “Library
Search Options” on page 47. The Other Options area contains the
following check boxes:
Automation
Indicates whether automatic library searching is
enabled.
AutoReport
Prints reports of the results of library searching with
user spectrum.
Apply Limits
Sets limits of m/z and peak abundance for spectral
comparison.
Use Constraints
Sets additional constraints on the search.
Use the Libraries page to specify the libraries used and the search order for
them. It is possible to keep different lists of active libraries for each of the
search modes.
The Automation page (see Figure 133) features the following:
Number of Hits to Set the number of spectra to be printed per page. The
Print
default value of 2 prints the unknown and the top
two matches on a single sheet of paper.
252
Include Spectrum
Plot In Report
Click these options to include spectrum, text of
intensities and masses in report.
Draw Structure in
Plots
Click this option to add a molecular structure to the
spectrum plot, if this is available.
Apply Maximum
Spectrum Length
Provide an upper mass limit for printed spectrum.
Return Focus to
Caller upon
Completion
Used in the context of an instrument control
program calling the search program.
Automatic
Search On
Click this check box for the Library Browser to carry
out a library search automatically when a spectrum is
exported from Qual Browser.
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5 Library Browser
Customizing a Search
Figure 133. Automation page of Library Search Options dialog
Limits Page
The Limits page lets you set limitations for the library search, including the
minimum peak abundance and the m/z range to be used for spectra
comparison.
Constraints Page
Use the Constraints page to define additional constraints on the search. For
example, limit the search to only compounds with names containing
specified letters (such as “ol” for alcohols), or specify that only certain
elements can be present in the compound.
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Library Browser
Customizing a Search
Including a User Library in
a Search
The default library for a search is the NIST/EPA/NIH Main Library
(mainlib), if it is present. Otherwise, the default is a demonstration User
library, nistdemo. You can add other libraries to the search and change the
order of searching on the Libraries page of the Library Search Options
dialog (see Figure 134).
Figure 134. Libraries page of the Library Search Options dialog
To access this page, click the Libraries tab of the Library Search Options
dialog. The Libraries page is divided into two panes:
254
Available Libs
Lists libraries defined within the Library Browser.
Include Libs
Lists the libraries used by the Library Browser in
searches, in the search order.
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5 Library Browser
Customizing a Search
To include a library in the search
1. Select the library name in the Available Libs list.
2. Click Add.
To exclude a library from a search
1. Select the library name in the Included Libs list.
2. Click the Delete button (large red X).
Order included libraries using the up and down arrows.
Limiting a Library Search
To examine or modify search constraints, click the Constraints tab of the
Library Search Options dialog.
Constraints are divided into six categories:
• Molecular Weight
• Name Fragment
• Elements Value
• Elements Present
• Peaks
• Other Databases
Consult the online Help for more information about the use of constraints.
Most of the constraints are identical to those available within a qualitative
Processing Method. See Chapter 2: Creating a Processing Method for
Qualitative Analysis.
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5
Library Browser
Managing User Libraries
Managing User
Libraries
Library Browser can create, manage and search User libraries. Use these
abilities to create reference libraries from your own spectra.
To create a User library
1. Export a spectrum from Qual Browser to start the new User library.
2. Choose Window > Librarian.
3. In the NIST MS Search 2.0 Librarian window (see Figure 135), select a
spectrum.
Figure 135. NIST MS Search 2.0 - Librarian window
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5 Library Browser
Managing User Libraries
4. Click Add in the Librarian window toolbar. The Choose Library To
Copy To dialog opens.
5. Type the name of the new User library.
6. Click OK.
The Browser creates the new User library and closes the dialog.
Editing a User Library
Entry
To edit a User library entry
1. Confirm that the library entry is in the Spec List.
If you have exported the spectrum from Qual Browser, Library Browser
automatically picks it up and loads it into the Spec List.
To put an existing library entry into the Spec List, refer to “Accessing a
User Library Entry” on page 260.
2. Choose Window > Librarian.
3. Select the library entry from the Spec List.
4. Click the Edit button to open the Spectrum Information dialog (see
Figure 136). Then, make the necessary modifications.
Use the Spectrum Information dialog to enter or to amend information
stored with a library spectrum. The dialog displays information for the
spectrum selected in the Spec List in the Librarian window.
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Library Browser
Managing User Libraries
Figure 136. Spectrum Information dialog
258
Name
Type a name for the spectrum.
Formula
Type the chemical formula, for example,
C12H8Cl6O.
Other Names
Enter synonyms for the compound.
Comments
Enter any comments about the spectrum.
Mol. Weight
NIST MS Search assigns a molecular weight based on
the formula. If no formula is available, enter the
molecular weight.
ID Number
Shows the ID number assigned to the library entry.
CAS Number
Shows the CAS number for the compound. (You
need not enter this information.)
Peaks
Shows the number of peaks in the mass list.
Add to Library
Choose this button to add the edited spectrum and
structure to an existing or new library.
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5 Library Browser
Managing User Libraries
Replace
Choose this button to replace the spectrum in the
library from which it came.
Add to List
Choose this button to add the edited spectrum to the
Spec List.
Attach Struct
Choose this button to obtain a structure from other
libraries.
Clipboard Struct
Choose this button to attach a structure from the
clipboard, if present.
To edit the mass list, use the Peaks Info area. Consult the Library Browser
online Help for information about this function.
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5
Library Browser
Managing User Libraries
Accessing a User Library
Entry
For existing Library entries, search for a library spectrum by Formula, ID
Number, Molecular Weight, Any Peaks, CAS Number, Sequential Method,
NIST Number, or Name. Use ID Search if other information is not
available, or for a spectrum exported from a Processing Method.
To search a library by ID Number
1. Choose Search > ID Number. This opens the ID Number Search
dialog (see Figure 137).
Figure 137. ID Number Search dialog
2. The Library Statistics area shows the number of available spectra. To
consider all spectra, specify a range in the ID Number box that is equal
to or larger than the ID range shown in the Library Statistics group box.
3. Select the appropriate User library in the Library drop down list.
4. Click the Search button. Xcalibur returns all entries within the specified
range.
5. To copy a spectrum to be edited into the Spec List from the Library
Search window, select the spectrum name in the Hit List, and use the
mouse to drag the selection to the Spec List window.
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5 Library Browser
Automated Compound Identification Using AMDIS
Automated Compound
Identification Using
AMDIS
The Automated Mass Spectral Deconvolution & Identification System
(AMDIS) is a computer program developed to reduce the effort involved in
identifying compounds by GC/MS while maintaining the level of reliability
associated with traditional analysis. AMDIS extracts spectra for individual
components from GC/MS data and uses these spectra to identify
compounds by matching these spectra with spectra in a reference library.
AMDIS uses an extension of the published “model peak” approach1 as the
basis for the calculations used to extract pure component spectra from
complex chromatograms. The model peak approach uses selected ion
chromatograms as models for component shape. Based on the shape,
individual mass spectral peak abundance profiles are extracted to produce a
“purified” spectrum.
AMDIS was developed at NIST with the support of the Defense Threat
Reduction Agency (DOD). NIST reports that the model peak method was
successfully used for target compound identification in a large-scale
EPA study2.
The first version of AMDIS was released September 1996, and AMDIS has
been distributed at no additional cost along with the NIST Mass Spectral
Database since January 1998. AMDIS is included with the Xcalibur
software and is also available through the NIST Internet site
(www.nist.gov/srd/).
AMDIS does the following:
• Performs an automated search of the NIST library
• Displays dual chromatographs
• Performs batch analyses
• Builds user defined libraries using either user provided GC/MS files or
the NIST Mass Spectral Database
• Provides compound class identification
• Tracks results of routine performance runs
1
Dromey, R. G; Stefi, M. J.; Reindfleisch, T. C; Duffield, A. M. Extraction of Mass Spectra Free of
Background and Neighboring Component Contributions from Gas Chromatography/Mass
Spectrometry Data. Anal. Chem. 1976 48 (9) 1368-1375.
2
Shackelford, W. M.; Cline, D. M.; Faas, L; Kurth, G. An evaluation of Automated Spectrum
Matching for Survey Identification of Wastewater Components by Gas Chromatography-Mass
Spectrometry. Analytica Chim. Acta 1983 146 25-27.
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Library Browser
Automated Compound Identification Using AMDIS
• Uses a library of mass spectra with or without retention indices to
identify compounds in a data file
AMDIS has two main windows: Results and Confirm. The Results window
contains only results and is most commonly used for routine analyses (see
Figure 138).
Figure 138. AMDIS Results window
The Results window can be thought of as being divided into three areas: the
Control panel (top left), the Results pane (top right), and the Library and
Settings pane (bottom).
The Control panel consists of the following buttons: Analyze, Help, Done,
Confirm, Print, and Load Results. You use these buttons to control the
program.
The Analyze button opens a window to select the type of analysis, the data
file to be analyzed, the target library file, and the calibration data or library.
The Help and Done buttons have the obvious functions. The Confirm
button takes you to the Confirm window. Use the Print button to print
different reports and information. Use the Load Results button to select
and display the results of a previous analysis.
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5 Library Browser
Automated Compound Identification Using AMDIS
The Results pane has four lists. They show the retention time for each
chemical identified, the name of the compound found, the component
parameters for the deconvoluted chromatographic peak, and the match
actors between the mass spectrum of the component and that of the library.
Use the Library and Settings pane to examine the library, compare the
library spectrum with the extracted spectrum, examine the settings for the
analysis, view the retention index standards and internal standards, examine
the QA/QC report or the S/N for the run, and set options for the printed
results.
The Confirm window shows the data used in identification in more detail
than does the Results window (see Figure 139).
Figure 139. AMDIS Confirm window
The Confirm window is divided into the Menu Bar/Button Bar (top),
Chromatogram (top most graphical display), Component Profile (middle
graphical display), Component/Target Information list (middle numeric
display), and Component/Target Mass Spectra (bottom graphical display).
The options available on the Menu Bar are File, Analyze, Mode, View,
Library, Options, Window, and Help. The Button Bar associated with the
Menu Bar has the buttons Run, Rescale, Info, and two arrows. The
Chromatogram display shows the ion chromatograms for each ion. The
Component Profile display shows the total ion current and the largest mass
chromatogram peaks over the region actually used in the deconvolution of
the component. The Component/Target Information list shows either
component or target data. The Component/Target Mass Spectra display
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Library Browser
Automated Compound Identification Using AMDIS
shows the raw mass spectrum (scan) and the deconvoluted spectrum at a
given retention time. Once data have been analyzed, this display also shows
the mass spectra of the component and the library.
AMDIS has many features and options not discussed here. Providing
information about these features and options is beyond the scope of this
manual. However, get a complete AMDIS manual through the NIST web
site noted above.
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Index
A
A/D card 143
Above Marked Position option button 229
Absolute peak constraint 60
Acquisition Queue 110
page 78
pausing 112
purging 113
resuming 112
Add Graphics dialog box 231
Add Programs To Tool Menu dialog box 238
Add Text dialog box 228
Add Tool dialog box 238
Adding
graphics to a Qual Browser view 229
heading to raw data graphical view in Qual Browser 231
peaks 157
plots to a view 124
spectra to a user library 51
text to a Qual Browser view 228
toolbar button to Qual Browser 120
Adductions 5
AMDIS 261
Amplify
command 227
toolbar 118
Analog 143
Annotating a plot
graphics 229
text 228
Append to user library 51, 244
Application programs 238
Apply Changes dialog box 15, 16
Applying
a layout to the active window 134
page changes in Processing Setup 15
Area label 153
Area noise factor 158
Area Noise Factor text box 31
Arranging
chromatogram plots 150
columns 82
map plots 207
spectrum plots 169
windows in Qual Browser 122
Thermo Electron Corporation
Auto Range option
Chromatogram view 156
spectrum 175
Autofilter command 148
Automated compound identification 261
Automating analysis 78
Autosampler 88
Avalon Event List 37
Avalon Event List dialog box 37
Avalon peak detection algorithm 12
Averaging, spectrum 163
Axis names
chromatogram 154, 175
map 210
spectrum 174, 177, 221
Axis page
Display Options dialog box
Chromatogram view 154
Map view 210
Spectrum view 174, 176, 221
B
Backdrop
chromatogram overlay 150
map overlay 207
spectrum overlay 169
Background subtract utility 235
Background subtraction
Left Region 43
Right Region group box 43
Spectrum List view 216
Spectrum view 178
Base File Name text box 87
Base peak 3
Base Peak label 152
Base Sample ID text box 87
Baseline Subtraction group box
below curve 145
chromatogram 145
flatten edges 145
overlay graph of fitted polynomial 146
polynomial order 145
tolerance 145
Baseline window 31, 158
Baselines 157
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Index: C
Batch Reprocess Setup dialog box 108
Below Marked Position option button 229
Boxcar smoothing
chromatogram 145
spectrum 165
spectrum list 215
Boxed (chromatogram) label 153
Boxed (spectrum) label 173
Bracket Type group box 89
C
Cancel Amplified Region command 227
Cascade command 122
Cell Information page 121, 135
Chromatogram Ranges dialog box 140
chromatograms 136
Ranges dialog box 162
spectra 136
Cell Size dialog box 131
Cell view, changing 132
Cells
active 20
adding graphics 229
adding text 228
Cell Information page 135
copy and paste 234
creating 130
defined 130
deleting 130
grid component 122
height 132
pinning 20
printing 233
removing text and graphics 231
width 131
Change Instruments in Use dialog box 105
Changing display options 23
Checking disk space 98
Chemical formulas
determining elemental composition 179
Chemical Ionization 5
Chromatogram
cursor actions 20
display options 149
trace type 26
Chromatogram command 139
Chromatogram ranges
Baseline Subtraction group box 145
Include Peaks group box 146, 166
Mass Precision group box 147, 167, 217
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Mass Tolerance group box 146, 147, 166
Smoothing group box 144
Chromatogram Ranges dialog box 140
Automatic Processing page 144
Ranges page 142
Chromatogram view
axes 154
backdrop 151
color 151
colors 151
labels 152
peak labels 152
plots, inserting and deleting 140
smoothing 145
style 149
CI 5
Clear All command 227
Clearing annotations 231
Colors
Chromatogram view 151
Map view 208
Spectrum view 170
Column Arrangement dialog 83
Columns in sequences
arranging 82
Combine option
Spectrum Enhancement page 42
Compare window 249
Compound identification, automated 261
Configuring the autosampler 88
Constrain peak width 31, 158
Constraining a library search 54, 255
Constraints 255
Copy Cell command 234
Copy Special command 234
Copy To Clipboard dialog box 234
CRC Handbook Of Data Of Organic Compounds database 56
Creating
layout in Qual Browser 133
sequences 84
Cursor actions
cells in Processing Setup 20
Qual Browser views 137
Customize Toolbar dialog box 119
Customizing
startup in Processing Setup 17
toolbar in Qual Browser 119
Cutoff threshold 46
Thermo Electron Corporation
Index: D
D
Databases 55
Deconvolution, automated 261
Delete Cells command 130
Delete Peaks command 158
Deleting
chromatogram plot 140
sequence row 93
spectrum plot 161
toolbar button in Qual Browser 120, 121
Density map 207
Details Of Selected Analysis dialog box 113
Detecting chromatogram peaks 156
Detector delay 25, 143
Detector type 25, 143, 163
Dialog boxes
Library Browser
ID Number Search 260
Library Search Property 251, 254
Spectrum Information 257
Change Instrument in Use 105
Column Arrangement 83
Disk Space 98
Export Sequence 100
Fill Down 91
Go to Line Number 94
Import Sequence 84
International 85
Match Position/Sample ID 95
New Sequence Template 86
Print Preview 98
Print Selection 98
Run Sequence 103
Select Directory 89
User Labels 83
Disk Space dialog box 98
Display Options dialog box
Chromatogram view 149
Map view 206
Spectrum List view 217
Spectrum view 168
Processing Setup
Apply Changes 15
Libraries 52
Options 23
Settings 17
Qual Browser
Add Graphics 231
Add Programs To Tool Menu 238
Add Text 228
Add Tool 238
Cell Size 131
Chromatogram Ranges 140
Copy To Clipboard 234
Customize Toolbar 119
Heading Editor 231
ICIS Peak Detection 158
Map Ranges 205
Options, in a Chromatogram view 149
Options, in a Map view 206
Options, in a Spectrum List view 217
Options, in a Spectrum view 168
Print 233
Properties 129
Sample Properties 127
Scan Filter Range 224
Scan Header Range 223
Search Properties 200
Spectrum List Ranges 215
Spectrum Ranges 162
Subtract Background 235
Toolbars 118
Queue Manager
Details of Selected Analysis 113
Sequence Setup
Batch Reprocess Setup 108
Thermo Electron Corporation
E
Editing
heading, in raw data graphical view 231
sequence 91
spectrum in a user library 257
Element constraints 57
Elemental composition
determining 179
selecting elements 184
selecting isotopes 184
spectrum lists 214
Elemental Composition page
about 179
figure 179
Elements in compound 58
Elevation angle
chromatogram overlay 150
map overlay 207
spectrum overlay 169
Enable check box
Peak Purity page 62
Enable Warnings command 16
Enhancing a spectrum 39
EPA Environmental Monitoring Methods Index database 56
Error Log report 225
European Index Of Commercial Chemical Substances database
56
Export Sequence dialog box 100
Export to NIST Library command 243
Exporting
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Index: F
report 64, 65
sequence 100
spectrum to Library Browser 243
spectrum to the Library Browser 204
F
File name 80
base 87
File Name text box 89
Fill
Map colors 208
Map style options 207
Fill Down command 91
Fill Down dialog box 91
Fine Chemical Index database 56
Fixing
spectrum plot scale 163
Flags
chromatogram labels 153
spectrum labels 171
Fragmentation patterns 5
Full Scan mode 8
G
Gaussian smoothing
chromatogram 145
spectrum list 215
Genesis Advanced Detection Options 37
Genesis Advanced Detection Options dialog box 37
Genesis peak detection algorithm 12
Go To Line Number dialog box 94
Graphics
adding to a plot 229
removing from a plot 231
Grid menu commands 130, 132
Grid toolbar buttons 130, 132
H
Heading, editing in raw data graphical view 231
Height Drawn group box 229
Height label 153
Hits list
interpreting 199
using 247
Home Page
Information view 110
Sequence Setup 79
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Hybrid similarity search 49, 203
I
ICIS Advanced Parameters 37
ICIS Advanced Parameters dialog box 37
ICIS Detection page, peak integration 30
ICIS peak detection algorithm 12
ICIS Peak Detection dialog box 158
ID Number Search dialog box 260
Identification 38
Identification Options 37
Identification page
advanced detection options 37
detector 25
detector delay 25
limit peaks 34
mass range 29
Retention Time window 30
scan filter 25
trace options 26
wavelength range 29
Identifying peaks in a chromatogram 24
Identity search options 48
Identity search parameter 201
Import Sequence dialog 84
Include Peaks group box 146, 166
Incremental Name Search window 248
Info Bar
Cell Information page 135
location 121
Peak List page 129
Sequence List page 127
Info Bar pages
cell information 121
mass spec 121
peak list 121, 121, 121
plots info 121
sequence list 121
Information view
Acquisition Queue page 110
Inj. Volume text box 90
Injection volume 81
Injections Per Sample text box 87
Insert Cells command 130
Inserting
chromatogram plot 140
sequence row 93
spectrum plot 161
Instrument method
report 225
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Index: J
sequence files 81
text box 87, 89
Intensity range
chromatogram 155
map 211
spectrum 175
International dialog 85
Interpreting data 116
Ionization 3
Isotopic distributions, simulating 186
Just Above Graph option button 229
molecular weight 55
name fragment 56
Library Search Options page
append to user library 51
figure 47
identity 48
options 49
similarity 49
Library Search Property dialog box 251, 254
Library Search window 245
Limit peaks 34
Limit Scan Wavelength check box
Peak Purity page 62
List separator character 101
Loss mass spectral peak constraint 59
L
M
Label Threshold parameter (spectrum) 173
Labels
chromatogram 152
chromatogram axes 154
map axes 210
spectrum 171
spectrum axes 174, 176, 221
Layouts
applying 134
creating 133
for new data 125
saving 134
Librarian window 250
Libraries
exporting spectra to 204
Libraries dialog box 52
Library Browser
about 242
exporting spectra to 243
GC/MS techniques 5
Library search
constraining 255
Library Browser 247
options in Library Browser 254
processing method search list 52
Qual Browser 198
selecting libraries 254
submitting spectra 198
Library Search Constraints page
databases 55
element constraints 57
elements in compound 58
figure 54
mass spectral peak constraints 59
Macros 66
Main Toolbar button 119
Map Ranges dialog box 205
Map view
axes 210
backdrop 208
color 208
colors 208
mass range 206
normalization 211
overlay 207
style 207
time range 206
using 205
Mass
chromatograms 8
parameter 29
spectrum labels 171
Mass ranges
Map view 206
plot types 143
Spectrum List view 215
split map 210
split spectrum 174, 177, 221
text box 29, 163
Mass spectral peak constraints 59
Mass Tolerance group box 146, 166
Match factor threshold 51
Match Position/Sample ID dialog box 95
Maximum hits search option 50
Maxmass mass spectral peak constraint 59
Molecular ion 3
Molecular weight
J
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Index: N
constraining a library search 55
search option 50
MS detectors 3
MSn Browser Information page
figure 191
using 191
MSn data, browsing 191
Options dialog box
Processing Setup 23
Options search parameter 201
Other Factor command 227
Other Search window 248
P
N
Name Fragment constraint 56
Names window 248
National Institute of Standards and Technology 242
Neutral loss 4
Neutral loss similarity search 49
New Sequence Template dialog box 86
NIH-NCI Inventory File database 56
NIST Browser 242
NIST library 55
NIST MS Search 242
NIST/EPA Gas Phase IR database 56
Noise Threshold text box
refine
Spectrum List view 215
Spectrum view 165
values 41
Normal Identity search option 48
Normal Mass Spectral Peak Constraint option 59
Normalization
chromatogram 155
map 211
spectrum 3, 175
spectrum list 220
Normalization page
Auto Range option 156
chromatogram view 155
Intensity Range text box 156
Number Of Samples text box 87
O
Offset (chromatogram) label 153
Offset (spectrum) label 173
Opening
raw file in Processing Setup 18
raw file in Qual Browser 124
results file in Qual Browser 128
sequence in Qual Browser 126
sequence in Sequence Setup 81, 81
Options command 200
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Xcalibur: Getting Productive with Qualitative Analysis
Pan commands 133
Path 81
Path text box 87, 89
Pausing the acquisition queue 112
PDA detector type
peak purity 61
Peak Coverage text box
Peak Purity page 62
Peak detection 156
manual 157
Peak height
constrain peak width 31, 158
Peak identification 158
Peak integration 30, 158
Peak labels
chromatogram 152
spectrum 171
Peak List page 121, 129
Peak noise factor 158
Peak Noise Factor text box 31
Peak Purity page
figure 61
Qual Browser 14
Peak Top Region text box 43
Peaks
baseline 157
deleting 158
detection 156
labeling 152, 171
Penalize Rare Compounds identity search 48, 202
Pinning 20, 122
Plot pane 248
Plot properties 163
Plots
adding 124
adding graphics 229
adding text 228
Autofilter command 148
chromatogram 140
overlaying (3D) 150
stacking (2D) 150
Chromatogram view 142
copy and paste 234
Thermo Electron Corporation
Index: Q
map 207
peak detection 156
printing 233
properties 142
range 143
removing text and graphics 231
replacing 124
scaling 133
spectrum 161
overlaying (3D) 169
stacking (2D) 169
Point-to-point peak profile
Chromatogram view 150
Spectrum view 168
Position 81, 95
Position (vial) text box 89
Print
from Qual Browser 233
methods 107
sequence 96
Print dialog box 233
Print Preview command 233
Print Preview dialog box 98
Print Selection dialog box 98
Probability factor 199
Probability threshold 51
Processing actions 107
Processing Method
exporting a spectrum 244
sequence column 81
text box in New Sequence Template 87
text box in Sequence Editor 89
Processing Setup
auto open raw file 17
buttons 15
Cancel button 15
customizing setup 17
load last processing method 17
OK button 15
Open Raw File command 18
Options dialog box 23
Programs view 66
Qual view 12
changing display options 23
chromatogram preview 13
Identification 38
Identification page 13, 24
Libraries dialog box 52
Library Search Constraints page 54
Library Search Options page 47
Spectrum Enhancement page 39
Spectrum preview 13
tabbed pages 14
Zoom commands 22
Thermo Electron Corporation
Reports view 63
Settings dialog box 17
Spectrum Enhancement page 39
window 13
Programs 106
Programs view 66
Properties dialog box 129
Q
Qual 122
Qualitative Processing option 107
Qualitative Reprocessing option 109
Qual Browser 116, 117
Chromatogram view 139
Elemental Composition page 179
Heading Editor 231
Map view 205
MSn Browser Information page 191
opening files 124
Report view 225
Scan Filter view 224
Scan Header view 223
Spectrum List view 213
Spectrum Simulation page 186, 186
Spectrum view 160
toolbar 118
Qual view
Auto Range command 22
Identification page 24
Peak Detection list box 25
Library Search Constraints page 54
Library Search Options page 47
Normalize Intensity command 22
Peak Detection menu algorithms 24
Processing Setup 13, 14
Spectrum Enhancement page 39
title bar format 14
toolbar 14
using the toolbar 22
Zoom commands 22
Qualitative analysis
about 2
initiating 107
qualitative analysis
reprocessing data 109
Quan
Quantitative Processing option 107
Quantitative analysis, initiating 107
Queue Manager window
updating the display 112
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Index: R
R
sample or sequence 102
Ranges
Chromatogram view 140
Spectrum view 162
Rank mass spectral peak constraint 59
Raw file 142
Reagent gas 5
Refine
algorithm 40, 41
Spectrum List view 215
Spectrum view 165
Region End (combine) 43
Region Width 43
Registry Of Toxic Effects Of Chemical Substances database 56
Relative ion constraint 60
Relative peak height threshold 37
Removing amplification of a plot 227
Removing programs from the Tool menu 238
Replacing plots 124
Replicate samples 88
Report view 225
Reports
exporting 64, 65
from processing method 107
sample 64
template 64, 65
Reports view 63
Reprocessing 108
Rescaling a preview display 22
Results
files, using in Qual Browser 128
Results Review 116
Resuming the acquisition queue 112
Retention Time
label 152, 152, 152
window 30
Reverse search
match factor threshold 51
option 50
Reverse Search check box 203, 203, 204
Reviewing data 116
Rotated
chromatogram label 153
spectrum label 173
Rows
deleting 93
inserting 93
RSI Matching factor 199
Run Sequence dialog box 103
Running
programs or macros 66
272
Xcalibur: Getting Productive with Qualitative Analysis
S
Sample ID text box 89
Sample Information report 225
Sample Information window 111
Sample Properties dialog box 127
Sample reports 64
Sample Type text box 89
Samples
ID 80, 95
name 81
replicate 88
running 102
type 80
unknowns 87
volume 81
weight 81
Saving
layout in Qual Browser 134
page parameters in Processing Setup 15
sequence in Sequence Setup 81
Scaling a plot 133
Scan Filter Range dialog box 224
Scan Filter view 224
Scan filters 25
Autofilter command 148
Chromatogram view 142
Map view 206
Spectrum List view 215
Scan Header Ranges dialog box 223
Scan Header view 223
Scan Number label 152
Scan Threshold text box
Peak Purity page 62
Search command 198
Search Properties dialog box
Search List page 200
Search Parameters page 201
Spectrum view 200
Search With MW
check box 203, 203
search option 50
Search, customizing 251
Select Directory dialog box 89
Select Isotopes dialog box
figure 184
using 184
Select top peaks 36
Selected Ion Monitoring 8
Thermo Electron Corporation
Index: S
Separator character 85
Sequence Editor toolbar 91
Sequence List page 121, 127
Sequence Setup view 78
Sequences
base sample ID 87
columns 80
creating automatically 86
creating manually 89
editing 91
exporting 100
importing 84
new 84
pausing 112
printing 96
priority 104
Qual Browser 126
queueing 110
resuming 112
running 103
samples 87
starting number 87
Shade display mode, Spectrum view 168
Shutdown 105
SI Matching factor 199
Signal-to-Noise label 152
SIM 9
Similarity search
Hybrid option 203
Neutral Loss option 203
Simple option 202
Similarity search options 49
Similarity search parameter 201
Simple similarity search 49
Skew angle
chromatogram overlay 150
map overlay 207
spectrum overlay 169
Smoothing
chromatogram 144
processing method 30
spectrum 164
spectrum list 215
Smoothing Points 31
Spec List
submitting a search from 247
transferring spectrum to Compare pane 247
Spectra
about 3
averaging 163
cursor actions 20
determining elemental composition 179
Thermo Electron Corporation
editing a user library entry 257
exporting to a library 204
library search 198
plots, inserting and deleting 161
simulating isotopic distributions 186
smoothing 165
Spectrum Enhancement page
refine 39
threshold 46
Spectrum Information dialog box 257
Spectrum List Ranges dialog box 215
Spectrum List view 213
normalization 220
number of masses 218
order of masses 218
smoothing 215
style 218
Spectrum lists
determining elemental composition 214
Spectrum Maximum, PDA plot type 143
Spectrum ranges
Include Peaks group box 164
Mass Precision group box 164
Mass Tolerance group box 164
Refine group box 164, 165
Smoothing group box 144, 165
Spectrum Ranges dialog box
Automatic Processing page 164
opening 162
Ranges page 163
Spectrum Simulation page
figure 186
using 186
Spectrum view
axes 174, 176, 221
backdrop 170
background subtraction 178
color 170
colors 170
customizing a library search 200
exporting to Library Browser 204
labels 171
library search 198
normalization 175
peak labels 171
search list 200
search parameters 200
style 168
using 160
Stacking
chromatogram plots 150
map plots 207
spectrum plots 169
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Index: T
Start instrument 104
Startup 105
Status Log report 225
Stick display mode
Chromatogram view 150
Spectrum view 168
Structure 4
Style
Chromatogram view 149
Map view 207
Spectrum List view 218
Spectrum view 168
Subtract Background dialog box 235
Subtract Spectra commands 178
Subtracting background spectra 178
Summary reports 65, 107
T
Tailing Factor 31
Tailing factor 31, 158
Text
adding to a plot 228
removing from a plot 231
Text Info pane 248
Thresholds, append to user library option 51
TIC 8, 143
Tile command 122
Time range 142
Map view 206
Spectrum List view 215
Spectrum List view, background subtraction 216
Spectrum view, background subtraction 164
split chromatogram 154
Toggle Peak Detection
In All Plots command 157
In This Plot command 156
Tool menu
adding/removing programs in Qual Browser 238
background subtract utility 235
Toolbar
amplify 227
dialog box 118
ToolTips display 118
Total Ion Current 8
Total wavelength scan 143
Toxic Substances Control Act Inventory database 56
Trace
analysis 9
combinations 26
type 26
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Xcalibur: Getting Productive with Qualitative Analysis
Tray type 88
Tune Method report 225
U
Updating Queue Manager 112
US Pharmacopoeia database 56
User Labels dialog 83
User libraries
appending spectra 51
creating 256
editing spectra 257
finding Xcalibur data 260
including in a search 254
maintenance 250
selecting 52
User-defined columns 81, 90
UV channel 143
V
Vial list 96, 97
Vial number 81, 88
Viewing
chromatogram 140
map 205
reports 225
scan filters 224
scan header 223
spectra 160
spectrum list 213
Views
adding graphics 229
adding text 228
Chromatogram 139
copy and paste 234
cursor actions in 137
Map 205
printing 233
removing text and graphics 231
Report 225
Scan Filters 224
Scan Header 223
Spectrum 160
Spectrum List 213
using interactively 137
W
Wavelength 29
Thermo Electron Corporation
Index: Z
Wavelength range 29, 143
Window size 41
refine
Spectrum List view 215
Spectrum view 165
Windows
arranging 122
Compare 249
Librarian 250
Library Browser 245
Library Search 245
Names 248
Other Search 248
Processing Setup 13
Qual Browser 116
Queue Manager 112
Sequence Setup 79
Z
Zoom commands 133
Thermo Electron Corporation
Xcalibur: Getting Productive with Qualitative Analysis
275
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