Waters PDA Software User Manual

Empower PDA Software
Getting Started Guide
34 Maple Street
Milford, MA 01757
71500031503, Revision A
NOTICE
The information in this document is subject to change without notice and should not be
construed as a commitment by Waters Corporation. Waters Corporation assumes no
responsibility for any errors that may appear in this document. This document is believed
to be complete and accurate at the time of publication. In no event shall Waters
Corporation be liable for incidental or consequential damages in connection with, or
arising from, the use of this document.
© 2002 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA.
ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE
REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE
PUBLISHER.
Millennium and Waters are registered trademarks, and Empower is a trademark of Waters
Corporation.
Microsoft, Windows, and Windows NT are registered trademarks of Microsoft Corporation.
All other trademarks or registered trademarks are the sole property of their respective
owners.
Table of Contents
Preface
....................................................................................... 10
Chapter 1
PDA Software Overview .................................................................. 15
1.1 What Is PDA Software? ........................................................ 15
1.2 Tutorial Overview................................................................... 17
1.3 Restoring the PDA Project .................................................... 18
Chapter 2
Viewing PDA Data ........................................................................... 23
2.1 Tutorial Overview................................................................... 23
2.2 Viewing Data in Review......................................................... 23
2.3 Displaying the 3D Plot........................................................... 29
2.4 Zooming In on Plots .............................................................. 31
2.5 Extracting a Chromatogram .................................................. 33
2.6 Extracting a Spectrum........................................................... 36
2.7 Annotating Chromatograms and Spectra.............................. 41
2.7.1 Adding Annotations.................................................... 42
2.7.2 Erasing Annotations................................................... 45
2.7.3 Annotation Tools ........................................................ 46
Chapter 3
Peak Purity Processing .................................................................... 47
3.1 Tutorial Overview................................................................... 47
3.2 Deriving Chromatograms ...................................................... 48
Table of Contents
3
3.3 Developing a PDA Processing Method ................................. 51
3.3.1 Viewing, Modifying and Saving the Method Set......... 65
3.3.2 Viewing the Peak Purity Calculation .......................... 67
3.4 Reviewing Peak Purity Results ............................................. 69
3.4.1 Using Spectrum Index ............................................... 69
3.4.2 Using the Results Window......................................... 75
Chapter 4
Library Matching .............................................................................. 80
4.1 Tutorial Overview................................................................... 80
4.1.1 Steps in Creating a Library ........................................ 80
4.1.2 Steps in Library Matching .......................................... 81
4.2 Creating a New Library ......................................................... 82
4.3 Matching Spectra to a Library ............................................... 89
4.3.1 Modifying an Existing Processing Method for Library
Matching .................................................................... 89
4.3.2 Creating a New Processing Method for Library
Matching .................................................................... 94
4.3.3 Performing Library Matching.................................... 107
4.4 Reviewing Library Matching Results ................................... 109
Table of Contents
4
Chapter 5
Printing Reports ............................................................................. 114
5.1 Previewing a Report............................................................ 114
5.2 Background Printing............................................................ 117
Appendix A
Default PDA Reports ...................................................................... 120
Index
..................................................................................... 140
Table of Contents
5
List of Figures
1-1
1-2
1-3
1-4
1-5
Empower Login Dialog Box............................................................ 18
Empower Pro Window ................................................................... 19
Configuration Manager .................................................................. 20
Restore Project Wizard - Start Software Page .............................. 21
Configuration Manager with Restored PDA_Default Project ......... 22
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
2-9
2-10
2-11
2-12
2-13
2-14
2-15
2-16
2-17
2-18
2-19
2-20
2-21
2-22
Empower Pro Window ................................................................... 24
Browse Project Dialog Box ............................................................ 24
Project Window.............................................................................. 25
Review with PDA Data, no Chromatogram Extracted .................... 26
Maximized Review Main Window in 3D Layout ............................. 27
Review Toolbar Buttons ................................................................. 28
Sample 3D Plot Window ................................................................ 30
Creating the Zoom Box.................................................................. 31
Zoomed View of the Contour Plot.................................................. 32
Scaling Tab .................................................................................... 33
Extracted Chromatogram at Wavelength 254 nm .......................... 34
Extracted Chromatogram at Wavelength 280 nm .......................... 35
Overlaid Chromatograms at Wavelengths 254 and 280 nm .......... 36
Extracted Spectrum at 1.388 Minutes............................................ 37
Overlaid Extracted Spectra ............................................................ 38
Spectra Table ................................................................................. 39
Normalized Spectra ....................................................................... 40
Viewing Spectrum Points: Absorbances Versus Wavelengths....... 41
Annotation Tools ............................................................................ 42
Annotation Object Properties Dialog Box for an Object ................. 43
Annotation Object Properties Dialog Box for Text .......................... 44
Annotation Example....................................................................... 45
List of Figures
6
3-1
3-2
3-3
3-4
3-5
3-6
3-7
3-8
3-9
3-10
3-11
3-12
3-13
3-14
3-15
3-16
3-17
3-18
3-19
3-20
3-21
3-22
3-23
3-24
3-25
3-26
3-27
3-28
3-29
3-30
3-31
Project Window of the PDA_Default Project .................................. 49
Review Main Window with a Max Plot Chromatogram .................. 50
Review Main Window with a Chromatogram at 254 nm ................ 51
Processing Method Wizard Dialog Box ......................................... 52
New Processing Method Dialog Box.............................................. 52
Integration - Peak Detection 1 Page, Full View.............................. 53
Setting the Peak Width Parameter................................................. 54
Integration - Peak Detection 2 Page .............................................. 55
Setting the Threshold Parameter ................................................... 56
Integration - Integration Region Page ............................................ 57
Integration - Peak Rejection Page ................................................. 58
Names and Retention Times Page ................................................ 59
PDA Purity/Matching Page ............................................................ 60
PDA Spectral Contrast Page.......................................................... 61
Setting the Noise Interval............................................................... 62
Processing Method Name Page .................................................... 63
Integrated Chromatogram in Review ............................................. 64
Method Set Editor Window ............................................................ 65
Method Set Tree Pane ................................................................... 66
Save Current Method Set Dialog Box ............................................ 66
Method Set Editor Window after Modifying and Saving................. 67
Purity Angle and Purity Threshold Values in the Peaks Table ....... 68
Apex Spectrum .............................................................................. 70
Apex Spectra Overlaid with Maximum Impurity Spectra................ 71
Maximum Impurity Spectrum with Peak 1 Spectra Zoomed .......... 72
Maximum Impurity Spectrum with Peak 2 Spectra Zoomed .......... 73
Maximum Impurity Spectrum with Peak 3 Spectra Zoomed .......... 74
Results Window ............................................................................. 75
Purity Plot ...................................................................................... 76
Plot Properties Dialog Box............................................................. 77
Purity Plot with Maximum Impurity Indicator.................................. 78
List of Figures
7
4-1
4-2
4-3
4-4
4-5
4-6
4-7
4-8
4-9
4-10
4-11
4-12
4-13
4-14
4-15
4-16
4-17
4-18
4-19
4-20
4-21
4-22
4-23
4-24
4-25
4-26
4-27
4-28
4-29
4-30
4-31
4-32
Steps in Library Matching .............................................................. 81
Selecting Data for a Library ........................................................... 82
Review Main Window..................................................................... 83
Open an Existing Method Set Dialog Box...................................... 84
Paraben Stds Chromatogram ........................................................ 85
Create a New Library Dialog Box .................................................. 86
Spectrum Review with Spectra Selected ....................................... 87
Add Spectrum to Library Dialog Box.............................................. 88
Project Window of the PDA_Default Project .................................. 89
Review Main Window..................................................................... 90
Integrated 254 nm Chromatogram of Mixture ................................ 91
Processing Method Window .......................................................... 92
PDA Library Search Tab ................................................................ 93
Processing Method Wizard Dialog Box ......................................... 94
New Processing Method Dialog Box.............................................. 95
Integration - Peak Detection 1 Page, Full View.............................. 96
Setting the Peak Width Parameter................................................. 97
Integration - Peak Detection 2 Page .............................................. 97
Setting the Threshold Parameter ................................................... 98
Integration - Integration Region Page ............................................ 99
Integration - Peak Rejection Page ............................................... 100
Names and Retention Times Page .............................................. 101
PDA Purity/Matching Page .......................................................... 102
PDA Spectral Contrast Page........................................................ 103
Setting the Noise Interval............................................................. 104
PDA Match Library Page ............................................................. 105
Processing Method Name Page .................................................. 106
Processed Chromatogram with Library Match Results................ 107
Integrated Chromatogram with Library Match Results ................ 108
Spectrum Index with Library Matching......................................... 109
Overlaid Library Match Spectra ................................................... 110
Results Window Displaying Library Match Plot ........................... 111
List of Figures
8
4-33
4-34
Library Match Plot and Library Match Tab ................................... 112
Triple Plot: Library Match ............................................................. 113
5-1
5-2
5-3
5-4
5-5
5-6
Project Window............................................................................ 114
Open Report Method Dialog Box................................................. 115
Preview of an Example Report .................................................... 116
Project Window............................................................................ 117
Background Processing and Reporting Dialog Box..................... 118
Sample Report............................................................................. 119
List of Figures
9
Preface
The Empower PDA Software Getting Started Guide describes the basics of how to use the
Empower™ PDA option to develop a PDA processing method for peak purity and library
matching and to review PDA results.
This guide is intended for a wide variety of users whose familiarity with computers and
software ranges from novice to expert.
Organization
This guide contains the following:
Chapter 1 describes the Empower PDA software.
Chapter 2 describes the basics of viewing PDA data.
Chapter 3 describes how to derive chromatograms and how to develop a peak purity
processing method.
Chapter 4 describes how to create a spectral library and match spectra to the library.
Chapter 5 describes the basics of printing reports.
Appendix A, Default PDA Reports shows examples of various PDA reports available in
Empower software.
Related Documentation
Waters Licenses, Warranties, and Support: Provides software license and
warranty information, describes training and extended support, and tells how Waters
handles shipments, damages, claims, and returns.
Online Documentation
Empower Help: Describes all Empower windows, menus, menu selections, and
dialog boxes for the base software and software options. Also includes reference
information and procedures for performing all tasks required to use Empower software.
Included as part of the Empower software.
Empower Read Me File: Describes product features and enhancements, helpful tips,
installation and/or configuration considerations, and changes since the previous
version.
Empower LIMS Help: Describes how to use the Empower LIMS Interface to export
results and import worklists.
10
Empower Toolkit Professional Help: Describes how to use the common-objectmodel, message-based protocol to communicate with the Empower software from a
third-party application.
Printed Documentation for Base Product
Empower Software Getting Started Guide: Provides an introduction to the Empower
software. Describes the basics of how to use Empower software to acquire data,
develop a processing method, review results, and print a report. Also covers basic
information for managing projects and configuring systems.
Empower Software Data Acquisition and Processing Theory Guide: Provides
theories pertaining to data acquisition, peak detection and integration, and quantitation
of sample components.
Empower System Installation and Configuration Guide: Describes Empower
software installation, including the stand-alone Personal workstation, Workgroup
configuration, and the Enterprise client/server system. Discusses how to configure the
computer and chromatographic instruments as part of the Empower System. Also
covers the installation, configuration, and use of acquisition servers such as the
32
LAC/E module, the busLAC/E™ card, and interface cards used to communicate with
serial instruments.
Empower System Upgrade and Configuration Guide: Describes how to add
hardware and upgrade the Empower software using an import-and-export upgrade
method.
Empower Software System Administrator’s Guide: Describes how to administer
the Empower Enterprise client/server system and Workgroup configuration.
Empower Software Release Notes: Contains last-minute information about the
product. Also provides supplementary information about specific Empower software
releases.
Printed Documentation for Software Options
Empower System Suitability Quick Reference Guide: Describes the basics of
the Empower System Suitability option and describes the equations used by the
System Suitability software.
Empower PDA Software Getting Started Guide: Describes the basics of how to
use the Empower PDA option to develop a PDA processing method and to review PDA
results.
Empower GC Software Getting Started Guide: Describes how to use the
Empower GC option to develop a GC processing method and to review GC results.
11
Empower GPC Software Getting Started Guide: Describes how to use the
Empower GPC option to develop a GPC processing method and to review GPC results.
Empower GPCV Software Getting Started Guide: Describes how to use the
Empower GPCV option to develop a GPCV processing method and to review GPCV
results.
Empower Light Scattering Software Getting Started Guide: Describes how to
use the Empower Light Scattering option to develop a light scattering processing
method and to review light scattering results.
Empower ZQ Mass Detector Software Getting Started Guide: Describes
installation, configuration, calibration, and tuning methods, as well as how to operate
the ZQ Mass Detector with Empower software.
Empower Chromatographic Pattern Matching Software Getting Started
Guide: Describes how to use the Chromatographic Pattern Matching option to develop
a pattern matching processing method and to review pattern matching results.
Empower Dissolution System Software Quick Start Guide: Describes how to
®
operate the Alliance Dissolution System using Empower software.
Empower Toolkit Programmer’s Reference Guide: Describes how to use the
common-object-model, message-based protocol to communicate with Empower
software from a third-party application.
Waters Integrity System Getting Started Guide: Describes features of the Waters
®
Integrity System and provides step-by-step tutorials that guide a user through the use
of the Empower Mass Spectrometry (MS) option.
Empower AutoArchive Software Installation and Configuration Guide:
Describes how to install and configure the Empower AutoArchive option.
Documentation on the Web
Related product information and documentation can be found on the World Wide Web.
Our address is http://www.waters.com.
Related Adobe Acrobat Reader Documentation
®
®
For detailed information about using Adobe Acrobat Reader, see the Adobe Acrobat
Reader Online Guide. This guide covers procedures such as viewing, navigating, and
printing electronic documentation from Adobe Acrobat Reader.
12
Printing This Electronic Document
Adobe Acrobat Reader lets you easily print pages, page ranges, or the entire document by
selecting File > Print. For optimum print quantity, Waters recommends that you specify a
®
PostScript printer driver for your printer. Ideally, use a printer that supports 600 dpi print
resolution.
Documentation Conventions
The following conventions can be used in this guide:
Convention
Usage
Purple
Purple text indicates user action such as keys to press, menu selections, and commands. For example, “Click Next to go to the next
page.”
Italic
Italic indicates information that you supply such as variables. It also
indicates emphasis and document titles. For example, “Replace
file_name with the actual name of your file.”
Courier
Courier indicates examples of source code and system output. For
example, “The SVRMGR> prompt appears.”
Courier Bold
Courier bold indicates characters that you type or keys you press in
examples of source code. For example, “At the LSNRCTL> prompt,
enter set password oracle to access Oracle.”
Underlined Blue
Indicates hypertext cross-references to a specific chapter, section,
subsection, or sidehead. Clicking this topic using the hand symbol
brings you to this topic within the document. Right-clicking and
selecting Go Back from the shortcut menu returns you to the originating topic. For example, “Section 4.1.2, Steps in Library Matching,
shows the steps used for library matching.”
Keys
The word key refers to a computer key on the keypad or keyboard.
Screen keys refer to the keys on the instrument located immediately below the screen. For example, “The A/B screen key on the
2414 Detector displays the selected channel.”
…
Three periods indicate that more of the same type of item can
optionally follow. For example, “You can store filename1,
filename2, … in each folder.”
>
A right arrow between menu options indicates you should choose
each option in sequence. For example, “Select File > Exit” means
you should select File from the menu bar, then select Exit from the
File menu.
13
Notes
Notes call out information that is helpful to the operator. For example:
Note: Record your result before you proceed to the next step.
Attentions
Attentions provide information about preventing damage to the system or equipment. For
example:
Attention: To avoid damaging the detector flow cell, do not touch the flow cell
STOP window.
Cautions
Cautions provide information essential to the safety of the operator. For example:
Caution: To avoid burns, turn off the lamp at least 30 minutes before removing it for
replacement or adjustment.
Caution: To avoid electrical shock and injury, turn off the detector and unplug the
power cord before performing maintenance procedures.
Caution: To avoid chemical or electrical hazards, observe safe laboratory practices
when operating the system.
14
Chapter 1
PDA Software Overview
1
This chapter describes basic features of Empower software for the PDA detector, an
overview of this tutorial, and the process for restoring sample PDA data on your
workstation or client. Once you have loaded the sample data, you can view and
manipulate the data in a variety of ways, as described in Chapter 2, Viewing PDA Data.
1.1 What Is PDA Software?
The Empower Photodiode Array (PDA) software enables you to use Empower software to
acquire and process spectral and chromatographic data. You use the PDA software with
®
the Waters 996 PDA Detector and/or the Waters 2996 PDA Detector.
Features of the PDA Software
The Empower System is a total chromatography and results management system that you
can adapt to your individual chromatography requirements. The Empower System consists
of the following components:
• A computer that runs Empower software. Basic hardware configurations are:
– Personal – A standalone workstation running Empower software
– Workgroup – A small network running Empower software with a maximum of four
computers
– Enterprise – A client/server network running Empower software
• Empower software
• Empower database
The Empower PDA software is an integrated part of Empower software. PDA data
acquisition, processing, and reporting with the PDA software requires use of the base LC
Empower software.
Features of the Base LC Empower Software
Empower software provides a graphical, icon-based user interface to acquire, process,
®
and manage chromatographic data. Empower software runs on Windows 2000
Professional and Windows XP Professional operating systems. Empower software also
supports multitasking operations, providing you with the ability to have multiple windows
What Is PDA Software?
15
open at the same time. You can view a real-time data acquisition run while simultaneously
producing summary results of previously acquired data.
The base LC Empower software provides tools for:
1
• Creating projects
• Configuring chromatographic systems
• Developing instrument methods to control chromatographic systems
• Acquiring data from samples and standards
• Developing a processing method to perform integration, calibration, and quantitation
• Processing data and obtaining results
• Creating report methods to generate custom reports
• Viewing and printing reports
• Backing up, deleting, restoring, and copying the contents of an individual project
PDA Software Functions
The Empower PDA software supports the following:
• PDA Instrument dialog box – Allows you to define control parameters for the 996
PDA Detector and the 2996 PDA Detector.
• Real-time PDA data acquisition displays – Allow you to plot the latest acquired
spectra and wavelengths in Run Samples for up to four individual real-time data
acquisition plots.
• 3D blank subtraction – Allows you to use a method set to subtract a labeled 3D
chromatogram (a solvent blank) from a second 3D chromatogram (a sample or
standard) and display the difference in Review. This feature is useful for subtracting
the effects of a mobile phase containing strong UV absorbing compounds.
• PDA calibration – Allows you to examine the wavelength calibration, the reference
spectrum and dark current, and the effects of exposure time on photodiode
saturation.
• PDA diagnostics – Allow you to run internal and external diagnostics on the 996
PDA Detector and the 2996 PDA Detector.
• Review – Allows you to view your chromatograms and spectra by using the Contour
plot, which allows you to extract, integrate, and process 2D chromatograms and
spectra (from the 3D data), and the Spectrum Index plot, which displays selected
spectra for the peaks shown in the Chromatogram plot. Displays the maximum
impurity for multiple PDA purity passes. Also displays the Review Results window
which displays purity and library match results.
PDA Software Overview
16
• Spectrum Review Spectral plot – Allows you to display library, peak, and extracted
spectra in Review, search against library spectra, create derived spectra, and create
and maintain spectral libraries.
• Spectrum Review tables – Allow you to display spectral and match results data in
Review, such as the:
– Spectra table with an image of all extracted spectra and peak apex spectra with
complete data on each spectrum including wavelength range and the resolution
used to acquire PDA data
– Library Match table with the results of the library search
– Spectrum Points table with the wavelength and absorbance data points
1.2 Tutorial Overview
This guide does not cover acquiring data, optimizing integration, generating calibration
curves, quantitating unknowns, obtaining results for 2D chromatograms, or performing
specific PDA procedures. For details on these topics, see the Empower Software Getting
Started Guide or the Empower Help.
This tutorial shows you how to:
• Restore PDA data from the Empower Software Options CD-ROM. This data is used
in all the examples in this guide.
• Extract chromatograms and spectra.
• Develop a processing method.
• Calculate peak purity and examine peak purity results.
• Build a UV spectral library and match unknown spectra to spectra in the library and
examine library match results.
• Generate a report.
Before You Begin
Note: This guide uses the Empower Pro interface. If you do not have access to this
interface, see your system administrator.
Before you perform the procedures described in this guide, make sure that:
• You installed or upgraded the Empower software as described in the Empower
System Installation and Configuration Guide or the Empower System Upgrade and
Configuration Guide.
• You reviewed and followed the basic operating procedures, including data acquisition,
described in the Empower Software Getting Started Guide.
Tutorial Overview
17
1
• You read the Waters 996 PDA Detector Operator’s Guide or the Waters 2996 PDA
Detector Operator’s Guide.
• Your printer is properly configured for use with Empower software.
1
1.3 Restoring the PDA Project
To run through the tutorial, you will restore the PDA example project, called PDA_Default.
This project contains the sample PDA data used in all the examples in this guide. Use the
Restore Project Wizard in Configuration Manager to restore the project data to your
computer. The PDA project is located on the Empower Options CD-ROM.
To restore the PDA project PDA_Default:
1. Start Empower software by double-clicking the Empower Login desktop icon.
Alternatively, select Start > Programs (for Windows XP, All Programs) >
Empower > Empower Login. The Empower Login dialog box appears
(Figure 1-1).
Personal System
Workgroup or Enterprise System
Figure 1-1 Empower Login Dialog Box
2. Enter your user name and password. If you do not know your user name or
password, see your system administrator.
3. If you are using an Empower Enterprise system, select the correct database from
the Database list. This list appears only when you are connected to a client/server
system.
4. Click Advanced and verify that the Requested Interface field is set to Pro. If you
cannot select the Empower Pro interface, see your system administrator.
5. Click OK. The Empower Pro window appears with the name of the logged-in user
displayed (Figure 1-2).
PDA Software Overview
18
Logged-in User
and Database
Name
Figure 1-2 Empower Pro Window
Restoring the PDA Project
19
1
6. Click Configure System (Figure 1-2) to open Configuration Manager with the
available projects displayed in the view table (Figure 1-3).
1
Restore Project(s) Tool
View
Table
Figure 1-3 Configuration Manager
7. Click
(Restore Project(s)) (Figure 1-3) to start the Restore Project Wizard
(Figure 1-4).
PDA Software Overview
20
8. Click the Browse button to select the path of your CD-ROM drive as shown in
Figure 1-4. The drive letter can differ from the one in Figure 1-4.
1
Browse Button
Figure 1-4 Restore Project Wizard - Start Software Page
9. Click Next and follow the instructions on each page to complete the wizard.
Note: If a message indicates that the restored project needs to be updated, click
OK.
10. Click Finish on the last page, Restore Display, to complete the restoration. The
restored project, PDA_Default, appears in the Configuration Manager Projects view
table (Figure 1-5). If the project does not appear, select View > Refresh.
11. Select File > Exit to close Configuration Manager.
Restoring the PDA Project
21
1
Figure 1-5 Configuration Manager with Restored PDA_Default Project
You are now ready to view the PDA data using Empower software.
PDA Software Overview
22
Chapter 2
Viewing PDA Data
This chapter provides step-by-step procedures for viewing chromatograms and spectra.
2.1 Tutorial Overview
This tutorial shows you the basics of using Empower software to view the data in the
PDA_Default project. Now that you have restored PDA sample data to your Empower
workstation, you can examine it in a variety of ways. The procedures in this chapter are the
building blocks for using the PDA software and are a foundation for the procedures in
Chapter 3, Peak Purity Processing, and Chapter 4, Library Matching. This chapter
describes the simple tasks you can do to view the PDA data that comes with Empower
software, including:
• Using Review and viewing the Contour plot
• Displaying the 3D plot
• Zooming in on plots
• Extracting a chromatogram
• Extracting a spectrum
• Annotating chromatograms and spectra
2.2 Viewing Data in Review
Review is the Empower software application that lets you view and manipulate 2D and 3D
chromatographic data brought in from the Project window. Review consists of menus,
toolbars, the Chromatogram plot, the Channel table, the Contour plot, the Spectrum
Review Spectral plot, and the Spectrum Review tables. Each item is explained in this
section.
To view a PDA data file in Review:
1. In the Empower Pro window (Figure 2-1), click Browse Project to open the Browse
Project dialog box (Figure 2-2).
Tutorial Overview
23
2
2
Figure 2-1 Empower Pro Window
Figure 2-2 Browse Project Dialog Box
2. In the Project to Browse list, select PDA_Default and click OK. The project window
opens (Figure 2-3).
Viewing PDA Data
24
Note: If PDA_Default does not appear in the Project to Browse list, check that you
restored it according to Section 1.3, Restoring the PDA Project.
Review Button
Channels Tab
2
Channels
View Table
Figure 2-3 Project Window
3. Click the Channels tab in the Project window, then select Mixture in the Channels
View table. Click
(Review) or select Tools > Review (Figure 2-3). Review
appears with the unprocessed data in a screen similar to the one in Figure 2-4. (If
your screen does not resemble Figure 2-4, select Window > Main Window.)
Viewing Data in Review
25
Spectrum
Review
Maximize
Button
Contour
Plot
2
Review
Main
Window
Figure 2-4 Review with PDA Data, no Chromatogram Extracted
4. If the Review window is not maximized, click
(maximize) (Figure 2-4). Maximize
both the main window and the daughter window.
Note: If you do not see the Contour plot and Spectrum Review, select View > 3D
Layout.
Figure 2-5 displays Review with the Review Main window maximized. The Contour
plot is an overhead view of the three-dimensional data file in which the x-axis plots
time and the y-axis plots wavelength. The chromatogram plot displays extracted
chromatograms. Spectrum Review displays extracted spectra.
Note: No chromatograms or spectra are extracted at this time.
Viewing PDA Data
26
Shortcut Tools
Contour
Plot
Review Menus
2
3D Channels Tab
3D Channels Table
Chromatogram
Plot
Spectra Table
Spectrum Review
Spectrum Review is Made Up of the Spectra
Table and the Spectra Plot
Figure 2-5 Maximized Review Main Window in 3D Layout
5. Click the 3D Channels tab at the bottom of Review (Figure 2-5) to view information
regarding the data file, such as sample name, type, date, time acquired, and so on.
6. Figure 2-6 shows the tools you can use as shortcuts in Review. This figure shows
the toolbar separated into two rows in order to label the tools.
Viewing Data in Review
27
Review Toolbar
Save Processing Method
Review Tools
Save All
Processing Method Wizard
Full View
Unzoom
Integrate
2
Review Main
Window
Calibrate
Quantitate
Processing Method
Previous 2D Channel
Results
Method Set
Next 2D Channel
Annotation Tools
Drop-Down List
Clear Annotation
Tool
Next 3D Channel
Previous 3D Channel
3D Plot
Apply Method Set
Empower Help
Print
Overlay
Extract Choices Drop-Down List
Extract Chromatogram
Extract Spectrum
Library Match
Maximum Impurity-Pass 4
Maximum Impurity-Pass 3
Maximum Impurity-Pass 2
Maximum Impurity-Pass 1
Peak Offsets
Peak Valleys
Peak Inflection
Peak Apex
Set Peak Width,
Set Threshold,
Set Minimum Area,
and
Set Minimum Height,
Respectively
Figure 2-6 Review Toolbar Buttons
Viewing PDA Data
28
2.3 Displaying the 3D Plot
The 3D plot provides a three-dimensional view of PDA data. The data is plotted on three
axes:
• X-axis – Represents time.
• Y-axis – Represents absorbance units.
• Z-axis – Represents wavelength.
You can rotate the 3D plot to view the data from the following different perspectives:
• The front view displays a chromatogram with time plotted on the x-axis and
absorbance units plotted on the y-axis.
2
• The side view displays the UV spectra with wavelength plotted on the x-axis and
absorbance units plotted on the y-axis.
• The top view displays a contour plot with time plotted on the x-axis and wavelength
plotted on the y-axis.
To display the 3D plot:
1. With the PDA data open in Review, click the Legend tab on the lower-right side of
Review to display the legend for the absorbance. The legend defines the colors
used in the Contour plot and the 3D plot.
2. Click
(3D Plot) or select Window > 3D Plot (Figure 2-6) to view the 3D plot.
3. You can rotate the handle on the 3D plot (Figure 2-7) to view it from one of the
following perspectives:
• Front – Move the handle to the upper center of the window.
• Side – Move the handle to the upper-right corner of the window.
• Top – Move the handle to the bottom center of the window.
Displaying the 3D Plot
29
Close
Button
Handle for Rotating 3D Plot
2
X-Axis Represents Time
Z-Axis Represents Wavelength
Y-Axis Represents Absorbance Units
Figure 2-7 Sample 3D Plot Window
4. To exit the 3D Plot window and return to the Review Main window, click
(Close)
(the lower close button) in the upper-right corner of the daughter (not the main)
window.
Viewing PDA Data
30
2.4 Zooming In on Plots
You can examine graphical plot features in detail by using a zoom box. You can zoom in on
the Chromatogram plot, Contour plot, and Spectral plot. You also use this technique to
view baseline noise in a chromatogram.
To create a zoom box, hold down the left mouse button and drag the mouse around the
features you want to enlarge. A box appears around the area of interest (Figure 2-8).
When the box is in the desired size and position, release the left mouse button.
Zoom Box
2
Figure 2-8 Creating the Zoom Box
Figure 2-9 shows the area after zooming.
Note: If you view the 3D plot after zooming on the Contour plot, the 3D plot is also zoomed
to the same region of data. There is no zoom capability directly in the 3D Plot window. See
Section 2.3, Displaying the 3D Plot, for information on viewing the 3D plot.
Zooming In on Plots
31
Full View Tool
Unzoom Tool
2
Figure 2-9 Zoomed View of the Contour Plot
Rescaling the Plot
You can revert to the unzoomed plot in one of the following ways:
• Full View – Reverts the plot to its original scale. To activate Full View, click
View) or right-click in the Contour plot and select Full View (Figure 2-9).
(Full
• Unzoom – Returns to the previous zoom when you have zoomed repeatedly. To
activate Unzoom, click
(Unzoom) or right-click in the Contour plot and select
Unzoom (Figure 2-9).
Viewing PDA Data
32
Saving Zoom Parameters
If you repeatedly want to see the same zoomed area, you can save the parameters of the
zoom box. To save the zoom parameters:
1. Right-click the desired plot, then select Properties to access the Plot Properties
dialog box.
2. Click the Scaling tab, then click Get Values from Plot to fill in values from the plot
in the Scaling section of the tab (Figure 2-10). Alternatively, enter the X-start, X-end,
Y-start, and Y-end values manually.
3. Click OK to apply the settings.
Note: For details on how to customize display settings, see the “Customizing the Display
of Data in Review” topic in the Empower Help.
Figure 2-10 Scaling Tab
2.5 Extracting a Chromatogram
Extracting a chromatogram manually allows you to see what the chromatogram looks like
at any wavelength across the collected wavelength range. You can extract chromatograms
from several wavelengths and then overlay them for further comparison.
Extracting a Chromatogram
33
2
To manually extract a chromatogram:
1. With the PDA data displayed in Review, select 254.0 from the Extract Choices list
to display a wavelength marker and an extracted 254.0-nm chromatogram in the
chromatogram plot (Figure 2-11).
Note: The 254.0 nm selection is a default value that automatically appears in the
list.
Extract Choices
Drop-Down List
Wavelength
Marker
2
2D Channels Tab
Spectra Tab
Figure 2-11 Extracted Chromatogram at Wavelength 254 nm
2. View a chromatogram at a different wavelength using one of these methods:
• In the Extract Choices list, select the empty choice at the top. Drag the
wavelength marker to the desired wavelength, for example, 280.0. As you drag
the wavelength marker, the chromatogram automatically updates in the
Chromatogram plot (Figure 2-12).
Viewing PDA Data
34
• In the Extract Choices list, select the empty choice at the top of the list.
Double-click the wavelength marker and enter the desired wavelength, for
example, 280.0, and press Enter (Figure 2-12).
• Enter the desired wavelength, for example, 280.0 in the Extract Choices list, and
click
(Extract Chromatogram) (Figure 2-12).
Extract Chromatogram
Tool
Extract Choices
Drop-Down List
Wavelength Marker
2
2D Channels Tab
Chromatogram Plot
Figure 2-12 Extracted Chromatogram at Wavelength 280 nm
3. To overlay the chromatograms, click
(Overlay) or select Plot > Overlay. The
overlaid chromatograms appear in the Chromatogram plot as shown in Figure 2-13.
The 2D Channels tab lists each extracted chromatogram. The Channel Description
field indicates the extracted wavelength (you might need to scroll to the right side of
the table to see this field).
Extracting a Chromatogram
35
4. Click
(Overlay) or select Plot > Overlay again to toggle back to a single
chromatogram. The extracted chromatogram that is selected in the 2D Channels tab
is displayed.
Overlay Tool
Two Markers Indicate a Chromatogram
Extracted at Two Different Wavelengths
2
Overlaid
Chromatograms
Channel
Description
Figure 2-13 Overlaid Chromatograms at Wavelengths 254 and 280 nm
2.6 Extracting a Spectrum
When you extract a spectrum manually, you can see what the spectrum looks like at any
time in the chromatographic run. You can extract a spectrum at several elution times and
then overlay them for further comparison. You can also use extracted spectra to build
spectral libraries.
Viewing PDA Data
36
To manually extract a spectrum:
1. With the PDA data displayed in Review, click
(Extract Spectrum) to display a
spectrum marker in the lower-left corner of the Contour plot and to display an
extracted spectrum in Spectrum Review.
2. Change the spectrum marker using one of the following methods:
• Drag the spectrum marker to the desired time, for example, 1.388 (the retention
time of the first peak apex). Notice as you drag the marker, the spectrum
automatically updates in Spectrum Review (Figure 2-14).
• Double-click the spectrum marker and enter the desired time, for example, 1.388,
then press Enter (Figure 2-14).
• Right-click the spectrum marker and select Edit. Enter the desired time, for
example, 1.388, then press Enter (Figure 2-14).
Extract Spectrum Tool
2
Spectrum Review
Spectrum
Marker
Figure 2-14 Extracted Spectrum at 1.388 Minutes
Extracting a Spectrum
37
3. Click
(Extract Spectrum) to extract another spectrum.
4. Change the spectrum marker to 2.159 (the retention time of the second peak apex).
The next spectrum is overlaid with the previous spectrum in Spectrum Review
(Figure 2-15).
2
Spectrum
Review
Figure 2-15 Overlaid Extracted Spectra
5. Click the Spectra tab on the lower-right side of the window (Figure 2-16) to view
information about the spectra in Spectrum Review.
Viewing PDA Data
38
Spectrum
Plot
2
Spectra
Table
Spectra Tab
Figure 2-16 Spectra Table
6. To view the overlaid spectra in a normalized fashion, double-click inside the
Spectrum plot. Note that the y-axis does not display absorbance units. Viewing the
normalized spectra corrects for concentration differences and allows you to
compare the UV spectra based on their shape differences (Figure 2-17).
You can restore the default scaling (autoscaling) by double-clicking again inside the
Spectrum plot.
Extracting a Spectrum
39
Normalized
Spectra
2
Spectra
Table
Select Check Boxes
Figure 2-17 Normalized Spectra
7. In the Spectra table below Spectral Review, clear all but one spectrum by selecting
the Select check box (Figure 2-17).
8. Scroll in the area containing the tabs below the Spectra table and click the
Spectrum Points tab to display the raw data points of the spectrum, as shown in
Figure 2-18.
Viewing PDA Data
40
2
Spectrum
Points
Table
Spectrum Points Tab
Scroll Here Until the Spectrum Points Tab Appears
Figure 2-18 Viewing Spectrum Points: Absorbances Versus Wavelengths
2.7 Annotating Chromatograms and Spectra
Empower software has a set of Annotation tools. In Review, these tools allow you to add
notes to chromatograms and spectra when you want to:
• Copy one to a document or presentation
• Print one for reference
Note: Annotations are not saved when you exit Review.
Annotating Chromatograms and Spectra
41
2.7.1 Adding Annotations
In the following example, you will annotate the chromatogram by drawing an arrow and
adding supporting text.
Drawing an Arrow
To draw an arrow on the chromatogram:
1. With the PDA data displayed in Review, select
Annotation Tools list (Figure 2-19).
(arrow tool) from the
Annotation Tools
Annotation Tools
Drop-Down List
2
Clear Annotations
Figure 2-19 Annotation Tools
2. Move the mouse to the Chromatogram plot. The pointer changes to an arrow tool.
3. Hold the left mouse button while drawing an arrow to the tallest peak. When you
finish drawing, release the mouse button.
Viewing PDA Data
42
4. Click the arrow to select it. The pointer changes to a pointing finger and the arrow is
selected (black handles appear on each end). When the arrow is selected, you can:
• Move it to another location by clicking a part of the arrow other than the black
handles and dragging it to the desired location.
• Rotate the arrow by holding one of the black handles and turning it in the desired
direction.
• Resize the arrow by pulling one of the black handles.
• Delete the arrow by pressing the Delete key.
If you do not want to change the arrow, click elsewhere in the Chromatogram plot to
deselect it.
5. Right-click the arrow and select Annotations > Properties to open the Annotation
Object Properties dialog box (Figure 2-20). From this dialog box you can change the
style, thickness, and/or color of the arrow.
Figure 2-20 Annotation Object Properties Dialog Box for an Object
Adding Text
To add text to the chromatogram:
1. From the Annotation Tools list, select
(text tool).
2. Move the mouse to the Chromatogram plot. The pointer changes to +abc.
Annotating Chromatograms and Spectra
43
2
3. Click the area of the plot where you want to add text to open the Annotation Object
Properties dialog box (Figure 2-21).
2
Figure 2-21 Annotation Object Properties Dialog Box for Text
4. In the Text field, type the text you want to add to the chromatogram. From this dialog
box you can also specify text properties such as alignment, font, and size. When you
are finished, click OK to add the text to the chromatogram.
5. Click the text to select it. The pointer changes to a pointing finger and the text is
selected (black handles appear on the corners). When the text is selected, you can:
• Move the text to another location by clicking a part of the text other than the black
handles and dragging to the desired location.
• Resize the text box (if all of your text is not displayed) by pulling one of the black
handles.
• Delete the text by pressing the Delete key.
If you do not want to make these changes to the text, click elsewhere in the plot to
deselect the text.
6. To change the text and/or the properties of the text, right-click the text and select
Annotations > Properties to open the Annotation Object Properties dialog box
Viewing PDA Data
44
Figure 2-22 shows an example of an annotation in the Chromatogram plot.
2
Figure 2-22 Annotation Example
2.7.2 Erasing Annotations
Click
(Clear Annotations) to remove all annotations. You can only remove individual
annotations by selecting them and pressing the Delete key.
Annotating Chromatograms and Spectra
45
2.7.3 Annotation Tools
The remaining Annotation tools, other than the text tool, function the same way as the
arrow. This list describes each Annotation tool and the action it performs or the object it
creates.
Tool
Action or Object
Draws a line.
Draws a line with an
arrowhead on one
end.
2
Draws a line with
arrowheads on both
ends.
Draws a rectangle.
Draws an ellipse.
Adds text.
Exiting Review
To exit Review:
1. Click
(Close) or select File > Exit (Figure 2-18). A dialog box warning that the
modified method set cannot be saved appears.
2. Click OK.
Next Steps
Now that you have examined the PDA_Default data in Review, you have the following
options:
• You can develop a processing method for peak purity, as described in Chapter 3,
Peak Purity Processing.
• You can create a spectral library and develop a processing method to match unknown
spectra against the library, as described in Chapter 4, Library Matching.
Viewing PDA Data
46
Chapter 3
Peak Purity Processing
This chapter provides a tutorial with step-by-step procedures for developing a PDA
processing method to determine peak purity.
3.1 Tutorial Overview
This tutorial familiarizes you with Empower PDA software tools and the procedures used
to develop a PDA processing method for determining peak purity. You calculate peak
purity to determine if a peak is spectrally homogeneous. Spectral heterogeneity can
indicate the presence of a coelution. A coelution of two or more spectrally distinct
compounds can produce a spectrally heterogeneous peak.
This tutorial shows you how to:
• Derive chromatograms.
• Develop a processing method to determine peak purity using the Processing Method
Wizard, which is where you define key parameters for integration and peak purity
determination.
• Examine peak purity results in Spectrum Index.
• View peak purity results in the Purity table and Purity plot of the Results window.
Steps to Compute Peak Purity
The following are the steps used to compute peak purity:
1. Bring the PDA data from the PDA_Default Project into Review (Section 3.2).
2. Derive chromatograms (Section 3.2).
3. Build a PDA processing method (Section 3.3).
Tutorial Overview
47
3
4. View results (Section 3.4).
• Peak apex spectrum
• Maximum impurity pass 1 spectrum
• Purity table
• Purity plot and points of maximum impurity
3.2 Deriving Chromatograms
Before developing a processing method that computes peak purity, you must derive a
chromatogram. A derived chromatogram is the two-dimensional chromatogram extracted
from the three-dimensional PDA data at the wavelength that you specify.
Note: A Max Plot chromatogram is a derived chromatogram which plots the maximum
spectral absorbance measured at each time point. It allows you to see all the
chromatographic peaks in the sample regardless of each peak’s lambda max (the
wavelength at maximum absorbance). The Max Plot chromatogram is useful in
determining the appropriate noise interval for your processing method. It is also necessary
to check the absorbance range of the Max Plot chromatogram. When obtaining purity
results, it is important that your sample does not exceed 1.0 AU in any region of the 3D
data (see step 4 in Section 3.2). However, data extracted at one particular wavelength is
typically used to set processing parameters when developing a processing method and for
quantitative purposes. Because Max Plot uses the absorbances from all wavelengths in
the range selected during data acquisition, it is not meaningful to use it to generate a
calibration curve and quantitate data.
To derive a chromatogram:
1. Open the Project window, which you opened in Chapter 2, Viewing PDA Data, by
selecting the PDA_Default project in the Windows taskbar.
Peak Purity Processing
48
3
2. In the Channels tab of the Project window (Figure 3-1), select Mixture. Click
(Review) or select Tools > Review. The Review window appears.
Review
Button
3
Figure 3-1 Project Window of the PDA_Default Project
3. From the Extract Choices list in Review, select MAX Plot to extract a Max Plot
chromatogram. The chromatogram appears in the Chromatogram plot (Figure 3-2).
Deriving Chromatograms
49
Select MAX Plot from the
Extract Choices Drop-Down List
3
Figure 3-2 Review Main Window with a Max Plot Chromatogram
4. Ensure that the absorbance of all peaks in the chromatogram is less than 1 AU
(absorbance unit). This criteria is necessary when calculating purity results in order
for these results to be valid. If the absorbance is less than 1 AU, proceed with the
next step. If the absorbance of any peak is greater than 1 AU, dilute the sample
appropriately and acquire it again.
5. From the Extract Choices list, select 254 to extract a chromatogram at 254.0 nm.
The Max Plot chromatogram is replaced with the newly extracted chromatogram
(Figure 3-3). We will use the 254-nm extracted channel to determine the optimal
integration parameters.
Peak Purity Processing
50
Select 254 from the
Extract Choices Drop-Down List
3
2D Channels Tab
Figure 3-3 Review Main Window with a Chromatogram at 254 nm
3.3 Developing a PDA Processing Method
Now that you have verified that your absorbance for each peak is less than 1 AU and you
have extracted a chromatogram at the desired wavelength, you can build a processing
method to integrate the chromatograms and assess peak purity. The simplest way to build
a PDA processing method is to use the Processing Method Wizard.
Note: This procedure assumes that the Use V3.0X Style Peak Width and Threshold
Determination system policy is enabled (the default setting). If this system policy is not
enabled on your system, the Processing Method Wizard will look slightly different. For
more information on this system policy, see the Empower Help.
Developing a PDA Processing Method
51
This procedure uses the traditional integration algorithm rather than the ApexTrack
integration algorithm. For more information on these processing algorithms, see the
Empower Software Acquisition and Processing Theory Guide or the ApexTrack
Integration: Theory and Application white paper (go to www.waters.com).
Note: Ensure that the 254-nm channel is selected in the Channels tab (Figure 3-3).
To build a PDA processing method:
1. Click
(Processing Method Wizard) or select File > New > Processing Method
to start the wizard and open the Processing Method Wizard dialog box (Figure 3-4).
3
Figure 3-4 Processing Method Wizard Dialog Box
2. Click Create a New Processing Method and click OK to open the New
Processing Method dialog box (Figure 3-5).
Figure 3-5 New Processing Method Dialog Box
Peak Purity Processing
52
3. From the Processing Type list, select PDA. If the Integration Algorithm list is present,
select Traditional. Make sure the Use Processing Method Wizard check box is
selected, then click OK to go to the Integration - Peak Detection 1 page (Figure 3-6).
Note: The Integration Algorithm list appears only if ApexTrack integration is enabled
in your system policies and ApexTrack is enabled in your PDA_Default project.
Peak Width
Zoom Box
3
Figure 3-6 Integration - Peak Detection 1 Page, Full View
This page allows you to set the peak width parameter in your processing method.
Peak width determines the number of points bunched together to act as a filter and
to produce a single point for peak detection (although all data points are used during
integration).
4. Follow the wizard instructions and use the mouse to draw a zoom box that encloses
the liftoff and touchdown of the narrowest peak of interest (peak 1 at 1.39 minutes).
The software displays the zoomed selection and determines the appropriate peak
width (Figure 3-7).
Developing a PDA Processing Method
53
Note: Right-click the plot to access the Full View/Unzoom shortcut menu selections.
If you are unsure which peak is the narrowest, you can check different peaks by
zooming and unzooming repeatedly on each peak. Each time you zoom, the
software calculates and displays the appropriate peak width. Zoom properly (and
perhaps multiple times) so that the peak start is at the beginning of the displayed
x-axis in the plot and the peak end is at the end of the displayed x-axis in the plot.
The peaks in this data have nearly identical widths and all yield a Peak Width setting
of 15 seconds when zoomed correctly. It therefore does not matter which peak is
chosen, however, some data does contain peaks of varying widths. In this case, the
appropriate peak should be used.
3
Figure 3-7 Setting the Peak Width Parameter
5. Click Next to go to the Integration - Peak Detection 2 page (Figure 3-8).
Peak Purity Processing
54
Figure 3-8 Integration - Peak Detection 2 Page
This page allows you to set the threshold parameter in your processing method
based on a selected area of baseline. Threshold is a slope setting that defines the
peak liftoff and touchdown points by determining whether the slope across three
data points exceeds the determined slope threshold. It is used to reject baseline
noise.
6. Follow the wizard instructions and use the mouse to draw a zoom box that encloses
the area of the baseline that contains the greatest amount of noise and no peaks of
Developing a PDA Processing Method
55
3
interest (approximately 0.2 to 1.2 minutes). The software displays the zoomed
selection and determines the appropriate threshold value (Figure 3-9).
Note: In general, you might need to zoom the baseline several times to ensure that
the selected area is free of peaks of interest.
3
Figure 3-9 Setting the Threshold Parameter
7. Click Next to go to the Integration - Integration Region page (Figure 3-10).
Peak Purity Processing
56
Figure 3-10 Integration - Integration Region Page
This page allows you to refine your integration by using an Inhibit Integration event
to specify a time region where no integration occurs.
8. If you want to inhibit integration, follow the wizard instructions and use the mouse to
draw a zoom box that encloses the area where you want integration to occur.
Integration is inhibited in the area outside of the zoom box. The software displays
the zoomed selection. Click Next to go to the Integration - Peak Rejection page
(Figure 3-11).
This step is not necessary with this particular data. If you do not want to inhibit
integration, do nothing in this dialog box and click Next.
Developing a PDA Processing Method
57
3
Figure 3-11 Integration - Peak Rejection Page
3
Use this page to reject small peaks that are not of interest. This step is not
necessary with this particular data as there is no unwanted baseline noise that is
integrated. Click Next to go to the Calibration - General page.
Note: To use this setting, select the smallest peak of interest to highlight it in red.
Select the Mimimum Area or Minimum Height check box to set the minimum
area or height to 95% of the selected peak.
When it is necessary to use the Minimum Area or Minimum Height parameter, you
typically use one or the other, but not both. For more information see the “Minimum
Area” and “Minimum Height” topics in the Empower Help.
9. Accept the default settings and click Next. A message indicates that channels
require channel names in order to perform cross-channel internal standard
processing. Click No in this dialog box. The Names and Retention Times page
appears (Figure 3-12).
Peak Purity Processing
58
Figure 3-12 Names and Retention Times Page
10. Click 1 to the left of the Name column in the first row. Press Shift and click 3 to the
left of the Name column in the third row. All rows are highlighted. Press Delete on
your keyboard to remove all entries from this table. Click Next on this page and
successive pages until the PDA Purity/Matching page appears (Figure 3-13).
Note: Entering component names and amounts is necessary only when you intend
to process standards, generate calibration curves, and quantitate unknowns. Since
this guide focuses on generating purity and library match data, this information is
unnecessary. For information about calibration and quantitation, see the Empower
Software Getting Started Guide or the Empower Help.
Developing a PDA Processing Method
59
3
Figure 3-13 PDA Purity/Matching Page
11. Ensure Yes is selected for the question “Do you wish to perform peak purity testing
on all peaks?”. For the question “Do you wish to match spectra against PDA library
spectra?” click No (Figure 3-13). Click Next to go to the PDA Spectral Contrast
page (Figure 3-14).
Peak Purity Processing
60
3
Figure 3-14 PDA Spectral Contrast Page
Use this page to set the noise interval by selecting a segment of the baseline that is
free of peaks. You must select a segment of the baseline that is at least one-half
minute in length.
Note: The MaxPlot check box is selected by default and allows you to display the
Max Plot chromatogram in the PDA Spectral Contrast page regardless of the type of
chromatogram that was extracted before starting the Processing Method Wizard. It
is recommended to use the Max Plot chromatogram when setting the noise interval
to ensure that this parameter is set to a region of the baseline where no
compound-related spectral absorbance occurs.
When the MaxPlot check box is selected, the software displays the Max Plot
chromatogram for the entire wavelength range in which the data was acquired. If
you need to view the Max Plot chromatogram without using the entire acquired
wavelength range, you must create a Max Plot chromatogram with the appropriate
Start and End Wavelength by creating a derived channel in your method set and
then applying the method set before starting the Processing Method Wizard. The
Start and End Wavelength Limits are set in the Purity tab of the processing method.
(These settings are not available when using the Processing Method Wizard. You
must view the processing method by clicking
(Processing Method) or by
selecting Window > Processing Method.)
It is often desirable to define the wavelength start range above 190 nm to eliminate
the effect of mobile phase absorption.
Developing a PDA Processing Method
61
3
12. Select the segment of the baseline from approximately 3.00 to 3.50 minutes
(Figure 3-15).
You can select the baseline area by using the mouse to zoom in on the desired area,
or you can specify the baseline area by typing the start and end time of the desired
area in the Noise Interval Start Time and Noise Interval End Time text boxes,
respectively.
When zooming, you can right-click and select Fullview or Unzoom to move
between zoom levels. Do this to view multiple sections of baseline before deciding
on the correct region for the noise interval.
For details about selecting the noise interval, see the “Determining the Noise
Interval” topic in the Empower Help.
3
Figure 3-15 Setting the Noise Interval
13. Click Next to go to the Processing Method Name page (Figure 3-16).
Peak Purity Processing
62
Figure 3-16 Processing Method Name Page
3
14. In the Method Name field, enter a processing method name, for example, Purity,
then click Finish. The processing method is saved and the Review Main window
appears with the chromatogram processed (Figure 3-17). The name of the
processing method is indicated in the right side of the status bar.
Note: The Empower PDA software automatically sets the Threshold Criteria in the
processing method to AutoThreshold. For details, see the “Threshold Criteria
Considerations for Spectral Contrast (PDA)” topic in the Empower Help.
This procedure assumes that the Automatically Apply Method Set and New Method
Set: Create a new method set when starting Review options are enabled in Review.
To verify or change these settings, select Options > Method Set Options. If the
Automatically Apply Method Set option is not enabled, your chromatogram will not
be automatically processed. To process it, click
(Apply Method Set) or select
Process > Apply Method Set.
Developing a PDA Processing Method
63
3
Integrated
Chromatogram
Click the Arrow to
Scroll Through
the Tabs
Name of Method Set and
Processing Method
Indicated Here
Figure 3-17 Integrated Chromatogram in Review
When working with a PDA processing method, you must specify an extracted channel(s)
and a processing method(s) to use when processing the extracted data. You also have the
ability to specify multiple extracted channels and to use different processing methods for
each, if desired. When you extract channels as you did in Section 3.2, Deriving
Chromatograms, they are automatically added to your method set. No further action is
required, however, it is recommended to verify the information in your method set and to
rename the derived channel from the generic name that the software assigns to a name
that is more meaningful to you.
Peak Purity Processing
64
3.3.1 Viewing, Modifying and Saving the Method Set
1. Click
(Method Set) or select Window > Method Set to open the Method Set
Editor window (Figure 3-18).
Method Set
3
Figure 3-18 Method Set Editor Window
Notice that the Max Plot and 254 nm (named Wvln Ch1) chromatograms that you
extracted earlier are listed in the Channel Name column of the Channel table. The
processing method that you just created has also been specified in this table for the
254-nm chromatogram.
2. Right-click the Wvln Ch1 derived channel in the tree pane on the left side of the
Method Set Editor window. Select Rename from the shortcut menu. Wvln Ch1 is
selected (Figure 3-19). Enter a new name for this derived channel, for example,
254nm.
Developing a PDA Processing Method
65
Figure 3-19 Method Set Tree Pane
3. Click the first row in the Channel table to select the Max Plot chromatogram
(Figure 3-18). Press Delete on your keyboard to remove this row from the table.
4. Select File > Save As > Method Set to open the Save current Method Set dialog
box (Figure 3-20).
3
Figure 3-20 Save Current Method Set Dialog Box
5. In the Name field, type a name, for example, Purity, then click Save. The name of
the method set appears in the title bar of the Method Set Editor window and should
look as in Figure 3-21.
Peak Purity Processing
66
Method Set
Name Appears in
Title Bar
3
Figure 3-21 Method Set Editor Window after Modifying and Saving
3.3.2 Viewing the Peak Purity Calculation
1. Click
(Review Main Window) or select Window > Main Window to return to
the Review Main window.
2. Click
(Apply Method Set) or select Process > Apply Method Set to extract
and process the channel specified in the method set you created (Figure 3-22).
Developing a PDA Processing Method
67
3
Scroll to View
Peaks Tab
Peaks
Tab
Peaks
Table
Scroll to the Right to Reveal Additional
Purity Angle and Purity Threshold
Values
Figure 3-22 Purity Angle and Purity Threshold Values in the Peaks Table
3. Select the scroll arrows where indicated at the bottom left of the window to make the
Peaks tab visible (Figure 3-22). To view the numeric results of the peak purity
calculation, click the Peaks tab at the bottom of the Review Main window.
4. Using the mouse, move the Chromatogram plot up or down by dragging the split bar
to size the Chromatogram plot and the Peaks table. The Peaks table displays the
Purity Angle and Purity Threshold values (Figure 3-22). If the Purity Angle and
Purity Threshold values are not visible, scroll within the Peaks table to reveal them.
5. If the Purity Angle is less than the Purity Threshold, the peak is spectrally
homogeneous. Scroll down in the table to view the values for every integrated peak.
Note that Peaks 1 and 2 are not spectrally homogeneous; whereas Peak 3 is
spectrally homogeneous. For details on interpreting peak purity results, see the
“Interpreting Peak Purity Results (PDA)” topic in the Empower Help.
Peak Purity Processing
68
3.4 Reviewing Peak Purity Results
Once you have calculated peak purity, you can view the results in different ways. For
example, you can examine the apex spectrum and the maximum impurity spectrum in
Spectrum Index (see Section 3.4.1). Alternatively, you can use the Results window to
examine the Purity plot and to view the peak purity results (see Section 3.4.2).
3.4.1 Using Spectrum Index
Spectrum Index allows you to view the apex spectra of the integrated peaks. To view
Spectrum Index:
1. In the Review Main window, click the Spectrum Index tab (Figure 3-23). The apex
spectrum for each integrated peak appears and additional Spectrum Index tools
appear in the toolbar if they were not already present.
Note: If the Spectrum Index tools do not appear, right-click the toolbar and select
Spectrum Index or select View > Toolbar > Spectrum Index.
3
Reviewing Peak Purity Results
69
Maximum Impurity Pass 1 Tool
Spectrum Index
Tab
Apex Spectrum
Spectrum
Index Tools
3
Figure 3-23 Apex Spectrum
2. Click
(Maximum Impurity - Pass 1) (Figure 3-23) to display the maximum
impurity spectrum in the Spectrum Index plot (Figure 3-24). You are now viewing the
apex spectrum overlaid with the spectrum within the integrated peak that differs
most from the apex spectrum. This is displayed for every integrated peak. The
dotted line in the Chromatogram plot indicates the location within each peak from
where these spectra are taken. The lambda max, which is the wavelength of the
maximum absorbance, for each spectrum is labeled in the Spectrum Index plot by
default.
Peak Purity Processing
70
Spectrum Index Plot
(y-axis is absorbance)
3
X-Axis Is Wavelength
Figure 3-24 Apex Spectra Overlaid with Maximum Impurity Spectra
3. In the Chromatogram plot, zoom in on the first peak and examine the overlaid
spectra and note the slight differences between the spectra (Figure 3-25). Recall
that this peak is not spectrally pure.
Reviewing Peak Purity Results
71
3
Scroll to the Right to Examine
Subsequent Peaks
Figure 3-25 Maximum Impurity Spectrum with Peak 1 Spectra Zoomed
4. Scroll to the right and examine the spectra from the second peak and note the
significant differences between the spectra (Figure 3-26). Recall that this peak is not
spectrally pure.
Peak Purity Processing
72
3
Figure 3-26 Maximum Impurity Spectrum with Peak 2 Spectra Zoomed
5. Scroll to the right and examine the spectra from the third peak (Figure 3-27). The
spectra overlay well.
The first and second peaks do not appear spectrally homogeneous and require
further investigation. The third peak appears spectrally homogeneous. This
validates the purity results discussed in step 5 of “Viewing the Peak Purity
Calculation”.
Reviewing Peak Purity Results
73
Results Tool
3
Figure 3-27 Maximum Impurity Spectrum with Peak 3 Spectra Zoomed
Peak Purity Processing
74
3.4.2 Using the Results Window
To view the Purity plot in the Results window:
1. Click
(Results) or select Window > Results Window (Figure 3-27) to open the
Results window (Figure 3-28). If necessary, maximize the Results window or size
the different panes by dragging the split bars.
Purity
Tab
3
Purity Plot Tab
Figure 3-28 Results Window
2. Click the Purity tab in the top pane and the Purity Plot tab in the bottom pane of
the Results window. The Purity plot (Figure 3-29) displays the currently active
(selected) chromatographic peak and plots the Purity Angle and the Purity
Threshold values across the entire peak. Note that the left y-axis is in absorbance
units and the right y-axis is in Spectral Contrast degrees. The Purity tab displays the
purity information for each peak in a numerical format. Select each peak using the
Peak list (Figure 3-29).
Reviewing Peak Purity Results
75
Peak List
Threshold
Angle
Purity Angle
3
Absorbance
Spectral Contrast
Degrees
Time
Figure 3-29 Purity Plot
For a spectrally homogeneous peak, the Purity Angle always remains below the
Threshold Angle. For the peak at 1.389 minutes, the Purity Angle is above the
Threshold Angle in the early region of the peak, which indicates that the peak is not
spectrally homogeneous in this region.
The Purity Plot plots the Spectral Contrast Angle between the peak apex spectrum
and all other peak spectra. For details, see the “Spectral Contrast Angle (PDA)”
topic in the Empower Help. The Purity Angle increases in the peak tails due to the
effects of baseline noise.
Peak Purity Processing
76
3. Select the next peak in the Peak list (Figure 3-29) to display the Purity plot for the
next peak at 2.165 minutes. The Purity Angle is above the Threshold Angle in the
early region of the peak, which indicates that the peak is not spectrally
homogeneous in this region.
4. Select the next peak in the Peak list (Figure 3-29) to display the Purity plot for the
next peak at 3.931 minutes. The Purity Angle is below the Threshold Angle
throughout the entire peak, which indicates that the peak is spectrally
homogeneous.
5. Right-click the Purity plot, then select Properties to open the Plot Properties dialog
box (Figure 3-30).
3
Figure 3-30 Plot Properties Dialog Box
6. In the Purity tab, select the Annotate Peak Apex/Max. Impurity check box, then
click OK. The point of maximum impurity appears in the Purity plot and is marked
with an “M” (Figure 3-31). The peak apex is marked with a vertical line drawn from
the apex of the peak perpendicular to the baseline.
Reviewing Peak Purity Results
77
Peak Apex
Point of Maximum Impurity
3
Figure 3-31 Purity Plot with Maximum Impurity Indicator
7. Save the result so you can print it in a report. To save the results, select File > Save
> Result. If Save > Result is not available, select Save > Processing Method
first.
8. Exit Review by clicking
(Close) or selecting File > Exit.
Peak Purity Processing
78
Next Steps
Now that you have developed a processing method to assess peak purity, you have the
following options:
• Modify the existing processing method to include library matching, as described in
Chapter 4, Library Matching.
• Modify the processing method for multicomponent peak purity. For details, see the
“Multicomponent Peak Purity Testing (PDA)” topic in the Empower Help.
• Print your results, as described in Chapter 5, Printing Reports.
3
Reviewing Peak Purity Results
79
Chapter 4
Library Matching
This chapter provides step-by-step tutorials describing the procedures for creating a
library and matching unknown or acquired spectra to spectra in a library.
4.1 Tutorial Overview
This tutorial familiarizes you with Empower software tools and procedures used to create a
library and to match spectra to a library. Library matching allows you to identify peaks by
comparing spectra from unknown peaks to spectra from known peaks. These tutorials
show you how to:
• Create a library from existing standards.
• Match unknown spectra to spectra in a library.
• Review library matching results.
Section 4.1.1, Steps in Creating a Library, lists the steps used to create a library.
Section 4.1.2, Steps in Library Matching, shows the steps used for library matching. If you
followed the tutorial in Chapter 3, Peak Purity Processing, you can use the processing
method that you developed and modify it to include library matching.
4.1.1 Steps in Creating a Library
4
The following steps in creating a library are outlined in Section 4.2:
1. Select PDA 3D Channels of data.
2. Bring data into Review.
3. Open an existing method set.
4. Create a new library.
5. Add spectra to the library.
Tutorial Overview
80
4.1.2 Steps in Library Matching
Start
Do You Want to
Do Library Matching and
Peak Purity?
Yes: With Peak Purity
No: Library Match Only
Bring PDA data into Review
(Section 4.3.1)
Bring PDA data into Review
(Section 4.3.1)
Derive a Chromatogram
(Section 4.3.2)
Open an Existing Method Set
(Section 4.3.1)
Set Integration Parameters
(Section 4.3.2)
Enable Library Matching
(Section 4.3.1)
Set Noise Interval
(Section 4.3.2)
Enable Library Matching
(Section 4.3.2)
4
Integrate Chromatogram and
Calculate Results
(Section 4.3.3)
View Results
(Section 4.4)
End
Figure 4-1 Steps in Library Matching
Library Matching
81
4.2 Creating a New Library
You must create a library of known spectra before you can match unknown spectra to
them. The first step in creating a library is to choose the spectra to add to the library. In this
procedure, you add the spectra for Paraben Stds, Phenone Stds, and Benzoate Stds to
the library.
To create a library:
1. In the Project window Channels view, which you opened in Chapter 2, Viewing PDA
Data, select Paraben Stds, Phenone Stds, and Benzoate Stds, then click
(Review) or select Window > Review (Figure 4-2) to open the Review Main window
(Figure 4-3).
Note: In Empower software, multiple items can be selected simultaneously by
selecting them in the table with a Ctrl-click (multiple noncontiguous rows) or a
Shift-click (multiple contiguous rows).
If the Review Main window is not displayed, click
select Window > Main Window (Figure 4-3).
(Review Main Window) or
Review Tool
4
Figure 4-2 Selecting Data for a Library
Creating a New Library
82
Review Main Window Tool
3D Channels Tab
Move the Split Bar Higher to
View More Rows in the 3D
Channels Table
Figure 4-3 Review Main Window
4
2. At the bottom of the Review Main window, click the 3D Channels tab (Figure 4-3).
Make sure Paraben Stds is selected.
3. To enlarge the 3D Channels table if necessary, use the mouse to drag the split bar
above the 3D Channels table higher on the screen, as shown in Figure 4-3.
4. Select File > Open > Method Set to open the Open an existing Method Set dialog
box (Figure 4-4).
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83
Figure 4-4 Open an Existing Method Set Dialog Box
5. In the Names list, select Purity or the method set that you created in Chapter 3,
Peak Purity Processing, then click Open. The Purity method set is automatically
applied to the data in the Review window. The chromatogram at 254 nm for Paraben
Stds appears in the Review Main window (Figure 4-5) and the apex spectra for the
peaks appear in Spectrum Review.
Note: If you did not create a method set, select PDA_Demo_MethSet. A method
set is required in order to extract a chromatogram during processing.
If the Automatically Apply Method Set check box is not enabled in the Method Set
Options dialog box (the default setting found by selecting Options > Method Set
Options), the method set is not automatically applied. To apply the method set,
click
(Apply Method Set) or select Process > Apply Method Set.
4
Creating a New Library
84
Apply Method Set Tool
Figure 4-5 Paraben Stds Chromatogram
6. Select Library > New Library to open the Create a new Library dialog box
(Figure 4-6).
4
Note: The retention times of the peaks do not match the retention times displayed in
the Spectra tab. The apex spectrum is extracted using a method that differs slightly
from that used to determine retention time.
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85
Figure 4-6 Create a New Library Dialog Box
7. In the Name field, type a name for the new library, for example, My Library, then
click Create to create the new library.
Adding Spectra to the Library
1. In the Spectra table, make sure that the check box in the Select column is only
selected for the two main peaks as shown in Figure 4-7. The two spectra appear in
Spectrum Review.
Note: When addding spectra to a library, choose your spectra by using the Select
column in the Spectra table. Spectra that do not have the Select field selected are
not added to the library.
Creating a New Library
86
4
Spectra Plot
Select Check Boxes
Spectra Table
Figure 4-7 Spectrum Review with Spectra Selected
2. Select Library > Add to library My Library to open the Add Spectrum to Library
dialog box (Figure 4-8).
3. In the Name text box, change the name from the default of Peak 2 to Methyl
Paraben, then click OK. The Add Spectrum to Library dialog box reappears for the
next spectrum.
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87
4
Figure 4-8 Add Spectrum to Library Dialog Box
4. Change the name from the default of Peak 3 to Ethyl Paraben in the Name text
box, then click OK. The spectra are added to the library and appear (unselected) in
the Spectra table.
5. In the 3D Channels table, select Phenone Stds. The processed chromatogram for
Phenone Stds appears. Repeat steps 1 and 2 for Peaks 2 and 3 in this sample.
6. In the Name text box, change the name from the default of Peak 2 to
Acetophenone, then click OK. The Add Spectrum to Library dialog box reappears
for the next spectrum.
7. Change the name from the default of Peak 3 to Propriophenone in the Name text
box, then click OK. The spectra are added to the library and appear (unselected) in
the Spectra table.
8. In the 3D Channels table, select Benzoate Stds. The processed chromatogram for
Benzoate Stds appears. Repeat steps 1 and 2 for Peaks 1 and 2 in this sample.
9. In the Name text box, change the name from the default of Peak 1 to Ethyl Paba,
then click OK. The Add Spectrum to Library dialog box reappears for the next
spectrum.
10. Change the name from the default of Peak 2 to Benzoic Acid in the Name text
box, then click OK. The spectra are added to the library and appear (unselected) in
the Spectra table.
11. Select File > Exit.
Creating a New Library
88
4
4.3 Matching Spectra to a Library
Now that you have created a spectral library, you can compare unknown spectra to spectra
in the library. As Figure 4-1 shows, you can create a processing method in one of the
following ways:
• If you plan to use peak purity and library matching, you can modify the existing
processing method that you created in Chapter 3, Peak Purity Processing, to include
library matching. See Section 4.3.3, Modifying an Existing Processing Method for
Library Matching.
• If you plan to do library matching only, you can create a new processing method for
library matching alone. SeeSection 4.3.2 Creating a New Processing Method for
Library Matching.
When you have finished creating a processing method, proceed to Section 4.3.3,
Performing Library Matching.
4.3.1 Modifying an Existing Processing Method for Library Matching
To modify the existing processing method:
1. In the Project window (Figure 4-9), which you opened in Chapter 2, Viewing PDA
Data, select Mixture from the Channels view, then click
(Review) or select
Tools > Review to open the Review Main window.
4
Figure 4-9 Project Window of the PDA_Default Project
Library Matching
89
The Review window appears with the unprocessed data (Figure 4-10).
Figure 4-10 Review Main Window
2. Select File > Open > Method Set to open the Open an existing Method Set dialog
box.
3. In the Names list, select Purity or the method set that you created in Chapter 3,
Peak Purity Processing, then click Open. The Purity method set is automatically
applied to the data in the Review window. The chromatogram at 254 nm for Mixture
appears in the Review Main window (Figure 4-11) and the apex spectra for the
peaks appear in Spectrum Review.
Note: If you did not create a method set, select PDA_Demo_MethSet. A method
set is required in order to extract a chromatogram during processing.
If the Automatically Apply Method Set check box is not enabled in the Method Set
Options dialog box (the default setting found by selecting Options > Method Set
Options), the method set is not automatically applied. To apply the method set,
click
(Apply Method Set) or select Process > Apply Method Set.
Matching Spectra to a Library
90
4
Processing Method Tool
Figure 4-11 Integrated 254 nm Chromatogram of Mixture
4. Click
(Processing Method) or select Window > Processing Method to open
the Processing Method window and modify the existing processing method for
library matching (Figure 4-12). If necessary, maximize the window.
Library Matching
91
4
PDA Library Search Tab
Figure 4-12 Processing Method Window
5. Click the PDA Library Search tab. In the Library list box, select the check box of
the library you want to search, for example, My Library (Figure 4-13).
Note: For information on setting a solvent angle, see the “Guidelines for Estimating
the Solvent Angle (PDA)” topic in the Empower Help. For this demonstration, we will
leave the solvent angle set to 1.0. In general, this value needs to be determined
empirically and set accordingly.
Matching Spectra to a Library
92
4
Library List Box
Figure 4-13 PDA Library Search Tab
6. Click
(Review Main Window) or select Window > Main Window to return to
the Review Main window.
7. To save the changes to the existing processing method, select File > Save >
Processing Method.
To save the modified method under a different name, select File > Save As >
Processing Method. Type a name in the Name field, for example, Purity and
Library Match, then click Save.
8. To save the modified method set under a different name, select File > Save As >
Method Set. Type a name in the text box, for example, Purity and Library Match,
then click Save.
Note: After modifying a processing method or method set, you should click
(Method Set) or select Window > Method Set and verify that the desired
processing method(s) are specified in the Channel table for the appropriate
channel(s). Save the method set if you make any modifications.
9. Proceed to Chapter 4.3.3, Performing Library Matching, to complete the steps for
library matching.
Library Matching
93
4
4.3.2 Creating a New Processing Method for Library Matching
If you plan to do library matching without peak purity, you can build a processing method
for library matching alone. The simplest way to build a PDA processing method is by using
the Processing Method Wizard.
Note: This procedure assumes that the Use V3.0X Style Peak Width and Threshold
Determination system policy is enabled (the default setting). If this system policy is not
enabled on your system, the Processing Method Wizard will look slightly different.
To build a PDA processing method for library matching:
1. Before you start, you must derive a chromatogram as described in Section 2.5,
Extracting a Chromatogram.
2. Click
(Processing Method Wizard) or select File > New > Processing Method
to start the wizard and open the Processing Method Wizard dialog box
(Figure 4-14).
Figure 4-14 Processing Method Wizard Dialog Box
4
Matching Spectra to a Library
94
3. Click Create a New Processing Method and click OK to open the New
Processing Method dialog box (Figure 4-15).
Figure 4-15 New Processing Method Dialog Box
4. From the Processing Type list, select PDA. If the Integration Algorithm list is present,
select Traditional. Make sure the Use Processing Method Wizard check box is
selected, then click OK to go to the Integration - Peak Detection 1 page
(Figure 4-16).
Note: The Integration Algorithm list appears only if ApexTrack integration is enabled
in your system policies and ApexTrack is enabled in your PDA_Default project.
4
Library Matching
95
Peak Width
Zoom Box
Figure 4-16 Integration - Peak Detection 1 Page, Full View
This page allows you to set the peak width parameter in your processing method.
Peak width determines the number of points bunched together to act as a filter and
to produce a single point for peak detection (although all data points are used during
integration).
5. Follow the wizard instructions and use the mouse to draw a zoom box that encloses
the liftoff and touchdown of the narrowest peak of interest (peak 1 at 1.39 minutes).
The software displays the zoomed selection and determines the appropriate peak
width (Figure 4-17).
Note: Right-click the plot to access the Full View/Unzoom shortcut menu selections.
If you are unsure which peak is the narrowest, you can check different peaks by
zooming and unzooming repeatedly on each peak. Each time you zoom, the
software calculates and displays the appropriate peak width. Zoom properly (and
perhaps multiple times) so that the peak start is at the beginning of the displayed
x-axis in the plot and the peak end is at the end of the displayed x-axis in the plot.
The peaks in this data have nearly identical widths and all yield a Peak Width setting
of 15 seconds when zoomed correctly. It therefore does not matter which peak is
chosen, however, some data does contain peaks of varying widths. In this case, the
appropriate peak should be used.
Matching Spectra to a Library
96
4
Figure 4-17 Setting the Peak Width Parameter
6. Click Next to go to the Integration - Peak Detection 2 page (Figure 4-18).
4
Figure 4-18 Integration - Peak Detection 2 Page
Library Matching
97
This page allows you to set the threshold parameter in your processing method
based on a selected area of baseline. Threshold is a slope setting that defines the
peak liftoff and touchdown points by determining whether the slope across three
data points exceeds the determined slope threshold. It is used to reject baseline
noise.
7. Follow the wizard instructions and use the mouse to draw a zoom box that encloses
the area of the baseline that contains the greatest amount of noise and no peaks of
interest (approximately 0.2 to 1.2 minutes). The software displays the zoomed
selection and determines the appropriate threshold value (Figure 4-19).
Note: In general, you might need to zoom the baseline several times to ensure that
the selected area is free of peaks of interest.
4
Figure 4-19 Setting the Threshold Parameter
8. Click Next to go to the Integration - Integration Region page (Figure 4-20)
Matching Spectra to a Library
98
Figure 4-20 Integration - Integration Region Page
This page allows you to refine your integration by using an Inhibit Integration event
to specify a time region where no integration occurs.
9. If you want to inhibit integration, follow the wizard instructions and use the mouse to
draw a zoom box that encloses the area where you want integration to occur.
Integration is inhibited in the area outside of the zoom box. The software displays
the zoomed selection. Click Next to go to the Integration - Peak Rejection page
(Figure 4-21).
4
This step is not necessary with this particular data. If you do not want to inhibit
integration, do nothing in this dialog box and click Next.
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99
Figure 4-21 Integration - Peak Rejection Page
Use this page to reject small peaks that are not of interest. This step is not
necessary with this particular data as there is no unwanted baseline noise that is
integrated. Click Next to go to the Calibration - General page.
Note: To use this setting, select the smallest peak of interest to highlight it in red.
Select the Mimimum Area or Minimum Height check box to set the minimum
area or height to 95% of the selected peak.
When it is necessary to use the Minimum Area or Minimum Height parameter, you
typically use one or the other, but not both. For more information see the “Minimum
Area” and “Minimum Height“ topics in the Empower Help.
10. Accept the default settings and click Next. A message indicates that channels
require channel names in order to perform cross-channel internal standard
processing. Click No in this dialog box. The Names and Retention Times page
appears (Figure 4-22).
Matching Spectra to a Library 100
4
Figure 4-22 Names and Retention Times Page
11. Click 1 to the left of the Name column in the first row. Press Shift and click 3 to the
left of the Name column in the third row. All rows are highlighted. Press Delete on
your keyboard to remove all entries from this table. Click Next on this page and
successive pages until the PDA Purity/Matching page appears (Figure 4-23).
Note: Entering component names and amounts is necessary only when you intend
to process standards, generate calibration curves, and quantitate unknowns. Since
this guide focuses on generating purity and library match data, this information is
unnecessary. For information about calibration and quantitation, see the Empower
Software Getting Started Guide or the Empower Help.
Library Matching
101
4
Figure 4-23 PDA Purity/Matching Page
12. For the question “Do you wish to perform peak purity testing on all peaks?”, click No.
For the question “Do you wish to match spectra against PDA library spectra?”, click
Yes. Click Next to go to the PDA Spectral Contrast page (Figure 4-24).
4
Matching Spectra to a Library 102
Figure 4-24 PDA Spectral Contrast Page
Use this page to set the noise interval by selecting a segment of the baseline that is
free of peaks. You must select a segment of the baseline that is at least one-half
minute in length.
Note: The MaxPlot check box is selected by default and allows you to display the
Max Plot chromatogram in the PDA Spectral Contrast page regardless of the type of
chromatogram that was extracted before starting the Processing Method Wizard. It
is recommended to use the Max Plot chromatogram when setting the noise interval
to ensure that this parameter is set to a region of the baseline where no
compound-related spectral absorbance occurs.
When the MaxPlot check box is selected, the software displays the Max Plot
chromatogram for the entire wavelength range in which the data was acquired. If
you need to view the Max Plot chromatogram without using the entire acquired
wavelength range, you must create a Max Plot chromatogram with the appropriate
Start and End Wavelength by creating a derived channel in your method set and
then applying the method set before starting the Processing Method Wizard. The
Start and End Wavelength Limits are set in the Purity tab of the processing method.
(These settings are not available when using the Processing Method Wizard. You
must view the processing method by clicking
(Processing Method) or by
selecting Window > Processing Method.)
It is often desirable to define the wavelength start range above 190 nm to eliminate
the effect of mobile phase absorption.
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103
4
13. Select the segment of the baseline from approximately 3.00 to 3.50 minutes
(Figure 4-25).
You can select the baseline area by using the mouse to zoom in on the desired area,
or you can specify the baseline area by typing the start and end time of the desired
area in the Noise Interval Start Time and Noise Interval End Time text boxes,
respectively.
When zooming, you can right-click and select Fullview or Unzoom to move
between zoom levels. Do this to view multiple sections of baseline before deciding
on the correct region for the noise interval.
For details about selecting the noise interval, see the “Determining the Noise
Interval” topic in the Empower Help.
By default, the Empower PDA software sets the Threshold Criteria to Noise plus
Solvent with the Solvent Angle set to one degree. For details, see the “Threshold
Criteria Considerations for Spectral Contrast (PDA)” topic in the Empower Help.
4
Figure 4-25 Setting the Noise Interval
14. Click Next to go to the PDA Match Library page (Figure 4-26).
Matching Spectra to a Library 104
Figure 4-26 PDA Match Library Page
15. Select the check box for the library(s) you want to match against, for example, My
Library, then click Next to go to the Processing Method Name page (Figure 4-27).
4
Library Matching
105
Figure 4-27 Processing Method Name Page
16. In the Method Name field, enter a processing method name, for example, Library
Matching Only, then click Finish. The processing method is saved and the Review
Main window appears with the Max Plot chromatogram processed. The name of the
processing method appears in the right side of the status bar.
When working with a PDA processing method, you must save both the processing method
and method set. You can then apply the saved method set to subsequent data to
determine library matching.
4
Saving the Method Set
1. Select File > Save As > Method Set to open the Save current Method Set
dialog box.
2. In the Name field, type a name, for example, Library Matching Only, then click
Save. The processing method is saved and the Review Main window appears with
the chromatogram processed (Figure 4-28). The name of the processing method is
indicated in the right side of the status bar.
Note: This procedure assumes that the Automatically Apply Method Set and New
Method Set: Create a new method set when starting Review options are enabled in
Review. To verify or change these settings, select Options > Method Set Options.
If the Automatically Apply Method Set option is not enabled, your chromatogram will
not be automatically processed. To process it, click
(Apply Method Set) or
select Process > Apply Method Set.
Matching Spectra to a Library 106
Name of Method Set and
Processing Method
Indicated Here
Figure 4-28 Processed Chromatogram with Library Match Results
4
4.3.3 Performing Library Matching
Now that you have created or modified a processing method, you can perform library
matching. Apply an appropriate method set to compute the library Match Angle and
Threshold Angle for each peak.
1. If the Review Main window is not displayed, click
select Window > Main Window (Figure 4-29).
2. Click
(Review Main Window) or
(Apply Method Set) or select Process > Apply Method Set.
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107
Integrate Tool
Scroll Here Until
the Peaks Tab
Appears
Apply Method
Set Tool
Review Main
Window Tool
Scroll to the Right to View
Library Match Results
Figure 4-29 Integrated Chromatogram with Library Match Results
3. Use the arrows at the bottom of the Peaks table to scroll until the Peaks tab appears
(Figure 4-29). Click the Peaks tab. The Peaks table displays the results of library
matching.
4. Scroll to the right of the Peaks table (Figure 4-29) to view the portion of the table that
lists the match results, if necessary.
A Match Angle less than a Match Threshold indicates a good match. A Match Angle
greater than the Match Threshold indicates a poor match. Match 1 is the closest
(best) match to a library spectrum; Match 2 is the next closest; Match 3 is the third
closest. For details, see the “Interpreting PDA Library Matching Results” topic in the
Empower Help.
Matching Spectra to a Library 108
4
4.4 Reviewing Library Matching Results
Once you have determined library matching, you can view the results in different ways. For
example, you can examine the apex spectra and the Triple plot by using the Spectrum
Index and the Results window, respectively.
To view the library matching results:
1. Click the Spectrum Index tab to view Spectrum Index in the Review Main window
(Figure 4-30).
Spectrum
Index Tab
Library Match Tool
4
Figure 4-30 Spectrum Index with Library Matching
2. Click
(Library Match) to overlay the library match spectrum with the peak
spectrum in the Spectrum Index plot (Figure 4-31).
Library Matching
109
Overlaid
Spectra
Overlaid Spectra
are Indicated by Retention
Time and are Color Coded
Results
Tool
Figure 4-31 Overlaid Library Match Spectra
By default, the black spectra are the library spectra and the blue spectra are the
peak apex spectra. If these types of spectra overlay well, this is a qualitative
indication of a good match.
Note: The colors displayed in your software can differ from those described here.
3. For another view of the library match results, click
(Results) or select
Window > Results to open the Results window, which contains the Library Match
table and Match Plot (Figure 4-32). If necessary, maximize the Results window or
size the different panes by dragging the split bars.
4. In the Results window, click the Match Plot tab (Figure 4-32). The Library Match
plot displays the peak spectrum overlaid with any possible matches to the library
spectra. The name of the library spectra is displayed in the upper-right side of the
plot. When you select a specific peak from the Peaks tab in the top pane of this
Reviewing Library Matching Results 110
4
window, the Match plot displays the corresponding match data for the selected peak.
Up to three matches can be displayed for each apex spectrum.
Library Match Tab
Names of
Library
Spectra
Match Plot Tab
4
Figure 4-32 Results Window Displaying Library Match Plot
5. Click the Library Match tab in the upper table (Figure 4-33). The Library Match tab
displays the match information for each peak in a numerical format. Select each
peak using the Peak drop-down list. Up to three matches can be displayed for each
apex spectrum.
Library Matching
111
Library Match Table
Triple Plot Tab
Library Match Plot
Figure 4-33 Library Match Plot and Library Match Tab
Note: You can normalize the spectra to visually check the match. Right-click in the
Library Match plot, then select Properties to open the Plot Properties dialog box.
Click the Scaling tab, then select Normalize Y, and click OK.
6. In the lower pane, click the Triple Plot tab (Figure 4-34). The Triple plot shows the
peak spectrum, the library spectrum, and the difference spectrum, which shows the
difference between the peak spectrum and the library spectrum. Again, you can
navigate between peaks using the Peak drop-down list in the Library Match tab or by
selecting the appropriate row in the Peaks tab.
Reviewing Library Matching Results 112
4
Close Button
Peak
Spectrum
Library
Spectrum
Difference
Between
Peak
and Library
Spectrum
Figure 4-34 Triple Plot: Library Match
7. Save the result so you can print it in a report. To save the result, select File > Save
> Result. If Save > Result is not available, select Save > Processing Method
first.
8. Exit Review by clicking
(Close) or selecting File > Exit.
Next Step
You can now proceed to Chapter 5, Printing Reports, to print your results.
Library Matching
113
4
Chapter 5
Printing Reports
When you want to print a report with PDA data (using Empower software), you can:
• Preview the report before printing it.
• Print in the background.
5.1 Previewing a Report
To preview a report before printing it:
1. Go to the Project window (Figure 5-1), which you opened in Chapter 2, Viewing PDA
Data. If the Project window is minimized in the Windows taskbar,
maximize it.
Preview Tool
Results Tab
5
Figure 5-1 Project Window
2. Click the Results tab (Figure 5-1) to view the Results view in the Project window.
Previewing a Report 114
3. Select the data you want to print, then click
(Preview) or select Tools >
Preview/Publisher to open the Open Report Method dialog box (Figure 5-2).
Figure 5-2 Open Report Method Dialog Box
4. Click the Use the following Report Method option button, then select PDA
Default from the list, and click OK to view a preview of the report (Figure 5-3).
5
Printing Reports
115
Print Tool
Annotation Tools
Drop-Down List
Annotation Erase Tool
Figure 5-3 Preview of an Example Report
5. From Preview, you have the following options:
• You can add annotations. For more information on using the Annotation tools,
see Section 2.7, Annotating Chromatograms and Spectra. When annotations are
added in Preview, they appear on the report when the report is saved and
subsequently printed. However, annotations do not become part of the report
method and are not saved with the method.
Note: To modify a method so that all reports that use that method contain the
annotations, use the drawing objects in the report group tree of Report Publisher.
For more information on these tools, see the Empower Help.
• If the report looks the way you want it, click
(Print) (Figure 5-3). The Print
dialog box appears. Make sure that the correct settings are selected, then click
OK. A report similar to the one in Figure 5-6 prints.
Previewing a Report 116
5
• If you want to change the way your report looks, you can modify it by using
Report Publisher. For details, see the “Report Publisher” topic in the Empower
Help.
• To exit without printing, click
(Close), or select File > Exit.
5.2 Background Printing
To print in the background:
1. Go to the Project window (Figure 5-4), which you opened in Chapter 2, Viewing PDA
Data. If the Project window is minimized in the Windows taskbar, maximize it.
Print Tool
Results Tab
Figure 5-4 Project Window
2. Click the Results tab. The Results view appears in the Project window.
3. Select the data you want to print, then click
(Print) in the Project window
(Figure 5-4). The Background Processing and Reporting dialog box appears
(Figure 5-5).
Printing Reports
5
117
Figure 5-5 Background Processing and Reporting Dialog Box
4. In the Reporting section, make sure that Print and Use specified report method
are selected.
5. From the drop-down list next to the Use specified report method option button,
select PDA Default, then click OK. A report similar to the one in Figure 5-6 prints.
5
Background Printing 118
PDA Default
System
Project Name:
SAMPLE
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By:
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
0.40
0.35
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Cpd 2 - 2.166
Cpd 1 - 1.387
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
Sample Set Name:
PDA_Default
0.30
Cpd 3 - 3.931
Reported by User :
AU
0.25
0.20
0.15
0.10
0.05
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes
PDA Result Table
Name
RT
Area
Purity (#1)
Angle
Purity (#1)
Threshold
Purity
Flag
Match (#1)
Spect. Name
Match (#1)
Angle
Match (#1)
Threshold
PDA Match
Flag
1
Cpd 1 1.387
2319720
0.303
0.233
Yes
Methylparaben
0.316
0.206
Yes
2
Cpd 2 2.166
1816222
1.049
0.220
Yes
Ethyl paba
0.325
0.207
Yes
3
Cpd 3 3.931
3059836
0.066
0.230
No
Propiophenone
0.049
0.209
No
Report Method: PDA Default
Printed 10:41:14 PM
4/24/2002
5
Page: 1 of 1
Figure 5-6 Sample Report
Printing Reports
119
Appendix A
Default PDA Reports
A
There are nine default reports available in the PDA Default project. These reports are
available for your direct use or could be used as a starting point to create a customized
report. These reports can be copied from the PDA Default project and pasted into other
projects. You can also transfer these reports by dragging them from one project and
dropping them into another project.
Examples of the sample reports are provided in this appendix. The following reports are in
the PDA Default project:
• 1 Pass Peak Purity Report
• 254 nm Extraction Example Report
• 3D Plot Report
• 4 Pass Peak Purity Report
• LC Default Individual Report
• PDA Default Report
• PDA Purity Lib Match Report
• PDA Purity Plot Report
• PDA Spectrum Index Plot Report
Default PDA Reports
120
A.1 1 Pass Peak Purity Report
A
1Pass Peak Purity
Reported by User :
System
Project Name:
SAMPLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
PDA_Default
INFORMATION
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:06:11 PM
PDA_Demo_Processing
Maxplot 200 350nm
PDA MaxPlot (200.0 nm to 350.0 nm)
Purity Plot
0.30
90.00
80.00
70.00
60.00
AU
50.00
0.20
40.00
Degrees
0.40
Cpd 1 - 1.387
Purity
Auto Threshold
30.00
0.10
20.00
10.00
0.00
0.00
1.30
1.40
1.50
1.60
1.70
1.80
1.90
Minutes
PA: 0.303 TH: 0.233
Peak: Cpd 1 Retention Time: 1.387
Purity
Auto Threshold
Report Method: 1Pass Peak Purity
Printed 10:34:38 PM
4/24/2002
Page: 1 of 3
Default PDA Reports
121
A
1Pass Peak Purity
Reported by User :
System
Project Name:
PDA_Default
Purity Plot
Purity
Auto Threshold
70.00
Cpd 2 - 2.166
0.25
AU
0.20
60.00
50.00
40.00
0.15
Degrees
0.30
30.00
0.10
20.00
0.05
10.00
0.00
0.00
2.00
2.10
2.20
2.30
Minutes
2.40
2.50
2.60
PA: 1.049 TH: 0.220
Peak: Cpd 2 Retention Time: 2.166
Purity
Auto Threshold
Report Method: 1Pass Peak Purity
Printed 10:34:38 PM
4/24/2002
Page: 2 of 3
Default PDA Reports
122
A
1Pass Peak Purity
Reported by User :
System
Project Name:
PDA_Default
Purity Plot
Cpd 3 - 3.931
Purity
Auto Threshold
0.35
0.30
0.25
30.00
25.00
AU
15.00
0.15
0.10
10.00
0.05
5.00
Degrees
20.00
0.20
0.00
0.00
3.70
3.80
3.90
4.00
4.10
Minutes
4.20
4.30
4.40
4.50
PA: 0.066 TH: 0.230
Peak: Cpd 3 Retention Time: 3.931
Purity
Auto Threshold
PDA Result Table
Report Method: 1Pass Peak Purity
Purity (#1)
Angle
Purity (#1)
Threshold
Name
RT
1
Cpd 1
1.387
0.303
0.233
2
Cpd 2
2.166
1.049
0.220
3
Cpd 3
3.931
0.066
0.230
Printed 10:34:38 PM
4/24/2002
Page: 3 of 3
Default PDA Reports
123
A.2 254 nm Extraction Example Report
A
254 nm Extraction Example
Reported by User :
System
Project Name:
SAMPLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
PDA_Default
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method :
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:06:11 PM
PDA_Demo_Processing
Maxplot 200_350nm
PDA MaxPlot (200.0 nm to 350.0 nm)
254 nm Chromatogram
0.30
AU
0.20
0.10
0.00
0.50
1.00
1.50
2.00
2.50
min
3.00
3.50
4.00
4.50
5.00
996 Extraction at 254 nm
Example Extraction Table
Time: 4.000 min
Channel: 996
Wavelength
(nm)
Absorbance
(au)
Wavelength
(nm)
Absorbance
(au)
Wavelength
(nm)
Absorbance
(au)
1
220.37
0.01562
2
221.55
0.01787
15
236.87
0.07518
16
238.05
0.07940
29
253.41
0.06030
30
254.60
3
222.72
0.02056
17
239.23
0.05359
0.08295
31
255.78
4
223.90
0.02371
18
0.04675
240.41
0.08582
32
256.96
5
225.08
0.02739
0.04002
19
241.59
0.08802
33
258.15
6
226.26
0.03372
0.03135
20
242.78
0.08946
34
259.33
7
0.02809
227.44
0.03558
21
243.96
0.08996
35
260.51
0.02323
8
228.62
0.04013
22
245.14
0.08941
36
261.70
0.01919
9
229.80
0.04506
23
246.32
0.08782
37
262.88
0.01592
10
230.97
0.05023
24
247.50
0.08525
38
264.07
0.01338
11
232.15
0.05539
25
248.68
0.08181
39
265.25
0.01147
12
233.33
0.06044
26
249.87
0.07751
40
266.44
0.01012
13
234.51
0.06545
27
251.05
0.07242
41
267.62
0.00921
14
235.69
0.07043
28
252.23
0.06662
42
268.81
0.00863
Report Method: 254 nm Extraction Example
Printed 10:35:47 PM
4/24/2002
Page: 1 of 2
Default PDA Reports
124
A
254 nm Extraction Example
Reported by User :
System
Project Name:
PDA_Default
Example Extraction Table
Time: 4.000 min
Channel: 996
Wavelength
(nm)
Absorbance
(au)
43
269.99
0.00828
44
271.18
0.00810
45
272.36
0.00802
46
273.55
0.00804
47
274.74
0.00809
48
275.92
0.00815
49
277.11
0.00822
50
278.30
0.00827
51
279.48
0.00827
52
280.67
0.00821
53
281.86
0.00808
54
283.05
0.00785
55
284.23
0.00758
56
285.42
0.00730
57
286.61
0.00695
58
287.80
0.00661
59
288.99
0.00622
60
290.17
0.00576
61
291.36
0.00526
62
292.55
0.00471
63
293.74
0.00414
64
294.93
0.00355
65
296.12
0.00302
66
297.31
0.00255
67
298.50
0.00211
68
299.69
0.00180
Report Method: 254 nm Extraction Example
Printed 10:35:47 PM
4/24/2002
Page: 2 of 2
Default PDA Reports
125
A.3 3D Plot Report
A
3D Plot
Reported by User :
System
Project Name:
SAMPLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
PDA_Default
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method :
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:06:11 PM
PDA_Demo_Processing
Maxplot 200_350nm
PDA MaxPlot (200.0 nm to 350.0 nm)
3D Plot
0.40
0.35
0.30
AU
0.25
0.20
0.15
0.10
0.05
0.00
250.00
300.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes
Report Method: 3D Plot
Printed 10:36:51 PM
4/24/2002
Page: 1 of 1
Default PDA Reports
126
A.4 4 Pass Peak Purity Report
A
4 Pass Peak Purity
Reported by User :
System
Project Name:
SAMPLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
Sample Set Name :
PDA_Default
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Purity Plot
60.00
40.00
20.00
0.00
1.40
1.60
1.80
Minutes
Minutes
PA: 0.303 TH: 0.233
PA: 0.062 TH: 0.234
40.00
AU
60.00
0.20
20.00
0.00
0.00
1.40
0.40
80.00
1.60
Purity
Auto Threshold
40.00
20.00
0.00
1.40
Minutes
Report Method: 4 Pass Peak Purity
60.00
0.00
1.80
PA: 0.026 TH: 0.235
80.00
Cpd 1 - 1.387
Purity
Auto Threshold
Degrees
Cpd 1 - 1.387
80.00
Degrees
1.80
Cpd 1 - 1.387
AU
0.20
1.60
Purity
Auto Threshold
0.00
0.00
1.40
0.20
20.00
0.00
0.40
AU
40.00
Degrees
60.00
Degrees
0.20
0.40
80.00
Cpd 1 - 1.387
AU
Purity
0.40
Auto Threshold
1.60
1.80
Minutes
PA: 0.019 TH: 0.235
Peak: Cpd 1 Retention Time 1.387
Purity
Auto Threshold
Printed 10:38:10 PM
4/24/2002
Page: 1 of 3
Default PDA Reports
127
A
4 Pass Peak Purity
Reported by User :
System
Project Name:
PDA_Default
Purity Plot
2.40
2.60
Minutes
PA: 0.119 TH: 0.220
0.30
0.00
2.20
2.40
0.10
Report Method: 4 Pass Peak Purity
20.00
10.00
0.00
2.60
0.00
2.00
Minutes
PA: 0.040 TH: 0.221
2.60
Purity
Auto Threshold
0.20
AU
10.00
Degrees
20.00
0.00
2.00
2.40
Minutes
Cpd 2 - 2.166
AU
0.10
2.20
PA: 1.049 TH: 0.220
Purity
Auto Threshold
0.20
0.00
2.00
Degrees
2.20
10.00
0.00
0.00
2.00
20.00
Degrees
0.10
20.00
0.00
0.30
0.20
AU
40.00
Purity
Auto Threshold
Cpd 2 - 2.166
0.10
Degrees
AU
0.20
60.00
Cpd 2 - 2.166
0.30
Purity
Auto Threshold
Cpd 2 - 2.166
0.30
2.20
2.40
2.60
Minutes
PA: 0.018 TH: 0.222
Peak: Cpd 2 Retention Time 2.166
Purity
Auto Threshold
Printed 10:38:10 PM
4/24/2002
Page: 2 of 3
Default PDA Reports
128
A
4 Pass Peak Purity
Reported by User :
System
Project Name:
PDA_Default
Purity Plot
4.20
4.40
4.00
4.20
4.40
Minutes
Minutes
PA: 0.066 TH: 0.230
PA: 0.033 TH: 0.231
0.00
4.00
4.20
0.20
20.00
10.00
0.00
0.00
3.80
30.00
Cpd 3 - 3.931
10.00
AU
20.00
Degrees
Cpd 3 - 3.931
0.20
Purity
Auto Threshold
30.00
4.40
Degrees
10.00
0.00
3.80
Purity
Auto Threshold
AU
20.00
Degrees
4.00
0.20
0.00
0.00
3.80
30.00
Cpd 3 - 3.931
10.00
0.00
AU
20.00
Degrees
0.20
Purity
Auto Threshold
30.00
Cpd 3 - 3.931
AU
Purity
Auto Threshold
0.00
3.80
4.00
Minutes
4.20
4.40
Minutes
PA: 0.018 TH: 0.232
PA: 0.016 TH: 0.232
Peak: Cpd 3 Retention Time 3.931
Purity
Auto Threshold
PDA Result Table
Purity (#1)
Angle
Purity (#1)
Threshold
Purity (#2)
Angle
Purity (#2)
Threshold
Purity (#3)
Angle
Purity (#3)
Threshold
Name
RT
1
Cpd 1
1.387
0.303
0.233
0.062
0.234
0.026
0.235
2
Cpd 2
2.166
1.049
0.220
0.119
0.220
0.040
0.221
3
Cpd 3
3.931
0.066
0.230
0.033
0.231
0.018
0.232
PDA Result Table
Purity (#4)
Threshold
Purity (#4)
Angle
1
0.235
0.019
2
0.222
0.018
3
0.232
0.016
Report Method: 4 Pass Peak Purity
Printed 10:38:10 PM
4/24/2002
Page: 3 of 3
Default PDA Reports
129
A.5 LC Default Individual Report
A
LC Default Individual Report
System
Project Name:
SAMPLE
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
0.40
0.35
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Cpd 2 - 2.166
Cpd 1 - 1.387
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
PDA_Default
0.30
Cpd 3 - 3.931
Reported by User :
AU
0.25
0.20
0.15
0.10
0.05
0.00
0.50
1.00
Peak Name
1.50
RT
2.00
Area
2.50
Minutes
% Area
3.00
4.00
4.50
5.00
Height
1 Cpd 1
1.387 2319720
32.24 431888
2 Cpd 2
2.166 1816222
25.24 281385
3 Cpd 3
3.931 3059836
42.52 357284
Report Method: LC Default Individual Report
3.50
Printed 10:39:53 PM
4/24/2002
Page: 1 of 1
Default PDA Reports
130
A.6 PDA Default Report
A
PDA Default
System
Project Name:
SAMPLE
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method :
Channel Name:
Proc. Chnl. Descr.:
0.40
0.35
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Cpd 2 - 2.166
Cpd 1 - 1.387
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
PDA_Default
0.30
Cpd 3 - 3.931
Reported by User :
AU
0.25
0.20
0.15
0.10
0.05
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes
PDA Result Table
Purity (#1)
Angle
Purity (#1)
Threshold
Purity
Flag
Match (#1)
Spect. Name
Match (#1)
Angle
Match (#1)
Threshold
PDA Match
Flag
Name
RT
Area
1
Cpd 1
1.387
2319720
0.303
0.233
Yes
Methylparaben
0.316
0.206
Yes
2
Cpd 2
2.166
1816222
1.049
0.220
Yes
Ethyl paba
0.325
0.207
Yes
3
Cpd 3
3.931
3059836
0.066
0.230
No
Propiophenone
0.049
0.209
No
Report Method: PDA Default
Printed 10:41:14 PM
4/24/2002
Page: 1 of 1
Default PDA Reports
131
A.7 PDA Purity Lib Match Report
A
PDA Purity Lib Match Report
Reported by User :
System
Project Name:
SAMPLE
Sample Name :
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Purity Plot
Library Match Plot
Purity
0.45
Auto Threshold
0.35
Cpd 1
Match #1 Angle 0.316 Methylparaben
90.00
Cpd 1 - 1.387
0.40
PDA_Default
80.00
70.00
0.30
AU
50.00
0.20
40.00
M
Degrees
60.00
0.25
0.15
30.00
0.10
20.00
0.05
10.00
0.00
0.00
1.40
1.60
Minutes
1.80
PA: 0.303 TH: 0.233
Peak: Cpd 1
Purity
Auto Threshold
220.00
240.00
260.00
280.00
300.00
320.00
340.00
nm
PDA Result Table
Name
RT
Area
1 Cpd 1 1.387 2319720
Purity
Angle
0.303
Report Method: PDA Purity Lib Match Report
Purity
Threshold
Purity
Flag
0.233 Yes
Match
Spect. Name
Methylparaben
Printed 10:43:13 PM
4/24/2002
Match
Angle
0.316
Match
Threshold
Match
Flag
0.206 Yes
Page: 1 of 3
Default PDA Reports
132
A
PDA Purity Lib Match Report
Reported by User :
System
Project Name:
SAMPLE
Sample Name :
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Purity Plot
Library Match Plot
0.30 Purity
Auto Threshold
60.00
40.00
0.15
Degrees
50.00
0.20
AU
Cpd 2
Match #1 Angle 0.325 Ethyl paba
70.00
Cpd 2 - 2.166
0.25
PDA_Default
30.00
0.10
20.00
0.05
M
10.00
0.00
0.00
2.00
2.20
2.40
2.60
Minutes
PA: 1.049 TH: 0.220
Peak: Cpd 2
Purity
Auto Threshold
220.00
240.00
260.00
280.00
300.00
320.00
340.00
nm
PDA Result Table
Name
RT
Area
1 Cpd 2 2.166 1816222
Purity
Angle
1.049
Report Method: PDA Purity Lib Match Report
Purity
Threshold
Purity
Flag
0.220 Yes
Match
Spect. Name
Match
Angle
Ethyl paba
Printed 10:43:13 PM
4/24/2002
0.325
Match
Threshold
Match
Flag
0.207 Yes
Page: 2 of 3
Default PDA Reports
133
A
PDA Purity Lib Match Report
Reported by User :
System
Project Name:
SAMPLE
Sample Name :
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Purity Plot
Library Match Plot
Cpd 3
Match #1 Angle 0.049 Propiophenone
Purity
Auto Threshold
Cpd 3 - 3.931
0.35
0.30
PDA_Default
30.00
25.00
0.25
AU
M
15.00
Degrees
20.00
0.20
0.15
10.00
0.10
0.05
5.00
0.00
0.00
3.80
4.00
4.20
4.40
Minutes
PA: 0.066 TH: 0.230
Peak: Cpd 3
Purity
Auto Threshold
220.00
240.00
260.00
280.00
300.00
320.00
340.00
nm
PDA Result Table
Name
RT
Area
1 Cpd 3 3.931 3059836
Purity
Angle
0.066
Report Method: PDA Purity Lib Match Report
Purity
Threshold
Purity
Flag
0.230 No
Match
Spect. Name
Propiophenone
Printed 10:43:13 PM
4/24/2002
Match
Angle
0.049
Match
Threshold
Match
Flag
0.209 No
Page: 3 of 3
Default PDA Reports
134
A.8 PDA Purity Plot Report
A
PDA Purity Plot
Reported by User :
System
Project Name:
SAMPLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume :
Run Time:
Sample Set Name :
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
PDA_Default
INFORMATION
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method :
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Purity Plot
Cpd 1 - 1.387
0.40
AU
0.30
0.20
80.00
60.00
M
40.00
0.10
Degrees
Purity
Auto Threshold
20.00
0.00
0.00
1.30
1.40
1.50
1.60
1.70
1.80
1.90
Minutes
PA: 0.303 TH: 0.233
Peak: Cpd 1 Purity Angle 0.303 Purity Threshold 0.233
Purity
Auto Threshold
Report Method: PDA Purity Plot
Printed 10:44:06 PM
4/24/2002
Page: 1 of 4
Default PDA Reports
135
A
PDA Purity Plot
Reported by User :
System
Project Name:
PDA_Default
Purity Plot
Purity
Auto Threshold
70.00
Cpd 2 - 2.166
0.25
AU
0.20
60.00
50.00
40.00
0.15
30.00
Degrees
0.30
0.10
20.00
M
0.05
10.00
0.00
0.00
2.00
2.10
2.20
2.30
2.40
2.50
2.60
Minutes
PA: 1.049 TH: 0.220
Peak: Cpd 2 Purity Angle 1.049 Purity Threshold 0.220
Purity
Auto Threshold
Report Method: PDA Purity Plot
Printed 10:44:06 PM
4/24/2002
Page: 2 of 4
Default PDA Reports
136
A
PDA Purity Plot
Reported by User :
System
Project Name:
PDA_Default
Purity Plot
0.30
0.25
AU
30.00
0.20
25.00
20.00
M
15.00
0.15
0.10
10.00
0.05
5.00
Degrees
Purity
Auto Threshold
Cpd 3 - 3.931
0.35
0.00
0.00
3.70
3.80
3.90
4.00
4.10
4.20
4.30
4.40
4.50
Minutes
PA: 0.066 TH: 0.230
Peak: Cpd 3 Purity Angle 0.066 Purity Threshold 0.230
Purity
Auto Threshold
Report Method: PDA Purity Plot
Printed 10:44:06 PM
4/24/2002
Page: 3 of 4
Default PDA Reports
137
A
PDA Purity Plot
Reported by User :
System
Project Name:
PDA_Default
PDA Result Table
Purity (#1)
Angle
Purity (#1)
Threshold
Match (#1)
Spect. Name
Match (#1)
Angle
Match (#1)
Threshold
Name
RT
1
Cpd 1
1.387
0.303
0.233
Methylparaben
0.316
0.206
2
Cpd 2
2.166
1.049
0.220
Ethyl paba
0.325
0.207
3
Cpd 3
3.931
0.066
0.230
Propiophenone
0.049
0.209
Report Method: PDA Purity Plot
Printed 10:44:06 PM
4/24/2002
Page: 4 of 4
Default PDA Reports
138
A.9 PDA Spectrum Index Plot Report
A
PDA Spectrum Index Plot
Reported by User :
System
Project Name:
SAMPLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
Sample Set Name :
PDA_Default
INFORMATION
Mixture
Unknown
4
1
5.00 ul
5.0 Minutes
Acquired By :
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
DEMO
1/5/1995 6:44:28 PM
PeakPurity
8/20/2001 10:05:42 PM
PDA_Demo_4Pass_Processing
PDA Max Plot 190.0 - 800.0
PDA MaxPlot (190.0 nm to 800.0 nm)
Spectrum Index Plo
Cpd 1 - 1.387
250.00
Cpd 2 - 2.166
nm
Cpd 3 - 3.931
nm
250.00
300.00
250.00
nm
300.00
1.38
300.00
2.16
289.0
3.93
257.0
244.0
AU
0.30
0.20
Cpd 2 - 2.166
Cpd 1 - 1.387
0.40
Cpd 3 - 3.931
221.5
0.10
0.00
0.50
1.00
1.50
2.00
2.50
Minutes
3.00
3.50
4.00
4.50
5.00
PDA Result Table
Name
RT
Area
Purity (#1)
Angle
Purity (#1)
Threshold
Purity
Flag
Match (#1)
Spect . Name
Match (#1)
Angle
Match (#1)
Threshold
PDA Match
Flag
1
Cpd 1
1.387
2319720
0.303
0.233
Yes
Methylparaben
0.316
0.206
Yes
2
Cpd 2
2.166
1816222
1.049
0.220
Yes
Ethyl paba
0.325
0.207
Yes
3
Cpd 3
3.931
3059836
0.066
0.230
No
Propiophenone
0.049
0.209
No
Report Method: PDA Spectrum Index Plot
Printed 10:45:14 PM
4/24/2002
Page: 1 of 1
Default PDA Reports
139
Index
Numerics
3D Channels table 27
enlarging 83
3D data 23, 26
3D plot 29
viewing 27
3D plot 29
colors 29
maximizing 30
rotating 30
tool 28
996/2996 Photodiode Array detector 15
Waters 2996 PDA Detector Operator’s
Guide 18
Waters 996 PDA Detector Operator’s
Guide 18
A
Absorbance
different wavelengths 40
legend 29
maximum 48
Acquisition time 27
Adding spectra to library 82
Annotation Object Properties dialog box 43
text 44
Annotation tools 46
arrow 42
Annotations
adding to chromatogram and spectra 42
Annotation Object Properties dialog box
43
description 41
erasing 45
selecting, resizing, rotating, deleting,
moving 43
I
N
D
E
X
text 43
tools 46
Apex spectrum 69, 70, 109
zoomed 71
Apply Method Set tool 28
AutoThreshold parameter 63
B
Background printing 117
Baseline noise 31, 76
C
Calculating peak purity 47
Calibration curves 17
Channels table 23
Channels View table 25
Chromatogram plot 23, 27, 31, 35
Chromatograms
at different wavelengths 33
deriving 48
displaying peaks 75
extracting 33
full view 32
integrated 64
marker 34
Max Plot 48
overlaid 35
plot 23, 27, 31, 35
zooming 31
Comparing spectra 39
Configuration Manager 20
Contour plot 23, 27
colors 29
zooming 31
Conventions, documentation 13
Index 140
Creating a library 82
assigning name 85
flowchart 80
D
Data
acquisition 17
acquisition time 27
displaying 23, 25
sample name 27
sample PDA 18
sample type 27
viewing 23
zooming 31
Default PDA reports
1Pass Peak Purity Report 121
254 nm Extraction Example Report 124
3D Plot Report 126
4 Pass Peak Purity Report 127
LC Default Individual Report 130
overview 120
PDA Default Report 131
PDA Purity Lib Match Report 132
PDA Purity Plot Report 135
PDA Spectrum Index Plot Report 139
Deriving chromatograms 48
Displaying
apex spectra 69
chromatograms at different wavelengths
34
Contour plot 23
library versus data file spectra 112
peaks 48, 75
Purity plot 75
three-dimensional data 29
Documentation
conventions 13
related 10
E
Empower 15
Empower Configurations
Enterprise 15
Personal 15
Workgroup 15
Empower Help tool 28
Empower PDA software,
prerequisites 17
Empower software
base LC software overview 15
features 15
icons 28
PDA software overview 16
Restore program 18
Empower System Installation and
Configuration Guide 17
Extract a Max/Tic Plot tool 50, 51
Extract Chromatogram tool 28, 34
Extracting
chromatograms 33
spectra 36
I
F
Flowcharts
library creation 80
library matching 81
Full View command 32
Full View tool 28
H
Hardware
for Empower system 15
Waters 996 or 2996 PDA Detector 18
Index 141
I
N
D
E
X
I
Icons. See Tools for Empower software
Integrate tool 28
Integrated chromatogram 64
Integration 17
Interpreting
library matching results 108
peak purity results 68
L
Lamda max 48, 70
Legend for absorbances 29
Library
adding spectra 82
creating 82
creating, flowchart 80
name 86
spectrum 112
See also Library matching
Library Match plot,
normalized 112
Library Match table 17, 110
Library Match tool 28
Library matching 80, 108
flowchart 81
performing 107
processing method for 89, 94
results 108, 109
selecting 92, 102
Login defaults 18
M
Markers, chromatogram 34
Match angle 108
Match threshold 108
Max Plot chromatogram 48
Maximizing
3D Plot window 30
Review 26
Review Main window 26
Maximum absorbance 48
Maximum impurity
point of 77
spectrum 70
Maximum Impurity Pass 1 tool 28
Method Set
opening 83, 90
saving 65, 106
Method Set tool 28
Modifying
processing method 80, 89, 91
reports 117
I
N
Noise interval 61, 103
Normalized
Library Match plot 112
spectra 39, 112
O
Opening
Method Set 83, 90
PDA_Default project 24
project window 48
Overlay tool 28, 35, 36
Overlaying
chromatograms 35
spectra 38
P
Password, default 18
PDA default reports 120
PDA detector 15
PDA Review window, accessing 25
PDA software 16
prerequisites 17
Index 142
I
N
D
E
X
PDA_Default project 18, 24
loading sample data 18
restored 21
Peak purity 47
interpreting results 68
processing method 52, 94
selecting 60
viewing results 69
Peaks 108
apex 77
viewing 48, 75
Peaks table 68, 108
Plot Properties dialog box 33
Plots
3D 29
Chromatogram 23, 27, 35
Contour 23, 27, 29
full view 32
Purity 75
Spectra 17, 23, 27, 37, 39, 86
Triple 112
zooming 31
Prerequisites for Empower PDA software 17
Preview tool 115
Previewing reports 115
Print tool 28
Printing 114
background 117
Report Preview 115
Processing method
for library matching 80, 89, 94
for peak purity 47, 51
modifying 80, 89, 91
name 62
saving 65, 106
tool 28
wizard 51, 94
Processing Method Wizard tool 28
Project window 23
minimized 48
opening 48
Review Data selection mode 24
with results 114, 117
Projects
opening 24, 48
PDA_Default 18, 21 , 24
restoring 18
Purity angle 68, 75, 76
Purity plot 75
Purity threshold 68, 75
I
Q
Quantitation 17
R
Related documentation 10
Report Publisher 117
Reports
default PDA 120
modifying 117
preview 116
printing 114
sample 119
Requirements for Empower PDA software 17
Restore
program 18
Project wizard 20
tool 20
Restoring projects 18
Results
library matching 108, 109, 110
peak purity 68
saving 113
tool 28
Results window
library matching 110
Project window 114, 117
Purity plot 75
with maximum impurity 77
Review
maximizing 26
menus 27
summary 16, 23
Index 143
I
N
D
E
X
tools 27
Review Main window 26
maximizing 26
parts of 27
peak purity results 68
tools 28
Rotating 3D plot 30
S
Sample
name 27
reports 118
type 27
Saving
method set 65 , 106
processing method 65, 106
results 113
zoom parameters 33
Scaling tab 33
Selecting
library matching 92, 102
peak purity testing 60
Setting noise interval 61, 103
Shortcuts 27, 28
Spectra
adding to library 82
apex 69, 109
comparing 39
comparing library versus data file 112
extracting 36
matching results 110
maximum impurity 70
normalized 39, 112
overlaid 38, 70, 71, 109
shape differences 39
zooming 31
Spectra plot 17, 23, 27, 37, 86
normalized 39
zooming 31
Spectra table 17, 27, 38, 40, 86
Spectral Contrast angle 76
Spectral homogeneity 47, 68
Spectrum Index 23, 69, 109
Spectrum matching. See Library matching
Spectrum Points table 17, 41, 42
I
Spectrum Review 17, 27
See also Spectral plot
System
password 18
T
Tables
3D Channels 27
Channels 23
Channels View 25
Library Match 17, 110
Peaks 68, 108
Spectra 17, 27, 38, 40, 86
Spectrum Points 17, 41, 42
Taskbar 48
Three-dimensional data
3D plot 29
in Contour plot 26
viewing 23, 27
Threshold
criteria 63, 104
interpreting 63, 104
Tools for Empower software 28
Triple plot 112
U
Unzoom tool 28
Unzooming windows 32
V
Viewing
apex spectra 69
chromatograms 34
data 23
Index 144
I
N
D
E
X
library versus data file spectra 112
matches to library 110
peaks 48, 75
projects 24
Purity plot 75
Report Preview 115
Spectrum Index 109
three-dimensional data 27
zooming and unzooming data 32
Views
full plot 32
saving parameters 33
zoomed 31
Y
Y-axis 26, 75
Z
I
Zoom box 31
Zoom parameters 33
Zooming
apex spectra 71
plots 31
W
Waters 2996 PDA Detector Operator’s Guide
18
Waters 996 PDA Detector Operator’s Guide
18
Waters 996/2996 PDA Detector 15
Wavelengths, marker for 34
Windows
full view 32
maximizing 26
Project 23, 24, 48, 114
rescaling 32
Results 75, 110, 114
Review Main 27
unzooming 32
Windows taskbar 48
Wizards
processing method 51, 94
restore project 20
X
X-axis 26, 75
Index 145
I
N
D
E
X
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