Data Analysis Software

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Data Analysis Software

for VISION

™

_{, BioCAD}

®

_{700E, SPRINT}

™

_{, and INTEGRAL}

®

Workstations

Version 3 Series Software

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Information in this document is subject to change without notice. Applied Biosystems 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 Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document.

ABI PRISM, Applied Biosystems, BioCAD, CytoFluor, GeneScan, INTEGRAL, POROS, Procise, and Tropix are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

AB (Design), Applera, Data Explorer, Expedite, FMAT, Mariner, PEG-PS, Pioneer, Proteomics Solution 1, SPRINT, VISION, and Voyager are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

ICAT is a trademark of the University of Washington in the U.S. and certain other countries, exclusively licensed to Applied Biosystems Group of Applera Corporation.

MDS is a registered trademark, and SCIEX is a trademark of MDS, Inc. TaqMan is a registered trademark of Roche Molecular Systems, Inc.

Microsoft, MS, MS-DOS, and Windows are registered trademarks of Microsoft Corporation. Pentium is a registered trademark of Intel Corporation.

All other trademarks are the sole property of their respective owners.

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Table of Contents

Chapter 1 Before You Begin

1.1 What You Will Learn ... 1-2 1.2 Data Analysis Software Overview ... 1-3 1.3 Guidelines for Accessing Files in the Data Analysis Module ... 1-6 1.4 Changes to Version 3.0 Software ... 1-8 1.5 Using the Sample Files Provided ... 1-9

Chapter 2 Analyzing Data

2.1 Overview ... 2-2 2.2 Analyzing Using Default Analysis Parameters ... 2-3 2.3 Adjusting Analysis Parameters ... 2-8 2.4 Background Analyzing ... 2-19

Chapter 3 Reporting Results

3.1 Overview ... 3-2 3.2 Generating a Report with Default Report Format Parameters ... 3-3 3.3 Customizing Report Format Parameters ... 3-4 3.4 Printing a Customized Report ... 3-9

Chapter 4 Generating Calibration Curves and

Quantitating Unknowns

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4.6 Entering Calibration Levels and Component Amounts ... 4-10 4.7 Associating Areas With Levels and Generating Calibration Curves ... 4-14 4.8 Adjusting the Report Format ... 4-18 4.9 Creating a Method File for Unknowns ... 4-20 4.10 Quantitating Unknowns ... 4-21

Appendix A Technical Support and Training

A.1 Contacting Technical Support ... A-2 A.2 Obtaining Technical Documents ... A-8 A.3 Obtaining Customer Training Information ... A-10

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1 Before You Begin

This chapter includes the following sections:

1.1 What You Will Learn ... 1-2 1.2 Data Analysis Software Overview ... 1-3 1.3 Guidelines for Accessing Files

in the Data Analysis Module ... 1-6 1.4 Changes to Version 3.0 Software ... 1-7 1.5 Using the Sample Data Files Provided ... 1-8

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1 1.1 What You Will Learn

The following table explains what you will learn in each chapter of the Data Analysis Getting Started Guide:

NOTE: This guide contains brief procedures. For more

detailed procedures and reference information, refer to the Data Analysis Software User’s Guide.

In … You will learn how to …

Chapter 2, Analyzing Data

• Analyze using default analysis parameters • Adjust and save analysis parameters • Background analyze

Chapter 3, Reporting Results

• Generate a report with default report format parameters

• Adjust report format parameters • Print a customized report

Chapter 4, Generating

Calibration Curves and Quantitating Unknowns

• Adjust processing parameters • Enter component information • Generate result files

• Enter calibration levels and component amounts • Associate areas with levels and generate calibration

curves

• Adjust the report format

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Data Analysis Software Overview

1 1.2 Data Analysis

Software Overview

Overview

Applied Biosystems Data Analysis software is part of your BioCAD®_{, VISION}™_{, or INTEGRAL}®_{software version 3.0 or}

later.

Applied Biosystems Data Analysis software includes two modules:

• Group Analysis—Allows you to view, compare, and

export chromatograms.

• Data Analysis—Allows you to process, calibrate, and

report data that you acquire on your SPRINT™_,

BioCAD 700E, VISION, or INTEGRAL 100Q Workstation.

For information on Group Analysis and Data Analysis functions not described in this guide, see the Data Analysis User’s Guide.

Analysis and

report

parameters

The Data Analysis module allows you to adjust the following parameters in a BioCAD, VISION, or INTEGRAL method file (.MET):

• Analysis parameters (.MTH)—Include:

• Processing parameters—Control peak detection,

integration, and report/replot printing

• Calibration parameters—Identify and calibrate

components analyzed

• Report format parameters (.RPT)—Determine

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1

Analysis (.MTH) and report format (.RPT) parameters are embedded in each method (.MET), data (.B*), and group (.GRO) file. Each of these files contains default analysis and report format parameters that you can use as is, or customize.

Figure 1-1 Analysis and Report Format Parameters within a Method File (.MET)

For more information, refer to the Data Analysis User’s Guide.

Assumption

Each BioCAD®_{SPRINT, BioCAD 700E, VISION}™_{, or}

INTEGRAL®_{workstation comes with a Getting Started Guide for}

the workstation. This Data Analysis Software Getting Started

Guide assumes you have already performed the procedures in

the Getting Started Guide provided with your workstation.

Windows

®

₉₅

_{The Data Analysis software is a Windows}®_{95 application and}

this guide assumes familiarity with Windows 95 conventions. See the Introducing Microsoft® Windows® 95 manual provided

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Data Analysis Software Overview

1 Data Analysis

editors and

windows

The Data Analysis module includes:

• Text Method Editor—Allows you to create or change

analysis parameters (processing and calibration). Changes are saved to the analysis section of a method file (.MET), data file (.B*), or group file (.GRO).

• Report Format Editor—Allows you to create or change

reporting parameters. Changes are saved to the report

format section of a method file (.MET), data file (.B*), or

group file (.GRO).

• Graphic Method Editor—Allows you to analyze a data file

and view its chromatogram and allows you to change the analysis parameters (processing and calibration) by graphically manipulating a chromatogram. Changes are saved to the analysis section of a method file (.MET), data file (.B*), or group file (.GRO).

• Fit Analysis Window—Allows you to plot the calibration

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1 1.3 Guidelines for Accessing Files

_{in the Data Analysis Module}

Before using the Data Analysis module, note the following: • You always open or select either a method file (.MET), data

file (.B*), or group file (.GRO) in the Method Editor, Group Analysis window, or Control Panel before accessing the Data Analysis module.

• You cannot open method files (.MET), data files (.B*), or group files (.GRO) directly within the Data Analysis module. • To access a different method file or data file, you must

return to the Group Analysis window or the Method Editor, then select another file and data channel to re-access the Data Analysis module.

• You can view only one data channel (UV #1, UV #2, or Auxiliary) at a time in the Data Analysis module. You select the data channel to view when you open a file.

• You can access analysis parameters (.MTH), report format parameters (.RPT), raw data (.RAW), or results (.RST) sections of a selected method file (.MET), data file (.B*), or group file (.GRO) from within the Data Analysis module. • Once in the Data Analysis module, do not save files as

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Guidelines for Accessing Files in the Data Analysis Module

1

NOTE: Analysis parameters (.MTH), report format

parameters (.RPT), and raw data (.RAW) are not stored as separate, permanent files, but as sections of a method or data file. You can access these sections of a method file or data file only through the Data Analysis module.

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1 1.4 Changes to Version 3.0 Software

Version 3.0

changes

If you have used earlier versions of Applied Biosystems Data Analysis software, major changes you need to know about before using the version 3.0 Data Analysis software are:

• No Analysis Parameters file (.ANP)—The software no

longer uses Analysis Parameter files (.ANP). Instead, each method file (.MET), data file (.B*), and group file (.GRO) includes three Analysis sections (.MTH), one for each data channel. Each analysis section (.MTH) contains processing (peak detection and integration) and calibration

parameters.

• Data channel references—The data channels are now

referred to as UV #1, UV #2, and Auxiliary, not as channels A, B, and C.

• Single data channel—You can view and change

parameters for one data channel at a time. You can set parameters for all three data channels because there are three analysis sections (.MTH) and three report format sections (.RPT) within a method file (.MET), data file (.B*), or group file (.GRO).

• Opening data files—You must open data files from the

Control Panel, Method Editor or Group Analysis module of the BioCAD, VISION, or INTEGRAL software. Doing so opens the Data Analysis module. You cannot open data files directly within the Data Analysis module.

For additional information on changes to version 3.0 software, see the Data Analysis User’s Guide.

Differences from

Turbochrom

Software

The Data Analysis module incorporates the basic data analysis features of PE Nelson™_{Turbochrom software, but does not}

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Using the Sample Files Provided

1 1.5 Using the

Sample Files Provided

Sample files

The following sample data files and group files are provided on your system to use as you perform the procedures in this guide:

• CATMAP.B06 • CALSTD.B00 • CALSTD.B01 • CALSTD.B02 • CALSTD.GRO • CALUNKNWN.B00 • CALUNKNWN.B01 • CALUNKNWN.GRO

Sample data files are located in the \GETTINGSTARTED directory for your system. For example, on a VISION system, data files are located in C:\VISION\GETTINGSTARTED.

CAUTION

The sample files were generated on a VISION Workstation and may not be compatible with BioCAD 700E, SPRINT, or INTEGRAL workstations. Do not try to export the sample data files or open the Method files (.MET): .MET or .CFG from these files on non-VISION systems.

Resetting the

default analysis

and report

parameters

If the sample files provided on your system have been used to perform the procedures in this guide, the analysis and report format sections may have been modified. If they do not contain the default values, your results will not match the results presented in this guide. You can reset the default parameters to ensure the files are the same as when they were used to generate the examples in this Getting Started Guide.

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1 Resetting the data

files

To reset the sample data files:

1. Display the Group Analysis window by selecting Group

Analysis from the Window menu in the Control Panel.

2. Select Open Group from the File menu.

3. Select CALSTD.GRO from the \GETTINGSTARTED directory.

4. Click OK.

The CALSTD.GRO file and its associated data files are displayed in the Group Analysis window.

5. From the Individual section of the Analysis menu, select

Replace Analysis/Report.

The Replace Analysis/Report Format Sections dialog box (Figure 1-2) appears.

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Using the Sample Files Provided

1

6. Select the GETTINGSTARTED directory from the Look in: drop-down list. If you do not see the directory, click the button to the right of the drop-down list.

7. Select Data Files from the Files of type: drop-down list.

8. Select as the destination files:

• CATMAP.B06 • CALSTD.B00 • CALSTD.B01 • CALSTD.B02

Hint: You can select multiple files by holding the Control

key and clicking the file names in the list box.

CAUTION

Do not select the CALUNKNWN files. These files should never be modified from their factory settings.

9. Select UV #1.

10. Select the Analysis source file check box and the

Report format source file check box. The default files

are automatically selected.

11. Click OK.

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1 Resetting the

group file

To reset the sample group file:

1. In the Group Analysis window, select Replace

Analysis/Report from the Group section of the Analysis

menu.

The Replace Analysis/Report Format Sections dialog box (Figure 1-2) appears.

2. Select the GETTINGSTARTED directory from the Look in: drop-down list. If you do not see the directory, click the button to the right of the drop-down list.

3. Select Group Files from the Files of type: drop-down list.

4. Select CALSTD.GRO as the destination file.

CAUTION

Do not select the CALUNKNWN files. These files should never be modified from their factory settings.

5. Select UV #1.

6. Select the Analysis source file check box and the Report

format source file check box. The default files are

automatically selected.

7. Click OK.

The default analysis (.MTH) and report format (.RPT) parameters are copied into the destination file.

For more

information

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2 Analyzing Data

This chapter includes the following sections:

2.1 Overview... 2-2 2.2 Analyzing Using

Default Analysis Parameters ... 2-3 2.3 Adjusting Analysis Parameters... 2-8 2.3.1 Viewing the Peak Report ... 2-8 2.3.2 Adjusting Noise and

Area Thresholds ... 2-10 2.3.3 Adjusting Bunching Factor ... 2-12 2.3.4 Setting Baseline Timed Events ... 2-14 2.3.5 Naming Peaks ... 2-16 2.3.6 Saving the Analysis Parameters ... 2-18 2.3.7 Replacing Analysis Parameters ... 2-18 2.4 Background Analyzing ... 2-19

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2 2.1 Overview

In this chapter

In this chapter, you will learn how to:

• Analyze data, interactively and in the background • Adjust and save analysis parameters

Peak detection

and integration

Peak detection and integration are the processes of extracting information from raw data.

When you analyze a data file, the Data Analysis software reads a raw data file and detects peaks and integrates according to the analysis parameters (.MTH) embedded in the method file (.MET) used to acquire the data.

The integrated data appears as baseline, peaks, and peak information in a chromatogram.

Adjusting

integration and

peak detection

(analysis)

parameters

You can change the way the software identifies baseline, peaks, and peak information by adjusting the analysis parameters in the Graphic Method Editor.

This chapter introduces the techniques you use to work with the Graphic Method Editor. After you develop integration

parameters that suit your needs, you can save them as part of the .MTH section of the data file, and use them for other analyses.

For more

information

For more information on analyzing, adjusting analysis

parameters, and the Graphic Method Editor, see the following sections of the Data Analysis User’s Guide:

• Chapter 4, Analyzing Data

• Chapter 6, Adjusting Processing Parameters

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Analyzing Using Default Analysis Parameters

2 2.2 Analyzing Using

Default Analysis Parameters

The simplest way to integrate the peaks in a chromatogram is to analyze the data file using the default analysis parameters. When you create a method file, it contains a set of default analysis parameters.

This section includes:

• Resetting default analysis and report parameters • Analyzing a chromatogram

• Reviewing the chromatogram • Opening additional files

Resetting

default analysis

and report

parameters

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2 Analyzing a

chromatogram

To analyze a data file using default analysis parameters: 8. Display the Group Analysis window by selecting Group

9. Select New Group from the File menu.

10. Select Select Files from the File menu.

The Select Data Files to Analyze dialog box appears.

Loading the

data file

11. From the \GETTINGSTARTED directory, select

CATMAP.B06 and click Add.

12. Click OK.

The chromatogram for CATMAP.B06 is displayed in the Group Analysis window (Figure 2-3).

Figure 2-3 Chromatogram for CATMAP.B06

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2 Selecting the

data file

14. Select Analyze Chromatogram from the Analysis menu or click on the ribbon at the top of the Group Analysis window.

The Open dialog box appears (Figure 2-4) with CATMAP.B06 as the selected data file.

Figure 2-4 Open Dialog Box

15. From the \GETTINGSTARTED directory, select the

CATMAP.B06 data file.

Selecting the

data channel

16. Select the UV #1 data channel and click OK.

NOTE: All procedures in this guide adjust parameters for

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2

The Graphic Method Editor is displayed.

CATMAP_B06_UV1.mth appears in the title bar. The software opens the raw data section (.RAW) associated with the selected data file and analyzes it using the default analysis parameters (.MTH) embedded in the data file. Then the software displays the chromatogram, showing the resulting integration and peak identification (Figure 2-5).

Figure 2-5 Chromatogram Analyzed with Default Analysis Parameters

Reviewing the

chromatogram

Use the mouse to click-drag and zoom in on the working chromatogram so that the peaks of interest are displayed. Right-click the mouse to return to the previous display. Detected peaks have a retention time peak label at the top of the working chromatogram.

Note that the default analysis parameters detect and integrate baseline noise as peaks. Section 2.3, Adjusting Analysis Parameters, introduces the most common ways you can improve peak detection and integration.

Reference

Working chromatogram

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2 Opening

additional files

You can not open additional data files (or other files) from within the Graphic Method Editor. If you try to open one of these files, an error message is displayed.

To open a different data file:

1. Close the Graphic Method Editor and return to the Group Analysis window.

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2 2.3 Adjusting Analysis Parameters

This section includes:

• Viewing the Peak Report

• Adjusting Noise and Area Thresholds • Adjusting Bunching Factor

• Setting baseline timed events • Naming peaks

• Saving analysis parameters • Replacing analysis parameters

In general, increasing the Noise and Area Thresholds and Bunching Factor minimizes the detection of baseline noise.

2.3.1 Viewing the Peak Report

The Peak Report displays peak information based on the most recent integration of the data. It lists each peak in retention time order, and displays its retention time, area, and other useful information.

To view the Peak Report:

1. In the Graphic Method Editor, select Peak Report from the Display menu.

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Adjusting Analysis Parameters

2

Figure 2-6 Peak Report

The Peak Report may contain many peaks. Use the scroll bar to the right of the Peak Report window to scroll up and down to the peaks of interest.

2. Click to close the Peak Report. Peak

Report

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2 2.3.2 Adjusting Noise and Area Thresholds

Adjust the Noise and Area Thresholds to eliminate noise spikes that are incorrectly detected as peaks.

To adjust Noise and Area Thresholds:

1. Select Noise/Area Threshold from the Process menu.

“RMS Noise” appears in the status bar at the bottom of the screen.

2. Click-drag the mouse to outline a section of noisy baseline on the working chromatogram.

The Noise/Area Threshold dialog box is displayed (Figure 2-7) containing the current Noise Threshold (NT) and Area Threshold (AT) in use and recommended new values based on the chromatogram.

Figure 2-7 Noise/Area Threshold Dialog Box

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2

3. For this exercise, type 50000 in the Suggested AT text box. Do not change the Suggested NT value.

4. Click OK.

The data file is re-integrated using the new thresholds (Figure 2-8).

Figure 2-8 Chromatogram After Adjusting Noise and Area Thresholds

If the chromatogram still includes some spurious peaks, you can continue to increase the Noise Threshold or Area Threshold until the noise peaks are no longer detected.

NOTE: If you inadvertently zoom in on a portion of the

chromatogram, right-click the mouse or select Entire

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2 2.3.3 Adjusting Bunching Factor

A higher Bunching Factor smooths out raw data, which helps prevent the software from identifying baseline noise as peaks.

NOTE: Setting Bunching Factor too high can lead to small,

unresolved peaks being smoothed out completely, so they are undetected.

To adjust the Bunching Factor:

1. In the Graphic Method Editor, select Sampling

Rate/Bunching Factor from the Process menu.

“Peak Width” appears in the status bar.

2. Click-drag the mouse to outline the narrowest peak in the working chromatogram.

For this exercise, outline the small left-most peak at 0.03 minutes.

The Sampling Rate/Bunching Factor dialog box is displayed (Figure 2-9) containing the current Bunching Factor (BF) in use and a recommended new value based on the chromatogram.

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2

NOTE: The Sampling Rate field is not supported.

Sampling Rate corresponds to the Data Rate you set in the configuration section of the method before you acquire data. You cannot adjust Sampling Rate after you have acquired the data.

3. Accept the recommended value by clicking OK.

The data file is reintegrated using the new Bunching Factor (Figure 2-10).

Figure 2-10 Chromatogram After Adjusting Bunching Factor

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2 2.3.4 Setting Baseline Timed Events

If integration is not satisfactory after adjusting the Noise and Area Thresholds and Bunching Factor, you can set baseline timed events to:

• Force integration of undetected peaks • Force baselines

• Manually integrate peaks • Move peak starts and ends

• Skim minor “rider” peaks which are not fully separated from major peaks

Timed events are described in detail in the Data Analysis

User’s Guide.

Disabling peak

detection

To set a baseline timed event to disable peak detection before the first peak of interest:

1. In the Graphic Method Editor, select Baseline Events from the Process menu.

The baseline timed events display appears between the working and reference chromatograms.

2. Select -P Disable Detection from the list.

3. Click on the working chromatogram to the left of the first unwanted peak. For this exercise, click to the left of the small left-most peak at 0.03 minutes.

A -P appears on the chromatogram (Figure 2-11).

4. Select +P Enable Detection from the list.

5. Click on the chromatogram to the left of the first peak of interest. For this exercise, click to the left of the tall peak at 1.32 minutes.

A +P appears on the chromatogram (Figure 2-11).

6. Click to close the baseline timed events display.

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2

Figure 2-11 Setting a Baseline Event to Disable Early Peak Detection

Deleting events

If added events are not effective or are not placed correctly, you can delete the events by doing one of the following:

• Select Delete Events from the menu bar of the baseline timed events display

• Click the Delete Event icon on the baseline timed events display

Peak detection enabled

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2 2.3.5 Naming Peaks

The peaks in the sample chromatogram are (in order) chymotrypsinogen A, cytochrome C, and lysozyme. To enter the peak names:

1. In the Graphic Method Editor, select Edit Components from the Calibration menu.

Peak 1 is selected.

2. In the Name text box, type chymotrypsinogen A.

3. Click Next to select the next peak.

The label CHYM appears below Peak 1.

4. Repeat step 2 and step 3 to name the remaining peaks

cytochrome C and lysozyme.

The Components dialog box should look like the one shown in Figure 2-12 after you enter the information above.

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2

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2 2.3.6 Saving the Analysis Parameters

To save the analysis parameters and peak name information you created in the previous sections:

1. In the Graphic Method Editor, select Save from File menu.

The changes you made are saved to the analysis section (.MTH) of the CATMAP.B06 data file.

The data file is analyzed and the results are stored in the results section (.RST) of the data file.

2. If the Audit Trail or Documentation dialog box is displayed, click OK to close the dialog box.

NOTE: Audit Trail and Documentation features are not

supported in this version of software.

3. Select Exit from the File menu to close the Graphic Method Editor, and return to the Group Analysis window.

2.3.7 Replacing Analysis Parameters

Instead of adjusting analysis parameters, you can copy analysis parameters from one file (source file) to another file (destination file). This procedure replaces the analysis parameters in the destination file.

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Background Analyzing

2 2.4 Background Analyzing

Individual

versus Group

Group Analysis provides two modes of background analysis: • Individual—Analyzes selected data files (up to 100) in

a group file using the individual analysis parameters stored in each data file

• Group—Analyzes selected data files (up to 100) in a

group file using a single set of analysis parameters stored in the group file (.GRO)

This section describes analyzing in Individual mode. For information on creating group files and analyzing in group mode, see the Data Analysis User’s Guide.

Analyzing

individual

To background analyze selected data files in Individual mode: 1. In Group Analysis, select Select Files from the File

menu.

The Select Data Files to Analyze dialog box appears. 2. From the \GETTINGSTARTED directory, select

CATMAP.B06.

3. Click Add and click OK.

The chromatogram for CATMAP.B06 is displayed in the Group Analysis window.

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2

5. Select Analyze Individual from the Analysis menu or click on the ribbon at the top of the Group Analysis window.

The Analyze dialog box appears (Figure 2-13).

Figure 2-13 Analyze Dialog Box

6. Select up to three data channels (UV #1, UV #2, Auxiliary) to analyze.

7. Click OK.

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3 Reporting Results

This chapter includes the following sections:

3.1 Overview ... 3-2 3.2 Generating a Report with

Default Report Format Parameters ... 3-3 3.3 Customizing Report Format Parameters .... 3-4 3.4 Printing a Customized Report ... 3-9

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3 3.1 Overview

In this chapter

In this chapter, you will learn how to: • Generate a report

• Customize Report Format (RPT) parameters • Print the customized report

For more

information

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Generating a Report with Default Report Format Parameters

3 3.2 Generating a Report

with Default Report Format

Parameters

Resetting

default analysis

and report

parameters

If the sample files provided with your system have been previously used to perform the procedures in this or the previous chapter, they may not contain default analysis and report format parameters.

To reset the default parameters, see “Resetting the default analysis and report parameters” on page 1-9.

Generating a

report with

default

parameters

To generate a report using the default report format parameters: 1. Display the Group Analysis window by selecting Group

2. Select New Group from the File menu. 3. Select Select Files from the File menu.

The Select Data Files to Analyze dialog box appears. 4. From the \GETTINGSTARTED directory, select

CATMAP.B06 and click Add.

5. Click OK.

The chromatogram for CATMAP.B06 is displayed in the Group Analysis window.

Printing the report

6. Select the Print check box at the top of the Group Analysis window. Deselect the Export check box.

8. Select UV #1 in the Analyze dialog box and click OK.

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3 3.3 Customizing

Report Format Parameters

Adjusting

To adjust the report format parameters embedded in the CATMAP.B06 data file:

1. In Group Analysis, select Adjust Report Format from the

Individual section of the Analysis menu.

2. Select UV#1 and click OK in the Adjust dialog box.

The default report format is displayed in the Report Format Editor (Figure 3-14). CATMAP.B06 appears in the title bar as CATMAP_B06_UV1.RPT.

.

Figure 3-14 Report Format Editor

Changing the

report title

3. Click the report title Default Report.

The Title dialog box appears.

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Customizing Report Format Parameters

3 Deleting columns

5. Click the Area/Height column of the report.

The Area/Height Ratio dialog box (Figure 3-15) appears.

Figure 3-15 Area/Height Ratio Dialog Box

6. Click Delete to delete the column.

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3 Adding a column

8. Select Component Name from the Report menu.

The Component Name dialog box appears (Figure 3-16).

Figure 3-16 Component Name Dialog Box

9. Type 2 for the column number and 25 for the column width.

10. Click Insert/Add.

Changing the

footer

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Customizing Report Format Parameters

3

The Footer dialog box appears (Figure 3-17).

Figure 3-17 Footer Dialog Box

12. Enter My QC Lab in the text area.

13. Hold down the Ctrl key and press the M key to start a new line.

14. Type We produce the right results and click OK.

15. If the Audit Trail or Documentation dialog box is displayed, click OK to close the dialog box.

NOTE: Audit Trail and Documentation features are not

supported.

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3

Figure 3-18 Finished Report

Saving

To save the report format parameters:

1. In the Report Format Editor, select Save from File menu.

The changes you made are saved to the report format section (.RPT) of the CATMAP.B06 data file.

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Printing a Customized Report

3 3.4 Printing a Customized Report

This section describes: • Printing a report

• Printing a report without the chromatogram

• Printing the chromatogram and report on one page • Rescaling a chromatogram before printing

Printing a report

To print a report using the report format parameters you adjusted in the previous section:

1. Select the Print check box at the top of the Group Analysis window. Deselect the Export check box.

3. Select UV#1 in the Adjust dialog box and click OK.

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3 Printing a report

without the

chromatogram

To print a report without the chromatogram:

1. In Group Analysis, select Adjust Analysis Params from the Individual section of the Analysis menu.

3. In the Text Method Editor, select Replot from the Process menu.

4. In the Replot tab, deselect Generate a separate replot and click OK.

Figure 3-19 Disabling “Generate a separate replot” to Suppress Chromatogram Printing

5. In the Text Method Editor, select Save from the File menu.

7. Select Exit from the File menu to return to Group Analysis.

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Printing a Customized Report

3 Printing the

chromatogram

and report on

one page

To print a chromatogram and report on one page:

1. In Group Analysis, select Adjust Report Format from the

Individual section of the Analysis menu.

2. Select UV#1 and click OK in the Adjust dialog box.

3. In the Report Format Editor, select the Options menu.

4. In the Report Format Options dialog box:

• Select None for System Header • Select Print Replot with Report • Deselect Formfeed Between Reports

Figure 3-20 Setting Report Format Parameters to Print Chromatogram and Report on One Page

Hint: Replot size controls the height of the chromatogram

Set to None

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3

6. In the Report Format Editor, select Save from the File menu.

8. Select Exit from the File menu to return to Group Analysis.

9. In Group Analysis, select Adjust Analysis Params from the Individual section of the Analysis menu.

11. In the Text Method Editor, select Replot from the Process menu.

12. In the Replot dialog box, deselect Generate a separate

replot and click OK.

13. In the Text Method Editor, select Save from the File menu.

15. Select Exit from the File menu to return to Group Analysis.

16. Perform the procedure in “Printing a report” on page 3-9 to print.

Rescaling a

chromatogram

before printing

The chromatogram automatically scales to the highest peak. You can rescale the chromatogram before printing by adjusting the following parameters in the Text Method Editor:

• Deselect Set Plot Limits to Data Limits • Select Absolute Scaling for Scaling Type • Enter appropriate Scaling Parameters

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4 Generating Calibration

Curves and Quantitating

Unknowns

This chapter includes the following sections:

4.1 Overview... 4-2 4.2 Opening the Sample Data Files... 4-4 4.3 Adjusting Processing Parameters ... 4-5 4.4 Entering Component Information... 4-7 4.5 Generating Result Files ... 4-9 4.6 Entering Calibration Levels

and Component Amounts... 4-10 4.7 Associating Areas With Levels

and Generating Calibration Curves ... 4-14 4.8 Adjusting the Report Format ... 4-18 4.9 Creating a Method File for Unknowns ... 4-20 4.10 Quantitating Unknowns ... 4-21

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4 4.1 Overview

In this chapter

In this chapter you will create a three-level calibration curve using the external standards method. Then you will use the curve to quantitate a set of unknowns.

NOTE: It is not necessary to prepare calibration standards

to perform the procedures in this chapter. The software includes sample data files that you can use to construct the calibration curve.

Overview

To generate calibration curves and quantitate unknowns:

Open sample data

files

1. Open the sample data files provided. (When performing a real calibration, you would prepare and run standards first, and then open their data files.) See Section 4.2, Opening the Sample Data Files.

Adjust analysis

parameters

2. In the Graphic Method Editor, adjust:

• Processing parameters (peak

detection/integration) for the standards. See Section 4.3, Adjusting

Processing Parameters.

• Enter component information for the standards. See Section 4.4, Entering Component Information.

Generate result

files

3. In the Group Analysis window, analyze the data files for the standards. The analysis generates result files (.RST) that contain the peak area information needed to generate the calibration curve. See Section 4.5, Generating Result Files.

Enter level and

amount information

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Overview

4 Associate areas

with levels and

generate

calibration curves

5. Associate peak areas with levels and generate the calibration curves. See Section 4.7, Associating Areas With Levels and Generating Calibration Curves.

Adjust

report format

6. In the Report Format Editor, enable the Identified

Components option and add a Raw Amount column to the report format. See Section 4.8, Adjusting the Report Format.

Create method file

for unknowns

7. In the Group Analysis window, save the method file (.MET) embedded in the group file (.GRO) as a

stand-alone method file (.MET). See Section 4.9, Creating a Method File for Unknowns.

Quantitate

unknowns

8. Run and quantitate unknowns using the method created in step 7. See Section 4.10, Quantitating Unknowns.

For more

information

For more information on adjusting calibration parameters, the Text Method Editor, and the Graphic Method Editor, see the following sections of the Data Analysis User’s Guide:

• Section 4.5, Generating Calibration Curves and Quantitating Unknowns

• Chapter 7, Adjusting Calibration Parameters

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4 4.2 Opening the Sample Data Files

Resetting

default analysis

and report

parameters

If the sample data and group files provided on your system have been previously used to perform the procedures in this chapter, they may not contain default analysis and report parameters. To reset the defaults, see “Resetting the default analysis and report parameters” on page 1-9.

Opening data

files

To open the sample data files provided:

1. Display the Group Analysis window by selecting Group

2. Select Open Group from the File menu.

3. Select CALSTD.GRO from the \GETTINGSTARTED directory.

4. Click OK.

The chromatograms for the selected files are displayed in the Group Analysis window (Figure 4-1).

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Adjusting Processing Parameters

4 4.3 Adjusting

Processing Parameters

In this section, you adjust the following processing parameters:

• Noise/Area Threshold • Bunching Factor

Adjusting

To adjust processing parameters for calibration standards: 1. In Group Analysis, click the CALSTD.B00 chromatogram

window to select it.

This standard chromatogram best represents the group. This chromatogram will be displayed when you adjust processing parameters in the Graphic Method Editor.

2. From the Group section of the Analysis menu, select

Adjust Analysis Plot.

3. Select UV #1 and click OK in the Adjust dialog box.

The Graphic Method Editor opens.

4. Select the Noise/Area Threshold from the Process menu. “RMS Noise” appears in the status bar.

5. Click-drag to outline a flat portion of the baseline between the first and second peaks in the working chromatogram.

6. In the Noise/Area Threshold dialog box, ignore the recommended values. Type 5 in the Suggested NT (Noise Threshold) text box.

7. Type 100 in the Suggested AT (Area Threshold) text box.

8. Click OK.

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4

11. Type 5 in the Bunching Factor text box.

12. Click OK.

13. Continue to Section 4.4, Entering

Component Information, before exiting the Graphic Method Editor.

NOTE: The values used in this procedure are specific to

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Entering Component Information

4 4.4 Entering

Component Information

After adjusting processing parameters, enter component information in the group file:

1. In the Graphic Method Editor, select Edit Components from the Calibration menu.

Peak 1 is selected by default.

2. In the Name text box, type Comp1.

3. In the Relative Window (%) text box, type 20.

4. Click Next.

The first five characters of the component name you typed appear under the first peak in the chromatogram display.

5. Repeat step 2 through step 4 for the other two components using the following information:

The Edit Components dialog box should look like Figure 4-2 after you enter the information above.

Name Relative Window (%)

Comp2 3

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4

Figure 4-2 Edit Components

6. Click to close the Edit Components dialog box.

7. Select Save from the File menu.

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Generating Result Files

4 4.5 Generating Result Files

To analyze data files for the standards and generate result files that contain the area information for the calibration: 1. In Group Analysis, deselect the Export and Print check

boxes in the ribbon at the top of the window.

2. Select Analyze Group from the Analysis menu.

3. Select UV #1 in the Analyze dialog box.

4. Select the Save Result Files for Calibration check box.

5. Click OK to analyze the sample data files and generate result files.

Result files are saved in the directory in which the data files are located (in this case, \GETTINGSTARTED), with the following names:

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4 4.6 Entering Calibration Levels and

Component Amounts

After entering component names, enter concentration information for each component level:

1. In Group Analysis, select Adjust Analysis Params from the Group section of the Analysis menu.

2. Select UV #1 and click OK in the Adjust dialog box.

The Text Method Editor opens.

3. In Text Method Editor, select Edit Component from the Components menu.

4. Click the Calibration tab (Figure 4-3).

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Entering Calibration Levels and Component Amounts

4

5. With Component 1 selected, select the following:

6. Leave Origin Treatment, Scaling, and Weighting at the default settings.

7. Enter the following in the calibration table. Press Enter to add rows to the table.

The Components dialog box should look like Figure 4-4 after you enter the information above.

Parameter Select

Calibration Type Use Curve

Response Area

Curve fit type 1st Order

Row of

Table Level Amount Area

1 1 0.1 Leave at default

2 2 0.25 Leave at default

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4

Figure 4-4 Components Dialog Box with Information for Component 1

8. Click Next to move to the next component in the list.

9. Repeat step 5 through step 8 for the other two components in the mixture. Enter the same level and amount information for each component.

10. Click OK to close the Components dialog box.

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Entering Calibration Levels and Component Amounts

4

Figure 4-5 Calibration Curve Before Area/Level Information Entered

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4 4.7 Associating Areas With Levels

and Generating

Calibration Curves

After entering calibration levels, associate areas contained in result files with calibration levels and generate the calibration curves:

1. In the Text Method Editor, select Calibrate from the Components menu.

2. In the Manual Calibration dialog box (Figure 4-6), click .

Figure 4-6 Manual Calibration Dialog Box— Associating Areas in Result Files with Calibration

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Associating Areas With Levels and Generating Calibration Curves

4

3. In the File Select dialog box (Figure 4-7), select GETTINGSTARTED from the Look in: list box.

4. Select the following three files by pressing the

Control key and single-clicking on the file names:

• CALSTD_B00_UV1_CAL.RST • CALSTD_B01_UV1_CAL.RST • CALSTD_B02_UV1_CAL.RST

Figure 4-7 File Select Dialog Box

5. Click Open.

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4

7. In the Level list box, select 1, select Replace to replace the existing replicates with the new replicates, and click Add.

The file is a added to the list box at the bottom of the dialog.

8. Click on the CALSTD_B01_UV1_CAL.RST file.

9. Select Level 2, Replace, and Add.

10. Click on the CALSTD_B02_UV1_CAL.RST file.

11. Select Level 3, Replace, and Add.

The Manual Calibration dialog should look like Figure 4-8.

Figure 4-8 Manual Calibration Dialog Box

12. Click OK.

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Associating Areas With Levels and Generating Calibration Curves

4

The calibration curve for Component 3 should look like Figure 4-9.

Figure 4-9 Calibration Curve for Component 3

13. Display the other curves by clicking on the

component name in the list at the left of the screen.

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4 4.8 Adjusting the Report Format

To adjust the report format to include calibration

information, enable the Identified Components option and add a column for raw amount:

1. In Group Analysis, select Adjust Report Format from the Group section of the Analysis menu.

2. Select UV #1 in the Adjust dialog and click OK.

3. In the Report Format Editor, select Component

Name from the Report menu.

4. Type 1 in the Column Number text box and click

Insert/Add.

The Component Name identifier is placed in column 1 position and the existing columns are shifted to the right.

5. Click the Normalized Area Percent column (column 7) and click Delete.

6. Repeat step 5 for the BL and Area/Height columns.

7. Select Raw Amount from the Report menu and type

2 in the Column Number text box.

8. Click Insert/Add.

9. Click the Default Report title in the report display and type Calibration Report in the Title dialog box. Click OK.

10. Select the Options menu.

11. Select the Identified Components check box.

12. Click OK.

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Adjusting the Report Format

4

Figure 4-10 Report Format for Calibration

15. Select Exit from the File menu to return to Group Analysis.

Copying

analysis/report

parameters from

group to data files

All of the information entered so far has been placed into the CALSTD.GRO file you opened on page 4-4.

To copy the calibration curve information and report format parameters from the group file into the data files, select

Replace All w/ Group Params from the Individual section of

the Analysis menu.

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4 4.9 Creating a Method File

for Unknowns

Overview

In the previous sections you adjusted the analysis (.MTH) and report format (.RPT) parameters of the group file (.GRO) created for the calibration standards. You can save the new parameters by creating a method (.MET) file from the group (.GRO) file. Then use this new method file when acquiring unknowns.

When you save the method information from a group file as a new method file, the new method file contains the adjusted analysis and report format parameters from the group file. For this Getting Started Guide exercise, we have already created a method file for you, using the parameters from the examples in this book. Proceed to Section 4.10, Quantitating Unknowns.

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Quantitating Unknowns

4 4.10 Quantitating Unknowns

Your system includes two sample unknown files

(CALUNKNWN.B00 and CALUNKNWN.B01) generated from the new method created in Section 4.9, Creating a Method File for Unknowns. In this section you use the information

contained in the methods of those data files to quantitate them.

For information on other strategies for quantitation, see the

Data Analysis User’s Guide.

Quantitating

1. In Group Analysis, select Open Group from the File menu.

2. Select CALUNKNWN.GRO and click OK.

CALUNKNWN.B00 and CALUNKNWN.B01 data files are displayed in the Group Analysis window.

3. Select the Print check box at the top of the Group Analysis window to print a report after analysis.

4. Deselect the Export check box.

5. Select Analyze Individual from the Analysis menu.

6. Select UV #1 in the Analyze dialog box and click OK.

Each data file is analyzed using the analysis

parameters stored in the data file. A report is printed for each data file using the report format parameters stored in the data file.

NOTE: If you select Analyze Individual, analysis and

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A

Technical Support and

Training

This appendix contains the following sections:

A.1 Contacting Technical Support ... A-2 A.2 Obtaining Technical Documents... A-8 A.3 Obtaining Customer Training Information... A-10

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A

A.1 Contacting Technical Support

Overview

You can contact Applied Biosystems for technical support: • By e-mail

• By telephone or fax

• Through the Applied Biosystems web site

NOTE: For information on obtaining technical documents

such as Applied Biosystems user documents, MSDSs, and certificates of analysis, see “Obtaining Technical

Documents” on page A-8.

By E-mail

You can contact technical support by e-mail for help in the product areas listed below.

Product/Product Area E-Mail Address

Genetic Analysis (DNA Sequencing) [email protected]

Sequence Detection Systems and PCR [email protected]

Protein Sequencing, Peptide, and DNA Synthesis

[email protected]

• Biochromatography

• Expedite (8900) DNA Synthesis System • PNA

• Pioneer Peptide Synthesis System • Proteomics Solution 1 (PS1) System • ICAT reagent

• FMAT 8100 HTS System • Mariner Mass Spectrometers • Voyager Mass Spectrometers

• CytoFluor®_{4000 Fluorescence Plate Reader}

[email protected]

LC/MS (Applied Biosystems/MDS Sciex) [email protected]

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Contacting Technical Support

A

By telephone or

fax (North

America)

To contact Applied Biosystems Technical Support in North America, use the telephone or fax numbers in the table below.

NOTE: To schedule a service call for other support needs,

or in case of an emergency, dial 1.800.831.6844, then press 1.

Product/Product Area Telephone Fax

ABI PRISM® 3700 DNA Analyzer 1.800.831.6844, then

press 8a

1.650.638.5981

DNA Synthesis 1.800.831.6844, press 2,

then press 1a

1.650.638.5981

Fluorescent DNA Sequencing 1.800.831.6844, press 2,

then press 2a

1.650.638.5981

Fluorescent Fragment Analysis

(including GeneScan®_{applications)} 1.800.831.6844, press 2, _{then press 3}a 1.650.638.5981 Integrated Thermal Cyclers

(ABI PRISM® 877 and Catalyst 800 instruments)

1.800.831.6844, press 2,

then press 4a

1.650.638.5981

ABI PRISM® 3100 Genetic Analyzer 1.800.831.6844, press 2,

then press 6a 1.650.638.5981 Peptide Synthesis (433 and 43x Systems) 1.800.831.6844, press 3, then press 1a 1.650.638.5981

Protein Sequencing (Procise®

Protein Sequencing Systems)

1.800.831.6844, press 3,

then press 2a

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A

PCR and Sequence Detection 1.800.762.4001, then

press:

1 for PCRa 2 for TaqMan®

applications and Sequence Detection Systems including ABI PRISM® 7700, 7900, and 5700a

6 for the 6700 Automated

Sample Prep Systema or 1.800.831.6844, then press 5a 1.240.453.4613 • Voyager MALDI-TOF Biospectrometry Workstations • Mariner ESI-TOF Mass

Spectrometry Workstations • Proteomics Solution 1 (PS1) System • ICAT reagent 1.800.899.5858, press 1, then press 3b 1.508.383.7855 Biochromatography (BioCAD®_,

SPRINT, VISION, and INTEGRAL®_{Workstations and}

POROS®_{Perfusion Chromatography}

Products)

1.800.899.5858, press 1,

then press 4b

1.508.383.7855

Expedite (8900) Nucleic Acid Synthesis Systems

1.800.899.5858, press 1,

then press 5b

1.508.383.7855

Peptide Synthesis (Pioneer and 9050 Plus Peptide Synthesizers)

1.800.899.5858, press 1,

then press 5b

1.508.383.7855

PNA Custom and Synthesis 1.800.899.5858, press 1,

then press 5b

1.508.383.7855

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A

By telephone or

fax (outside North

America)

To contact Applied Biosystems Technical Support or Field Service outside North America, use the telephone or fax numbers below.

FMAT 8100 HTS System

CytoFluor®_{4000 Fluorescence Plate}

Reader 1.800.899.5858, press 1, then press 6b 1.508.383.7855 Chemiluminescence (Tropix) 1.800.542.2369 (U.S. only), or 1.781.271.0045c 1.781.275.8581 LC/MS

(Applied Biosystems/MDS Sciex)

1.800.952.4716 1.508.383.7899

a. 5:30 A.M. to 5:00 P.M. Pacific time. b. 8:00 A.M. to 6:00 P.M. Eastern time. c. 9:00 A.M. to 5:00 P.M. Eastern time.

Product/Product Area Telephone Fax

Region Telephone Fax

Eastern Asia, China, Oceania

Australia (Scoresby, Victoria) 61 3 9730 8600 61 3 9730 8799

China (Beijing) 86 10 64106608 or 86 800 8100497

86 10 64106617

Hong Kong 852 2756 6928 852 2756 6968

Korea (Seoul) 82 2 5936470/6471 82 2 5936472

Malaysia (Petaling Jaya) 60 3 79588268 603 79549043

Singapore 65 896 2168 65 896 2147

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A

Europe Austria (Wien) 43 (0)1 867 35 75 00 43 (0)1 867 35 75 11 Belgium 32 (0)2 532 4484 32 (0)2 582 1886 Denmark (Naerum) 45 45 58 60 00 45 45 58 60 01 Finland (Espoo) 358 (0)9 251 24 250 358 (0)9 251 24 243 France (Paris) 33 (0)1 69 59 85 85 33 (0)1 69 59 85 00 Germany (Weiterstadt) 49 (0) 6150 101 0 49 (0) 6150 101 101 Italy (Milano) 39 (0)39 83891 39 (0)39 838 9492 Norway (Oslo) 47 23 12 06 05 47 23 12 05 75 Portugal (Lisboa) 351.(0)22.605.33.14 351.(0)22.605.33.15

Spain (Tres Cantos) 34.(0)91.806.1210 34.(0)91.806.12.06

Sweden (Stockholm) 46 (0)8 619 4400 46 (0)8 619 4401

Switzerland (Rotkreuz) 41 (0)41 799 7777 41 (0)41 790 0676

The Netherlands (Nieuwerkerk a/d IJssel)

31 (0)180 392400 31 (0)180 392409 or 31 (0)180 392499

United Kingdom (Warrington, Cheshire)

44 (0)1925 825650 44 (0)1925 282502

European Managed Territories (EMT)

Africa, English speaking (Johannesburg, South Africa)

27 11 478 0411 27 11 478 0349

Africa, French speaking (Paris, France)

33 1 69 59 85 11 33 1 69 59 85 00

India (New Delhi) 91 11 653 3743 91 11 653 3744

91 11 653 3138

Poland, Lithuania, Latvia, and Estonia (Warszawa)

48 22 866 4010 48 22 866 4020

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A

Through the

Applied

Biosystems web

site

To contact Technical Support through the Applied Biosystems web site:

1. Go to www.appliedbiosystems.com

2. Click Services & Support at the top of the page, then click Frequently Asked Questions.

3. Click Contact Support in the contents list at the left of the screen.

4. Click your geographic region for the product area of interest.

5. In the Personal Assistance form, enter the requested information and your question, then click Ask Us RIGHT

NOW.

6. In the Customer Information form, enter the requested information, then click Ask Us RIGHT NOW.

For all other EMT countries not listed (Central and southeast Europe, CIS, Middle East, and West Asia

44 1925 282481 44 1925 282509

Japan

Japan (Hacchobori, ChuoKu, Tokyo) 81 3 5566 6230 81 3 5566 6507

Latin America

Caribbean countries, Mexico, and Central America 52 55 35 3610 52 55 66 2308 Brazil 0 800 704 9004 or 55 11 5070 9654 55 11 5070 9694/95 Argentina 800 666 0096 55 11 5070 9694/95 Chile 1230 020 9102 55 11 5070 9694/95 Uruguay 0004 055 654 55 11 5070 9694/95

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A

A.2 Obtaining Technical Documents

Overview

You can obtain technical documents, such as Applied

Biosystems user documents, MSDSs, certificates of analysis, and other related documents for free, 24 hours a day. You can obtain documents:

• By telephone

• Through the Applied Biosystems web site

Ordering

documents by

telephone

To order documents by telephone:

1. From the U.S. or Canada, dial 1.800.487.6809, or from outside the U.S. and Canada, dial 1.858.712.0317. 2. Follow the voice instructions to order documents (for

delivery by fax).

NOTE: There is a limit of five documents per fax request.

Obtaining

documents

through the

web site

To view, download, or order documents through the Applied Biosystems web site:

1. Go to www.appliedbiosystems.com/

2. At the top of the page, click Services & Support at the top of the page, then click Documents on Demand. 3. In the search form, enter and select search criteria, then

click Search at the bottom of the page. 4. In the results screen, do any of the following:

• Click to view a PDF version of the document.

• Right-click , then select Save Target As to download a copy of the PDF file.

• Select the Fax check box, then click Deliver

Selected Documents Now to have the document

faxed to you.

• Select the Email check box, then click Deliver

Selected Documents Now to have the document

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Obtaining Technical Documents

A

NOTE: There is a limit of five documents per fax request,

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A

A.3 Obtaining Customer Training

Information

To obtain Applied Biosystems training information, go to

www.appliedbiosystems.com, click Services & Support at