Data Analysis Software

Transcription

1 Data Analysis Software for VISION, BioCAD 700E, SPRINT, and INTEGRAL Workstations Version 3 Series Software Getting Started Guide DRAFT August 10, :47 pm DASgsg_Title.fm

2 Copyright 1998, 2001, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. 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. Printed in the USA, 06/2001 Part Number Rev. A DRAFT August 10, :47 pm DASgsg_Title.fm

3 Table of Contents Table of Contents Chapter 1 Before You Begin 1.1 What You Will Learn Data Analysis Software Overview Guidelines for Accessing Files in the Data Analysis Module Changes to Version 3.0 Software Using the Sample Files Provided Chapter 2 Analyzing Data 2.1 Overview Analyzing Using Default Analysis Parameters Adjusting Analysis Parameters Background Analyzing Chapter 3 Reporting Results 3.1 Overview Generating a Report with Default Report Format Parameters Customizing Report Format Parameters Printing a Customized Report Chapter 4 Generating Calibration Curves and Quantitating Unknowns 4.1 Overview Opening the Sample Data Files Adjusting Processing Parameters Entering Component Information Generating Result Files Data Analysis Software Getting Started Guide iii

4 Table of Contents 4.6 Entering Calibration Levels and Component Amounts Associating Areas With Levels and Generating Calibration Curves Adjusting the Report Format Creating a Method File for Unknowns Quantitating Unknowns 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 Index iv Applied Biosystems

5 1 Before You Begin 1 This chapter includes the following sections: 1.1 What You Will Learn Data Analysis Software Overview Guidelines for Accessing Files in the Data Analysis Module Changes to Version 3.0 Software Using the Sample Data Files Provided Data Analysis Software Getting Started Guide 1-1

6 Chapter 1 Before You Begin What You Will Learn The following table explains what you will learn in each chapter of the Data Analysis Getting Started Guide: In Chapter 2, Analyzing Data Chapter 3, Reporting Results Chapter 4, Generating Calibration Curves and Quantitating Unknowns You will learn how to Analyze using default analysis parameters Adjust and save analysis parameters Background analyze Generate a report with default report format parameters Adjust report format parameters Print a customized report 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 Create a method file for unknowns Quantitate unknowns NOTE: This guide contains brief procedures. For more detailed procedures and reference information, refer to the Data Analysis Software User s Guide. 1-2 Applied Biosystems

7 Data Analysis Software Overview 1.2 Data Analysis Software Overview 1 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. Analysis and report parameters For information on Group Analysis and Data Analysis functions not described in this guide, see the Data Analysis User s Guide. 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 parameters included in a printed report Data Analysis Software Getting Started Guide 1-3

8 Chapter 1 Before You Begin 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. *.MET System Configuration UV #1.MTH UV #2.MTH AUX.MTH Instrument Control.RPT.RPT.RPT Figure 1-1 Analysis and Report Format Parameters within a Method File (.MET) Assumption Windows 95 For more information, refer to the Data Analysis User s Guide. 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. 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 with the Windows 95 software, for more information. 1-4 Applied Biosystems

9 Data Analysis Software Overview 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 curve for any component that has amount and response data in the analysis section of the associated method file. 1 Data Analysis Software Getting Started Guide 1-5

10 Chapter 1 Before You Begin 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 separate.mth,.rpt, or.rst files. These file types cannot be used by the BioCAD, VISION, or INTEGRAL software. 1-6 Applied Biosystems

11 Guidelines for Accessing Files in the Data Analysis Module 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. 1 Results (.RST) can be stored as separate files for the purpose of creating a calibration curve. Report formats (.RPT) can be stored as separate files for the purpose of selecting optional reports in analysis parameters. Data Analysis Software Getting Started Guide 1-7

12 Chapter 1 Before You Begin 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. Differences from Turbochrom Software For additional information on changes to version 3.0 software, see the Data Analysis User s Guide. The Data Analysis module incorporates the basic data analysis features of PE Nelson Turbochrom software, but does not include instrument control features. The differences between Turbochrom software and Applied Biosystems Data Analysis software are described in the Data Analysis User s Guide. 1-8 Applied Biosystems

13 Using the Sample Files Provided 1.5 Using the Sample Files Provided 1 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. The following two procedures show you how to reset the default parameters in the analysis and report format sections of the sample data files and the sample group file. Data Analysis Software Getting Started Guide 1-9

14 Chapter 1 Before You Begin 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. Figure 1-2 Replace Analysis/Report Format Sections Dialog Box 1-10 Applied Biosystems

15 Using the Sample Files Provided 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 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 # Select the Analysis source file check box and the Report format source file check box. The default files are automatically selected. 11. Click OK. The default analysis (.MTH) and report format (.RPT) parameters are copied into the destination files. Data Analysis Software Getting Started Guide 1-11

16 Chapter 1 Before You Begin 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. For more information 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 on the types of files you can copy parameters from, see the Data Analysis User s Guide Applied Biosystems

17 2 Analyzing Data 2 This chapter includes the following sections: 2.1 Overview Analyzing Using Default Analysis Parameters Adjusting Analysis Parameters Viewing the Peak Report Adjusting Noise and Area Thresholds Adjusting Bunching Factor Setting Baseline Timed Events Naming Peaks Saving the Analysis Parameters Replacing Analysis Parameters Background Analyzing Data Analysis Software Getting Started Guide 2-1

18 Chapter 2 Analyzing Data 2.1 Overview 2 In this chapter Peak detection and integration Adjusting integration and peak detection (analysis) parameters For more information In this chapter, you will learn how to: Analyze data, interactively and in the background Adjust and save analysis parameters 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. 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 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 Chapter 9, Adjusting Analysis Parameters Graphically 2-2 Applied Biosystems

19 Analyzing Using Default Analysis Parameters 2.2 Analyzing Using Default Analysis Parameters Resetting default analysis and report 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 If the sample files provided with your system have been previously used to perform the procedures in this 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 Data Analysis Software Getting Started Guide 2-3

20 Chapter 2 Analyzing Data Analyzing a chromatogram To analyze a data file using default analysis parameters: 8. Display the Group Analysis window by selecting Group Analysis from the Window menu in the Control Panel. 2 Loading the data file 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. 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.B Deselect the Export and the Print check boxes. 2-4 Applied Biosystems

21 Analyzing Using Default Analysis Parameters 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. 2 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 the UV #1 data channel. You can adjust parameters for a different data channel by selecting a different channel when you select the data file. Data Analysis Software Getting Started Guide 2-5

22 Chapter 2 Analyzing Data 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). Reference chromatogram Working chromatogram 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. 2-6 Applied Biosystems

23 Analyzing Using Default Analysis Parameters 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. 2. Repeat the procedure starting on page Data Analysis Software Getting Started Guide 2-7

24 Chapter 2 Analyzing Data 2.3 Adjusting Analysis Parameters 2 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 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. The Peak Report replaces the reference chromatogram at the top of the window (Figure 2-6). 2-8 Applied Biosystems

25 Adjusting Analysis Parameters Peak Report Working chromatogram 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. Data Analysis Software Getting Started Guide 2-9

26 Chapter 2 Analyzing Data Adjusting Noise and Area Thresholds 2 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. Noisy section of baseline Figure 2-7 Noise/Area Threshold Dialog Box 2-10 Applied Biosystems

27 Adjusting Analysis Parameters 3. For this exercise, type 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). 2 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 Chromatogram from the Display menu to return to the display shown in Figure 2-8. Data Analysis Software Getting Started Guide 2-11

28 Chapter 2 Analyzing Data Adjusting Bunching Factor A higher Bunching Factor smooths out raw data, which helps prevent the software from identifying baseline noise as peaks. 2 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. Figure 2-9 Sampling Rate/Bunching Factor Dialog Box 2-12 Applied Biosystems

29 Adjusting Analysis Parameters 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). 2 Figure 2-10 Chromatogram After Adjusting Bunching Factor Most chromatograms will be correctly integrated based on the changes you make to Noise Threshold, Area Threshold, and Bunching Factor only. You can continue to adjust Noise and Area Thresholds, and Bunching Factor, until you obtain acceptable peak detection. Data Analysis Software Getting Started Guide 2-13

30 Chapter 2 Analyzing Data Setting Baseline Timed Events 2 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 Disabling peak detection Timed events are described in detail in the Data Analysis User s Guide. 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. Peak detection is disabled between the -P and +P timed events (Figure 2-11) Applied Biosystems

31 Adjusting Analysis Parameters Peak detection enabled Peak detection disabled 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 Data Analysis Software Getting Started Guide 2-15

32 Chapter 2 Analyzing Data Naming Peaks 2 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 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. 5. Click to close the Edit Components display Applied Biosystems

33 Adjusting Analysis Parameters 2 Figure 2-12 Components Dialog Box With Peak Names Data Analysis Software Getting Started Guide 2-17

34 Chapter 2 Analyzing Data 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. 2 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 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. To replace analysis parameters, follow the procedure in Resetting the default analysis and report parameters on page 1-9, and use the change buttons to select files other than the default files for Analysis source and Report format source Applied Biosystems

35 Background Analyzing 2.4 Background Analyzing Individual versus Group Analyzing individual 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. 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.B Click Add and click OK. The chromatogram for CATMAP.B06 is displayed in the Group Analysis window. 4. Deselect the Print and Export check boxes at the top of the Group Analysis window. 2 Data Analysis Software Getting Started Guide 2-19

36 Chapter 2 Analyzing Data 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). 2 Figure 2-13 Analyze Dialog Box 6. Select up to three data channels (UV #1, UV #2, Auxiliary) to analyze. 7. Click OK. The CATMAP.B06 data file is analyzed and the results are stored in the results section (.RST) of the data file Applied Biosystems

37 3 Reporting Results 3 This chapter includes the following sections: 3.1 Overview Generating a Report with Default Report Format Parameters Customizing Report Format Parameters Printing a Customized Report Data Analysis Software Getting Started Guide 3-1

38 Chapter 3 Reporting Results 3.1 Overview In this chapter For more information In this chapter, you will learn how to: Generate a report Customize Report Format (RPT) parameters Print the customized report For more information on customizing reports and the Report Format Editor see the Data Analysis User s Guide Applied Biosystems

39 Generating a Report with Default Report Format Parameters 3.2 Generating a Report with Default Report Format Parameters Resetting default analysis and report parameters Generating a report with default 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. To generate a report using the default report format parameters: 1. Display the Group Analysis window by selecting Group Analysis from the Window menu in the Control Panel. 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. 3 Printing the report 6. Select the Print check box at the top of the Group Analysis window. Deselect the Export check box. 7. Select Analyze Individual from the Analysis menu or click on the ribbon at the top of the Group Analysis window. 8. Select UV #1 in the Analyze dialog box and click OK. The data file is analyzed and the report prints. Data Analysis Software Getting Started Guide 3-3

40 Chapter 3 Reporting Results 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. 3. Figure 3-14 Report Format Editor Changing the report title 3. Click the report title Default Report. The Title dialog box appears. 4. Type the new title Cation Exchange Analysis Report and click OK. 3-4 Applied Biosystems

41 Customizing Report Format Parameters 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 3 6. Click Delete to delete the column. 7. Repeat step 5 and step 6 for the Norm. Area column and the BL column. Data Analysis Software Getting Started Guide 3-5

42 Chapter 3 Reporting Results Adding a column 8. Select Component Name from the Report menu. The Component Name dialog box appears (Figure 3-16). 3 Figure 3-16 Component Name Dialog Box Changing the footer 9. Type 2 for the column number and 25 for the column width. 10. Click Insert/Add. 11. Select Footer from the User Notes menu. 3-6 Applied Biosystems

43 Customizing Report Format Parameters 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 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. The Report Format Editor dialog box should look like Figure Data Analysis Software Getting Started Guide 3-7

44 Chapter 3 Reporting Results 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. 2. If the Audit Trail or Documentation dialog box is displayed, click OK to close the dialog box. 3. Select Exit from the File menu to close the Report Format Editor, and return to the Group Analysis window. 3-8 Applied Biosystems

45 Printing a Customized Report 3.4 Printing a Customized Report Printing a 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 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. 2. Select Analyze Individual from the Analysis menu or click on the ribbon at the top of the Group Analysis window. 3. Select UV#1 in the Adjust dialog box and click OK. A customized report and chromatogram are printed. 3 Data Analysis Software Getting Started Guide 3-9

46 Chapter 3 Reporting Results 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. 2. Select UV#1 in the Adjust dialog box and click OK. 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. 3 Figure 3-19 Disabling Generate a separate replot to Suppress Chromatogram Printing 5. In the Text Method Editor, select Save from the File menu. 6. If the Audit Trail or Documentation dialog box is displayed, click OK to close the dialog box. 7. Select Exit from the File menu to return to Group Analysis. 8. Perform the procedure in Printing a report on page 3-9 to print Applied Biosystems

47 Printing a Customized Report 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 Set to None 3 Deselect Select Figure 3-20 Setting Report Format Parameters to Print Chromatogram and Report on One Page Hint: Replot size controls the height of the chromatogram plot on the page. 5. Click OK. Data Analysis Software Getting Started Guide 3-11

48 Chapter 3 Reporting Results 6. In the Report Format Editor, select Save from the File menu. 7. If the Audit Trail or Documentation dialog box is displayed, click OK to close the dialog box. 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. 10. Select UV#1 in the Adjust dialog box and click OK. 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 In the Text Method Editor, select Save from the File menu. 14. If the Audit Trail or Documentation dialog box is displayed, click OK to close the dialog box. 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 For more information, see the Data Analysis User s Guide Applied Biosystems

49 4 Generating Calibration Curves and Quantitating Unknowns 4 This chapter includes the following sections: 4.1 Overview Opening the Sample Data Files Adjusting Processing Parameters Entering Component Information Generating Result Files Entering Calibration Levels and Component Amounts Associating Areas With Levels and Generating Calibration Curves Adjusting the Report Format Creating a Method File for Unknowns Quantitating Unknowns Data Analysis Software Getting Started Guide 4-1

50 Section 4 Generating Calibration Curves and Quantitating Unknowns 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. 4 Overview Open sample data files Adjust analysis parameters Generate result files Enter level and amount information To generate calibration curves and quantitate unknowns: 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. 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. 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. 4. In the Text Method Editor, enter calibration levels and component amounts in the group file. See Section 4.6, Entering Calibration Levels and Component Amounts. 4-2 Applied Biosystems

51 Overview Associate areas with levels and generate calibration curves Adjust report format Create method file for unknowns Quantitate unknowns For more information 5. Associate peak areas with levels and generate the calibration curves. See Section 4.7, Associating Areas With Levels and Generating Calibration Curves. 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. 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. 8. Run and quantitate unknowns using the method created in step 7. See Section 4.10, Quantitating Unknowns. 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 Section 9.6, Working with Components 4 Data Analysis Software Getting Started Guide 4-3

52 Section 4 Generating Calibration Curves and Quantitating Unknowns 4.2 Opening the Sample Data Files Resetting default analysis and report parameters Opening data files 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. To open the sample data files provided: 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 chromatograms for the selected files are displayed in the Group Analysis window (Figure 4-1). 4 Figure 4-1 Calibration Standards in Group Analysis 4-4 Applied Biosystems

53 Adjusting Processing Parameters 4.3 Adjusting Processing Parameters Adjusting In this section, you adjust the following processing parameters: Noise/Area Threshold Bunching Factor 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 Type 100 in the Suggested AT (Area Threshold) text box. 8. Click OK. 9. Select Sampling Rate/Bunching Factor from the Process menu. Peak Width appears in the status bar. 10. Click-drag to outline the base of the middle peak in the working chromatogram. Data Analysis Software Getting Started Guide 4-5

54 Section 4 Generating Calibration Curves and Quantitating Unknowns 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 this sample chromatogram. For different chromatograms, you need different values, depending upon baseline noise and other factors Applied Biosystems

55 Entering Component Information 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 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: Name Relative Window (%) Comp2 3 Comp3 3 4 The Edit Components dialog box should look like Figure 4-2 after you enter the information above. Data Analysis Software Getting Started Guide 4-7

56 Section 4 Generating Calibration Curves and Quantitating Unknowns Figure 4-2 Edit Components 4 6. Click to close the Edit Components dialog box. 7. Select Save from the File menu. 8. If the Audit Trail or Documentation dialog box is displayed, click OK to close the dialog box. 9. Select Exit from the File menu to return to the Group Analysis window. 4-8 Applied Biosystems

57 Generating Result Files 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: CALSTD_B00_UV1_CAL.RST CALSTD_B01_UV1_CAL.RST CALSTD_B02_UV1_CAL.RST 4 Data Analysis Software Getting Started Guide 4-9

58 Section 4 Generating Calibration Curves and Quantitating Unknowns 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). 4 Figure 4-3 Components Dialog Box (Calibration Tab) 4-10 Applied Biosystems

59 Entering Calibration Levels and Component Amounts 5. With Component 1 selected, select the following: Parameter Calibration Type Response Curve fit type Select Use Curve Area 1st Order 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. Row of Table Level Amount Area Leave at default Leave at default Leave at default The Components dialog box should look like Figure 4-4 after you enter the information above. 4 Data Analysis Software Getting Started Guide 4-11

60 Section 4 Generating Calibration Curves and Quantitating Unknowns Figure 4-4 Components Dialog Box with Information for Component 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. Component 3 should be highlighted in the components list and a flat calibration curve should be displayed in the graph on the right of the screen (Figure 4-5) Applied Biosystems

61 Entering Calibration Levels and Component Amounts Figure 4-5 Calibration Curve Before Area/Level Information Entered 11. Continue to Section 4.7, Associating Areas With Levels and Generating Calibration Curves, before exiting the Text Method Editor. 4 Data Analysis Software Getting Started Guide 4-13

62 Section 4 Generating Calibration Curves and Quantitating Unknowns 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. 4 Figure 4-6 Manual Calibration Dialog Box Associating Areas in Result Files with Calibration Levels 4-14 Applied Biosystems

63 Associating Areas With Levels and Generating Calibration Curves 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 4 Figure 4-7 File Select Dialog Box 5. Click Open. The selected result files are listed at the top of the Manual Calibration dialog box. 6. Click the CALSTD_B00_UV1_CAL.RST file. Data Analysis Software Getting Started Guide 4-15

64 Section 4 Generating Calibration Curves and Quantitating Unknowns 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 Figure 4-8 Manual Calibration Dialog Box 12. Click OK. The Area/Height column of the calibration table automatically fills with the information from the.rst files Applied Biosystems