Empower Software Data Acquisition and Processing Theory Guide 34 Maple Street Milford, MA 01757 71500031209, Revision B NOTICE The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, the use of this document. © 2002 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER. Alliance, Integrity, Millennium, and Waters are registered trademarks, and Empower, LAC/E, SAT/IN, and ZQ are trademarks of Waters Corporation. 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Table of Contents Preface ....................................................................................... 15 Chapter 1 Data Acquisition ............................................................................... 21 1.1 Overview............................................................................... 21 1.1.1 What Is Data Acquisition?.......................................... 21 1.1.2 What Is Processing?.................................................. 21 1.2 Integration Methods .............................................................. 22 1.2.1 Commonalities Between ApexTrack and Traditional Integration.................................................................. 22 1.3 Analog-to-Digital Conversion ................................................ 22 1.3.1 Data Conversion........................................................ 23 1.3.2 Data Transfer and Storage......................................... 23 1.4 Detection Sampling Rates .................................................... 24 1.4.1 Determining the Optimum Sampling Rate................. 25 1.4.2 Displaying the Data Points......................................... 25 1.5 Effects of Data Acquisition Rate on Disk Space ................... 25 1.6 Reference.............................................................................. 26 Chapter 2 ApexTrack Integration ...................................................................... 27 2.1 Features and Capabilities ..................................................... 27 2.1.1 ApexTrack Features................................................... 27 2.1.2 How ApexTrack Performs Integration......................... 28 2.1.3 Summary of Processing Method Parameters............ 28 Table of Contents 3 2.1.4 Summary of Timed Events ........................................ 31 2.1.5 Integration Peak Labels in ApexTrack........................ 32 2.2 Apex Detection...................................................................... 33 2.2.1 Detecting Apices........................................................ 34 2.2.2 Apex Detection Parameters....................................... 34 2.2.3 Obtaining the Second Derivative Plot........................ 35 2.2.4 Detecting the Peak..................................................... 36 2.2.5 Resolved Peaks and Shoulder Peaks........................ 36 2.2.6 Round Peaks ............................................................. 37 2.2.7 Second Derivative Apex and Inflection Point Data in the Peaks Table............................................. 38 2.3 Baseline Location.................................................................. 39 2.3.1 How ApexTrack Determines the Slope Difference Threshold .................................................................. 39 2.3.2 How ApexTrack Locates the Baseline for an Isolated Peak .......................................................................... 41 2.3.3 How ApexTrack Locates the Preliminary Baseline for a Cluster................................................ 42 2.3.4 How ApexTrack Determines the Final Cluster Baseline ........................................................ 43 2.3.5 Effect of Liftoff % and Touchdown % on Baseline Location..................................................................... 43 2.3.6 Effect of Changing Liftoff % and Touchdown % on Cluster Peaks ............................................................ 45 2.4 Determination of Peak Boundaries ....................................... 46 2.4.1 Sequence of Operations............................................ 47 Table of Contents 4 2.5 Computation of Integration Results....................................... 47 2.5.1 Peak Area.................................................................. 47 2.5.2 Peak Height ............................................................... 47 2.5.3 Retention Time .......................................................... 48 2.5.4 Retention Time and Height Values for a Manually Adjusted Peak ........................................................... 49 2.6 Peak Width Parameter.......................................................... 50 2.6.1 Peak Width in ApexTrack ........................................... 50 2.6.2 Auto-Peak Width........................................................ 51 2.6.3 Using Auto-Peak Width.............................................. 52 2.6.4 Effect of Variation of Peak Width Parameter.............. 52 2.7 Detection Threshold Parameter ............................................ 53 2.7.1 Baseline Noise in ApexTrack...................................... 53 2.7.2 AutoThreshold ........................................................... 54 2.7.3 Using AutoThreshold ................................................. 55 2.8 Peak Detection Events.......................................................... 56 2.8.1 Inhibit Integration Event............................................. 56 2.8.2 Detect Shoulders Event............................................. 57 2.8.3 Allow Negative Peaks Event...................................... 58 2.8.4 Set Events ................................................................. 60 2.9 Peak Integration Events........................................................ 66 2.9.1 Valley-to-Valley Event ................................................ 66 2.9.2 Gaussian Skim Event ................................................ 70 2.9.3 Merge Peaks Event for GPC, GPCV, GPC-LS, and GPCV-LS............................................................ 78 2.9.4 Set Liftoff % Event ..................................................... 79 2.9.5 Set Touchdown % Event............................................ 80 Table of Contents 5 2.10 Incompatible Events for ApexTrack ..................................... 80 2.11 When Timed Events Are Active .......................................... 81 2.12 References.......................................................................... 81 Chapter 3 Traditional Integration ...................................................................... 82 3.1 Peak Detection...................................................................... 82 3.1.1 Performing Data Bunching......................................... 83 3.1.2 Determining Peak Start ............................................. 84 3.1.3 Determining Preliminary Peak Apex.......................... 85 3.1.4 Determining Peak End............................................... 85 3.1.5 Determining Peak Width and Threshold Values ........ 86 3.1.6 Inhibiting Integration .................................................. 90 3.2 Peak Integration.................................................................... 91 3.2.1 Determining Fused Peaks ......................................... 91 3.2.2 Constructing the Baseline.......................................... 93 3.2.3 Calculating Peak Retention Time, Height, and Area........................................................................... 95 3.2.4 Peak Rejection Criteria.............................................. 97 3.3 Peak Detection Events.......................................................... 98 3.3.1 Allow Negative Peaks Event...................................... 98 3.3.2 Set Liftoff Event ....................................................... 100 3.3.3 Set Touchdown Event.............................................. 100 3.3.4 Set Peak Width Event.............................................. 101 3.4 Peak Integration Events...................................................... 101 3.4.1 Integration Peak Labels ........................................... 102 3.4.2 Force Baseline Events............................................. 103 Table of Contents 6 3.4.3 Forward Horizontal Events....................................... 106 3.4.4 Reverse Horizontal Events ...................................... 110 3.4.5 Valley-to-Valley Event .............................................. 114 3.4.6 Force Drop Line Event............................................. 115 3.4.7 Force Peak Event..................................................... 117 3.4.8 Skim Events............................................................. 118 3.4.9 Set Minimum Height Event ...................................... 123 3.4.10 Set Minimum Area Event....................................... 123 3.5 Incompatible Events............................................................ 124 3.6 References.......................................................................... 127 Chapter 4 Peak Matching and Quantitation of Sample Components ............. 128 4.1 Peak Matching .................................................................... 128 4.1.1 Calculating the Match Difference............................. 129 4.1.2 Choosing the Optimal Peak Match .......................... 129 4.1.3 Shifting RT and RT Windows................................... 130 4.2 Quantitation......................................................................... 132 4.2.1 Quantitation by Calibration ...................................... 132 4.2.2 Quantitation Without Calibration.............................. 133 4.2.3 Quantitation Using Sample Weight and Dilution .................................................................... 133 4.2.4 Quantitation Using Injection Volume........................ 135 4.2.5 Quantitation Using Responses Other than Peak Area and Height ............................................. 136 4.2.6 External and Internal Standard Quantitation ........... 137 4.2.7 External Standard Quantitation ............................... 137 Table of Contents 7 4.2.8 Internal Standard Quantitation with Separate Standard and Unknown Samples............................ 140 4.2.9 Internal Standard Quantitation Without Separate Standard and Unknown Samples (RF Internal Standard)............................................. 145 4.3 Calibration Curve Fit Types................................................. 148 4.3.1 Single-Level Calibration Curve ................................ 149 4.3.2 Multilevel Calibration Matrix Operations .................. 151 4.3.3 Multilevel Calibration Curves ................................... 154 4.3.4 Multilevel Forced-Through-Zero Calibration Curves..................................................................... 165 4.3.5 Weighting................................................................. 166 4.3.6 Statistics .................................................................. 168 4.4 References.......................................................................... 171 Appendix A Processing Codes .......................................................................... 172 Appendix B Data Processing System Policies................................................... 202 B.1 Use v3.0X Style Peak Width and Threshold Determination .................................................................... 202 B.2 Use v2.XX Style Retention Time Calculations ................... 204 B.3 Prompt User to Save Manual Changes Made in Review............................................................................... 204 B.4 Calculate % Deviation of Point from Curve ....................... 204 Table of Contents 8 Appendix C Getting Started: Processing with ApexTrack................................... 206 C.1 Starting Empower Software ............................................... 206 C.2 Setting Up Peak Integration .............................................. 207 C.3 ApexTrack Integration for the Empower Database ........... 207 C.4 ApexTrack Integration in Projects ..................................... 209 C.4.1 Enabling ApexTrack in a New Project .................... 209 C.4.2 Enabling ApexTrack in an Existing Project ............. 211 C.5 ApexTrack Integration in a Processing Method ................. 212 C.5.1 Creating a New Processing Method with ApexTrack ............................................................... 212 C.5.2 Enabling ApexTrack in an Existing Processing Method .................................................................... 213 C.5.3 Changing the Integration Algorithm in a Processing Method.................................................. 214 C.6 Summary of Integration Events ......................................... 216 C.7 Peak Labels in Result ........................................................ 216 C.8 Manual Integration Guidelines ........................................... 219 C.8.1 Adding or Deleting Peaks ....................................... 219 C.8.2 Moving Peak Starts and Ends................................. 219 C.8.3 Moving Vertical Drops............................................. 220 Index ..................................................................................... 221 Table of Contents 9 List of Figures 1-1 Data Acquisition Process...............................................................23 1-2 Acquired Data Points.....................................................................24 2-1 ApexTrack Default Processing Method..........................................29 2-2 ApexTrack Timed Events ...............................................................31 2-3 Gaussian Peak and its Second Derivative.....................................35 2-4 Baseline Resolved Peak, Valley Boundary, Shoulder Boundary, and Round Boundary ...................................................36 2-5 Fused Peaks with Valley (Left) and Fused Round Peaks (Right)............................................................................................38 2-6 Setting Baseline Parameters.........................................................39 2-7 Inflection Point Baseline.................................................................40 2-8 Computing Slope Differences........................................................40 2-9 Computing the Final Baseline........................................................41 2-10 Example of Search for Baseline in a Tailed Peak ..........................42 2-11 Preliminary Baselines in Cluster Peaks.........................................42 2-12 Cluster Baselines...........................................................................43 2-13 Touchdown of Largest Peak...........................................................44 2-14 Touchdown of 1/10 Peak................................................................45 2-15 Touchdown of 1/100 Peak..............................................................45 2-16 Peak Width Definitions in a Gaussian Peak...................................51 2-17 Second Derivative Showing Baseline Noise..................................53 2-18 Example of Peak-to-Peak Baseline Noise.....................................54 2-19 Automatic Measurement of Baseline Noise...................................55 2-20 Manual Measurement of Baseline Noise.......................................55 2-21 Inhibit Integration Event.................................................................57 2-22 Detect Shoulders Event (Example)................................................58 2-23 Example of Negative Peak Detection ............................................59 2-24 Set Detection Threshold Event......................................................61 List of Figures 10