Agilent ChemStation Understanding Your ChemStation Understanding Your Agilent ChemStation Agilent Technologies
Notices © Agilent Technologies, Inc. 2004, 2005-2008 No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.
In This Guide... In This Guide... This guide describes various concepts of the Agilent ChemStation. It is intended to increase your understanding of how the ChemStation works. For information on using the ChemStation please refer to the general help system and the Online help "Tutorial". 1 Agilent ChemStation Features This chapter describes the main components and features of the ChemStation. 2 Methods This chapter describes the concepts of methods and how to work with them.
In This Guide... 7 Calibration This chapter describes Calibration in the ChemStation. 8 Automation This chapter describes the concepts of automation. It explains how to work with sequences in ChemStation, what happens when a sequence is run and how to customize sequences. 9 Data Review, Reprocessing and Batch Review This chapter describes the possibilities to review data and how to reprocess sequence data.
Contents Contents 1 Agilent ChemStation Features 9 General Description 11 ChemStation Hardware 14 About the ChemStation Software 15 Instrument Control 31 Documentation 32 The ChemStation Directory Structure 34 Navigation Pane 38 2 Methods 41 What is a Method? 42 Parts of a Method 43 Status of Methods 46 Creating Methods 48 Editing Methods 49 Method Directory Structure 51 What Happens When a Method is Run? Method Operation Summary 57 3 Data Acquisition 59 What is Data Acquisition? Data Files 61 Onlin
Contents Principle of Operation 79 Peak Recognition 80 Baseline Allocation 89 Peak Area Measurement 102 Integration Events 105 Manual Integration 110 5 Quantification 113 What is Quantification? 114 Quantification Calculations 115 Correction Factors 116 Uncalibrated Calculation Procedures 118 Calibrated Calculation Procedures 119 ESTD Calculation 120 Norm% Calculation 122 ISTD Calculation 123 6 Peak Identification 127 What is Peak Identification? 128 Peak Matching Rules 129 Types of Peak Identification
Contents 8 Automation 159 What is Automation? 161 What is a Sequence/Sequence Template? 162 Preferences - Sequence Tab 163 Sequence Parameters 165 Sequence Table 166 Creating Sequences (Sequences and Sequence Templates) 167 Working with Sequences (Sequences and Sequence Templates) 169 Sequence Log File 172 What Happens When a Sequence is Run? 173 Sequence Data File Structure (Unique Folder Creation ON) 175 Data File Naming in a Sequence 176 Postsequence Operation 178 Automatic Recalibration 179 Specifyin
Contents Reporting Custom Field Values 219 Report Styles 220 Other Report Style Parameters 223 Report Destination 224 Sequence Summary Reporting 226 11 Evaluating System Suitability 231 Noise Determination 235 Calculation of Peak Symmetry 240 System Suitability Formulae and Calculations 242 General Definitions 243 Performance Test Definitions 244 Definitions for Reproducibility 250 Internally Stored Double Precision Number Access 255 12 System Verification 259 Verification and Diagnosis Views The GLPsa
Understanding Your Agilent ChemStation 1 Agilent ChemStation Features General Description 11 Additional Instrument Modules 12 Additional Data Evaluation Modules 12 Data Evaluation-only Products 13 ChemStation Hardware 14 About the ChemStation Software 15 Operating System 15 Methods and Sequences 15 System Configuration 15 Data Model 16 File Naming Conventions 16 Software User Interface 18 Data Acquisition 19 Data Analysis — Display 20 Data Analysis — Integration 21 Data Analysis — Quantification 21 Data
1 Agilent ChemStation Features In This Guide... Navigation Buttons 38 ChemStation Explorer 38 This chapter describes the main components and features of the ChemStation.
1 Agilent ChemStation Features General Description General Description The ChemStations for GC, LC, LC/MSD, CE, CE/MSD, and A/D systems are instrument control, data acquisition and data evaluation systems for • Agilent 7890A Gas Chromatographs, • Agilent 6890N, 6890Plus and 6890A Gas Chromatographs, • Agilent 6850 Gas Chromatographs, • 5890 Series II Gas Chromatograph • Agilent 1100/1200 Series modules and systems for LC, • Agilent 1100 Series LC/MSD, Agilent 6100 Series Single Quad LC/MSD • 1090 Series
1 Agilent ChemStation Features General Description • a single instrument analog-to-digital (A/D) ChemStation for analog data acquisition with external event control, product number G2072BA. The instrument control capability of the ChemStation software may be expanded by purchasing additional instrument data acquisition and control modules to allow multiple instrument, mixed technique configurations.
1 Agilent ChemStation Features General Description configured, no more than two diode array detectors are supported on one ChemStation, and the number of supported instruments is restricted to three. When the ChemStation for LC/MS is used to control the Agilent 1100/1200 Series LC/MS module (optionally with one Agilent 1100/1200 Series LC or 1090 Series II LC), no other instruments are supported on the PC.
1 Agilent ChemStation Features ChemStation Hardware ChemStation Hardware For details of ChemStation hardware, see Installing Your ChemStation manual.
Agilent ChemStation Features About the ChemStation Software 1 About the ChemStation Software Operating System The ChemStation requires Microsoft Windows XP Professional SP3 or Windows Vista Business SP1 operating system. The ChemStation Control Charts feature requires MicroSoft Excel. Methods and Sequences The analytical method fully describes how a particular separation is performed.
1 Agilent ChemStation Features About the ChemStation Software Data Model The ChemStation software is designed around a data model based on a memory structure called a register. Registers are multipurpose structures that can hold analytical data and information for both two-dimensional information (for example, time/intensity) and three-dimensional information (for example, time/intensity/wavelength).
Agilent ChemStation Features About the ChemStation Software 1 • COMx (where x is a number from 1 to 9) • LPT1x (where x is a number from 1 to 9) Also avoid these names followed by an extension (e.g. Nul.txt). NOTE English, Japanese, and Chinese operating systems are used to test naming conventions. Agilent cannot give a support statement for non-English operating systems and their special characters.
1 Agilent ChemStation Features About the ChemStation Software NOTE The toolbars displaying data file/sequence/method names have been resized to display up to 18 characters. All ChemStation logbooks report system messages in an extended format and information strings are printed over multiple lines. Certain reports, e.g. Sequence report, may truncate filenames to fit all information onto the report template.
Agilent ChemStation Features About the ChemStation Software 1 allow rapid access to instrument parameters and an animated graphical overview of the status of each analysis as it proceeds. The schematic instrument diagram may be turned off if it is not required, to save memory and other Windows resources.
1 Agilent ChemStation Features About the ChemStation Software During an analysis, the complete functionality of the ChemStation can be used through the offline copy. While acquisition is running, the Data Analysis part of the online session of an instrument is not accessible, and data review has to be performed in the offline copy. A snapshot function is available for users who wish to start processing data before the analysis is completed.
Agilent ChemStation Features About the ChemStation Software 1 • user-defined annotations may be interactively added to the display, with the selection of font, size, text rotation and color (once defined, the annotations may be graphically moved, edited or deleted), • copy the display to the Windows clipboard in both metafile and bitmap format, • a pick mode function to display the values of individual data points in detector units, and • export of time/intensity digitized points to the Microsoft Windows
1 Agilent ChemStation Features About the ChemStation Software Data Analysis — Data Review, Data Reprocess and Batch Review The following two additional toolsets are available within the Data Analysis view : • Navigation Table • Batch Review The Navigation Table makes possible several key graphical operations: • standard table configuration features, such as sorting, drag-and-drop options, column selection, item grouping to specify a preferred navigation table configuration • right mouse click functions to
1 Agilent ChemStation Features About the ChemStation Software Data Analysis — Specialized Reporting Advanced reporting capabilities are also included in the ChemStation for applications that require a more specialized set of reports. These include statistics on separation quality, reports that include trend analysis between samples and user-defined report layouts. System Suitability Reports System suitability reports enable users to report system performance parameters for individual analyses.
1 Agilent ChemStation Features About the ChemStation Software The Extended Performance style adds plots of each individual peak graphically showing the peak start and stop times, half width and baseline.
1 Agilent ChemStation Features About the ChemStation Software organize them in information sections and graphically adjust their relative position size and orientation of each defined element. The individual sections may be added, deleted, reordered and nested. The user may define headers and footers to appear on every page, time stamps for the report and page numbering in the page x of y format. The information included in the report may be any ChemStation or user-defined parameter.
1 Agilent ChemStation Features About the ChemStation Software The ChemStation includes commands and functions to support the dynamic data exchange (DDE) standard of the Microsoft Windows platform as both a DDE client and a DDE server. The command set includes commands to establish and terminate connections, transfer information in both directions and execute remote functions. Customization The ChemStation can be customized using the powerful command set.
1 Agilent ChemStation Features About the ChemStation Software table can be configured by the user. The user can jump between individual cells in the table and copy, cut or paste individual cells or entire rows or series of rows in order to build sequences efficiently and quickly. Samples may be identified in the sequence table as unknowns, calibration or control sample types.
1 Agilent ChemStation Features About the ChemStation Software Good Laboratory Practice The ChemStation is developed to internationally recognized design and development standards and has a number of features specifically to help users operating in a regulated environment. These features are in the area of complete method specification and verification that the methods are fit for their intended use, to check the operation of their system and ensure the traceability, originality and quality of the data.
1 Agilent ChemStation Features About the ChemStation Software The ChemStation may be configured for restricted access for two user access levels, an operator and manager level. The manager level may be password protected and allows access to the complete ChemStation functionality. The operator level restricts the user to key functionality and executing defined analytical methods.
1 Agilent ChemStation Features About the ChemStation Software All reports have time stamps and traceable page numbering (page x of y pagination style). The user may select the level of detail in each report ranging from simple summary reports to complete system details (see the Reporting section above).
1 Agilent ChemStation Features Instrument Control Instrument Control The instrument control capability of the ChemStation may be expanded through the purchase of additional instrument modules to allow multiple instrument, mixed technique configurations. For further information, see the handbook(s) supplied with the additional ChemStation modules.
1 Agilent ChemStation Features Documentation Documentation The documentation set contains specific sections on: • Installing and learning the ChemStation software, • Using the ChemStation software, • Understanding the principles of how the software works, and • Customizing the ChemStation.
Agilent ChemStation Features Documentation 1 Understanding the Principles The Understanding Your ChemStation manual documents the principles of the software operation and the algorithms used in the data manipulations. Customization Sophisticated users who wish to customize the operation of the ChemStation, or who want to build in additional features, may do so by writing macros.
1 Agilent ChemStation Features The ChemStation Directory Structure The ChemStation Directory Structure The following example shows the directory structure of the ChemStation. It comprises generic directories that are shared by all configured instruments and instrument-specific directories. The software installation program creates a subdirectory of the ChemStation directory (by default CHEM32) for each configured instrument with the instrument number.
Agilent ChemStation Features The ChemStation Directory Structure 8=:B(' 1 G:EHINA: 8DG: E>8IJG:H AVc\jV\Z ]ZaeZcj &%') -%% IddaWVgHjeedgi;^aZh A8 <8 8: BH >FI 9G>K:GH AVc\jV\Z -%% IddaWVgHjeedgi;^aZh <8> GVe^Y & I:BE 96I6 9:BD B:I=D9H 9:BD H:FJ:C8: K:G>;N heZXa^Wh Figure 1 ChemStation Directory Structure Understanding Your Agilent ChemStation 35
1 Agilent ChemStation Features The ChemStation Directory Structure Table 3 36 ChemStation subdirectories Directory Contents Chem32 The directory comprises the programs to configure and start the ChemStation software. It must be part of the PATH variable. This directory is added automatically by the installation program unless you provide an alternative. REPSTYLE Used for report templates defined using the Report Template Editor.
1 Agilent ChemStation Features The ChemStation Directory Structure Table 3 ChemStation subdirectories Directory Contents SEQUENCE Comprises the default path for sequence templates. The sequence templates in these directories have a .S extension. Additional sequence paths can be added using Preferences, see “Preferences - Sequence Tab” on page 163 and “Sequence Parameters” on page 165. VERIFY Comprises data files, methods, and the results of data processing stored in register (.REG) files.
1 Agilent ChemStation Features Navigation Pane Navigation Pane A Navigation Pane, available on the left side of all ChemStation views, is designed to speed access to many key ChemStation elements, as well as enabling quick switching between views. The Navigation Pane contains the tree-based ChemStation Explorer and a configurable button area.
Agilent ChemStation Features Navigation Pane Table 4 1 Navigation Pane Items Navigation Buttons ChemStation Explorer Elements Verification (LC and LC/MS) Verification view-specific shortcuts Diagnosis (LC and LC/MS) Diagnosis view-specific shortcuts Tune (LC/MS) Tune view-specific shortcuts Understanding Your Agilent ChemStation 39
1 40 Agilent ChemStation Features Navigation Pane Understanding Your Agilent ChemStation
Understanding Your Agilent ChemStation 2 Methods What is a Method? 42 Parts of a Method 43 Method Information 43 Instrument Control 43 Data Analysis 43 Run-Time Checklist 45 Status of Methods 46 Stored Methods 46 Current Method 47 Creating Methods 48 Editing Methods 49 Method Parts to Edit 49 Method Directory Structure 51 What Happens When a Method is Run? 52 Method Operation 52 Pre-run Command or Macro (Run Time Checklist) 53 Data Acquisition (Run Time Checklist) 53 Data Analysis (Run Time Checkli
2 Methods What is a Method? What is a Method? A method comprises all the parameters for acquisition and data analysis together with pre- and post-run tasks for a given sample, if they are needed. The available methods (*.m) files are visible in the ChemStation Explorer. For quick and easy navigation, you can add additional method locations to the ChemStation Explorer selection tree using the Paths tab of the Preferences dialog box.
Methods Parts of a Method 2 Parts of a Method A method is identified by a name of up to forty alphanumeric characters. The file name always has the .M extension to identify it as a method. Methods are stored as directories that contain individual files relating to the components of the method. Each method comprises four components: • method information, • instrument control, • data analysis, and • run-time checklist.
2 Methods Parts of a Method Signal Details Defines signals and their properties used for data evaluation. Integration Events Defines timed events that will occur at specific retention/migration times on a chromatogram/electropherogram. These timed events can be used to change the way the signal is integrated. Peak Identification Defines data processing parameters associated with the identification of peaks in the chromatogram/electropherogram.
Methods Parts of a Method 2 Run-Time Checklist Defines which parts of the method are executed when the method is run. You can use the run-time checklist to: • acquire, store and process data to produce a report, • execute only a portion of the method, • acquire and store data without analyzing it, • reanalyze existing data files, • use your own macros for data analysis, pre- and post-run processing, and • save the analysis results in a register for GLP purposes.
2 Methods Status of Methods Status of Methods A method can exist in two states: as a stored method, or as the current loaded method. Stored Methods These are methods stored on the computer disk. Stored methods have a name with up to forty alphanumeric characters followed by the extension .M.
2 Methods Status of Methods selected. Changes to this method (for example, timed integrations events) are specific to the associated data file, and are not propagated to the sequence method or the master method. The navigation table offers an update possibility for individual and sequence methods to their corresponding sequence or master method by right-mouse click on the method line item.
2 Methods Creating Methods Creating Methods Creating a new method always means modifying the current method and saving the modifications under a new method name. Be aware that when the current method is changed, the disk version remains unchanged until you save your changes. You have a choice of how to create a method. You can create a method to do either one or all parts of an analysis. For example, you can create a method to do only data acquisition.
2 Methods Editing Methods Editing Methods You can edit an existing method using the Edit Entire Method item of the Method menu. You are guided through all the method dialog boxes and at the end you can save the method.
2 Methods Editing Methods • Instrument Control depends on the configuration, and can comprise, for example: • oven parameters, • injector parameters, and • detector parameters. • Data Analysis comprises: • signal details, • integration parameters, • quantification parameters, • calibration parameters, • custom field setup parameters, and • reporting parameters. • Run Time Checklist comprises: • the parts of the method that will be executed.
Methods Method Directory Structure 2 Method Directory Structure A method comprises a group of files stored in the method directory. The methods subdirectory comprises all the method subdirectories that have a .M extension. Additional methods subdirectories can be added using the preference settings. Method files with the .MTH extension contain parameter sets and are in UNICODE format. The file INFO.MTH comprises the method control parameters.
2 Methods What Happens When a Method is Run? What Happens When a Method is Run? The run-time checklist dialog box specifies the parts of the method to execute when a run is started. There are eight parts to the run-time checklist: • pre-run command or macro, • data acquisition, • standard data analysis, • analysis method for second signal (GC only), • customized data analysis, • save GLP data, • post-run command or macro, and • save copy of method with data (RUN.M).
Methods What Happens When a Method is Run? 8]ZbHiVi^dc hiVijh >c_ZXi^dc HVkZ GJC#B dei^dcVa HVkZ 68F#B YZ[Vjai 2 HVkZ 96#B YZ[Vjai 9ViV ZkVajVi^dc >c_ZXi^dc VcY ^chigjbZci gjc GVl YViV [^aZ XadhZY HiVijh aZkZah Edhigjc bVXgd EgZgjc bVXgd EgZgjc Gjc bZi]dY hiVgiZY k^V 8]ZbHiVi^dc bZcj I^bZ Figure 3 Method Operation Pre-run Command or Macro (Run Time Checklist) If a pre-run command or macro is specified, it is executed before the analysis is started.
2 Methods What Happens When a Method is Run? Data Analysis (Run Time Checklist) When the stop-time has elapsed, the analysis is finished and all rawdata is stored on the computer’s hard disk. The data analysis part of the software starts when all the rawdata is stored. Integration • chromatogram/electropherogram objects in the signal are integrated as specified in the Integration Events dialog box. • The start of the peak, the peak apex, retention/migration time and the end of the peak are determined.
Methods What Happens When a Method is Run? 2 Peak Purity Checking (ChemStations for LC 3D, CE, CE/MS and LC/MS Systems Only) For a peak with UV-Visible spectra, you can calculate a purity factor for that peak and store it in a register. Peak purity may be determined automatically at the end of each analysis as part of the method, if the Check Purity box is checked when specifying an automated library search or when selecting an appropriate report style. See Understanding Your Spectral Module for details.
2 Methods What Happens When a Method is Run? These data are saved only if the Save GLP Data feature is activated by checking the check box in the runtime checklist. You can review, but not edit, GLP data in the data analysis menu of the ChemStation. Postrun Command or Macro (Run Time Checklist) If a postrun command or macro is specified it is executed after the data evaluation, for example, copying data to a disk for data backup.
Methods Method Operation Summary 2 Method Operation Summary The following list shows the flow of the method operation when all parts of the Run Time Checklist are selected. 1 Prerun Command Macro Does a task before the analysis is started. 2 Data Acquisition Does injector program. Injects sample. Acquires raw data. Stores data. 3 Save Copy of Method with Data (RUN.M) - optional by Run Time Checklist 4 Save Copy of Method with Data (ACQ.
2 58 Methods Method Operation Summary Understanding Your Agilent ChemStation
Understanding Your Agilent ChemStation 3 Data Acquisition What is Data Acquisition? Data Files 60 61 Online Monitors 63 Online Signal Monitor 63 Online Spectra Monitor 63 Logbook 64 Status Information 65 ChemStation Status 65 Status Bar 65 System Diagram 66 This chapter describes the concepts of Data Acquisition, data files, logbook, and more.
3 Data Acquisition What is Data Acquisition? What is Data Acquisition? During data acquisition, all signals acquired by the analytical instrument are converted from analog signals to digital signals in the detector. The digital signal is transmitted to the ChemStation electronically and stored in the signal data file. The available data (*.d) files are visible in the ChemStation Explorer.
Data Acquisition Data Files 3 Data Files A data file comprises a group of files, by default stored in the DATA directory as a subdirectory with a data file name and a .D extension. A data file name is can be defined manually using 40 characters including the extension. Each file in the directory follows a naming convention. Additional data directories can be added using the Preferences settings. Table 6 Data files Name Description *.CH Chromatographic/electropherographic signal data files.
3 Data Acquisition Data Files The method can be stored with the result files (run.m). In such cases the method directory is stored as a subdirectory in the data file directory, using the option “Save method with Data” in the Run-Time-Checklist. For data acquired with ChemStation B.02.01 revision and higher, each data folder (*.D) contains the following two methods folders • acquisition method (ACQ.M) for each individual data file • data analysis method (DA.
3 Data Acquisition Online Monitors Online Monitors There are two types of online monitors, the online signal monitor and the online spectra monitor. Online Signal Monitor The online signal monitor allows you to monitor several signals and, if supported by the associated instrument, instrument performance plots in the same window. You can conveniently select the signals you want to view and adjust the time and absorbance axis. For detectors that support this function a balance button is available.
3 Data Acquisition Logbook Logbook The logbook displays messages that are generated by the analytical system. These messages can be error messages, system messages or event messages from a module. The logbook records these events irrespective of whether they are displayed or not. To get more information on an event in the logbook double-click on the appropriate line to display a descriptive help text.
3 Data Acquisition Status Information Status Information ChemStation Status The ChemStation Status window shows a summary status of the ChemStation software.
3 Data Acquisition Status Information System Diagram If supported by the configured analytical instruments (for example, for the Agilent 1100/1200 Series modules for LC or the Agilent 6890 Series GC) you can display a graphical system diagram for your ChemStation system. This allows you to quickly check the system status at a glance. Select the System Diagram item from the View menu of the Method and Run Control View to activate the diagram. It is a graphical representation of your ChemStation system.
Understanding Your Agilent ChemStation 4 Integration What is Integration? 69 What Does Integration Do? 70 The ChemStation Integrator Algorithms Integrator Capabilities 71 71 Overview 73 Defining the Initial Baseline 73 Tracking the Baseline 74 Allocating the Baseline 75 Identifying the Cardinal Points of a Peak Definition of Terms 77 Cardinal Points 77 Solvent Peak 77 Shoulder (front, rear) Slope 78 Principle of Operation 76 78 79 Peak Recognition 80 Peak Width 80 Peak Recognition Filters 81 Bunc
4 Integration Status Information Peak Valley Ratio 92 Tangent Skimming 94 Unassigned Peaks 99 Peak Separation Codes 99 Peak Area Measurement 102 Determination of the area 102 Units and Conversion Factors 104 Integration Events 105 Initial Events 105 Timed Events 108 Autointegrate 108 Manual Integration 110 This chapter describes the concepts of integration the ChemStation integrator algorithms. It describes the integration algorithm, integration and manual integration.
4 Integration What is Integration? What is Integration? Integration locates the peaks in a signal and calculates their size. Integration is a necessary step for: • quantification, • peak purity calculations (ChemStations for LC 3D, CE, CE/MS and LC/MS Systems only), and • spectral library search (ChemStations for LC 3D, CE, CE/MS and LC/MS Systems only).
4 Integration What Does Integration Do? What Does Integration Do? When a signal is integrated, the software: • identifies a start and an end time for each peak and marks these points with vertical tick marks, • finds the apex of each peak; that is, the retention/migration time, • constructs a baseline, and • calculates the area, height and peak width for each peak. This process is controlled by parameters called integration events.
4 Integration The ChemStation Integrator Algorithms The ChemStation Integrator Algorithms The ChemStation integrator algorithm is the second revision of a new generation aimed at improved ruggedness, reliability and ease-of-use.
4 Integration The ChemStation Integrator Algorithms • the ability of baseline correction parameters (non signal related), • integrator control commands defining retention/migration time ranges for the integrator operation. • peak shoulder allocation through the use of second derivative or degree of curvature calculations, • improved sampling of non-equidistant data points for better performance with DAD LC data files that are reconstructed from DAD spectra.
Integration Overview 4 Overview To integrate a chromatogram/electropherogram the integrator: 1 defines the initial baseline, 2 continuously tracks and updates the baseline, 3 identifies the start time for a peak and marks this point with a vertical tick mark, 4 finds the apex of each peak and prints the retention/migration time, 5 identifies the end time for the peak, and marks this point with a vertical tick mark, 6 constructs a baseline, and 7 calculates the area, height, and peak width for each peak.
4 Integration Overview Before the integrator can integrate peaks, it must establish a baseline point. At the beginning of the analysis, the integrator establishes an initial baseline level by taking the first data point as a tentative baseline point. It then attempts to redefine this initial baseline point based on the average of the input signal. If the integrator does not obtain a redefined initial baseline point, it retains the first data point as a potential initial baseline point.
Integration Overview 4 Allocating the Baseline The integrator allocates the chromatographic/electropherographic baseline during the analysis at a frequency determined by the peak width value. When the integrator has sampled a certain number of data points, it resets the baseline from the initial baseline point to the current baseline point. The integrator resumes tracking the baseline over the next set of data points and resets the baseline again.
4 Integration Overview Identifying the Cardinal Points of a Peak The integrator determines that a peak may be starting when potential baseline points lie outside the baseline envelope, and the baseline curvature exceeds a certain value, as determined by the integrator’s slope sensitivity parameter. If this condition continues, the integrator recognizes that it is on the up-slope of a peak, and the peak is processed. Start 1 Slope and curvature within limit: continue tracking the baseline.
Integration Definition of Terms 4 Definition of Terms Cardinal Points eZV` VeZm ed^ci d[ ^c[aZXi^dc WVhZa^cZ ed^ci ]dg^odciVa XddgY^cViZ d[ ZaVehZY i^bZ ed^ci d[ ^c[aZXi^dc WVhZa^cZ ed^ci kZgi^XVa XddgY^cViZ d[ ]Z^\]i Figure 5 Cardinal points Cardinal points are the points chosen by the integrator to define and quantify a peak. Baseline points, valley points, peak apex, and points of inflection are designated cardinal points and saved.
4 Integration Definition of Terms Shoulder (front, rear) Shoulders occur when two peaks elute so close together that no valley exists between them, and they are unresolved. Shoulders may occur on the leading edge (front) of the peak, or on the trailing edge (rear) of the peak. When shoulders are detected, they may be integrated either by tangent skim or by drop-lines.
Integration Principle of Operation 4 Principle of Operation >c^i^Va eVgVbZiZgh 9Z[^cZ ^c^i^Va WVhZa^cZ >c^i^Va^oVi^dc IgVX` VcY gZhZi WVhZa^cZ EZV` XajhiZg hiVgi 7VhZa^cZ igVX`^c\ I^bZY ZkZcih >YZci^[n eZV` XddgY^cViZh 9ZiZXi WVhZa^cZ EZV` XajhiZg ZcY 8ajhiZg YZiZXi^dc 8dchigjXi eZV` 9ZiZXi h`^bbZghQmY* HidgZ eZV`h ^c EZV` IVWaZ Figure 6 8VaXjaViZ eZV` hiVi^hi^Xh 8ajhiZg ZkVajVi^dc Integrator Flow Diagram Understanding Your Agilent ChemStation 79
4 Integration Peak Recognition Peak Recognition The integrator uses several tools to recognize and characterize a peak: • peak width, • peak recognition filters, • bunching, • peak recognition algorithm, • peak apex algorithm, and • non-Gaussian calculations (for example tailing, merged peaks). Peak Width During integration, the peak width is calculated from the peak area and height: Width = Area/Height or, if the inflection points are available, from the width between the inflection points.
4 Integration Peak Recognition In Figure Peak Width Calculation, the total area, A, is the sum of the areas a1, a2, a3 and a4. Fs is the front slope at the inflection point, Rs is the rear slope at the inflection point. If either inflection point is not found, the peak width is defined as: Width = Adjusted area / Adjusted height The peak width setting controls the ability of the integrator to distinguish peaks from baseline noise.
4 Integration Peak Recognition To obtain good performance from the recognition filters, the peak width must be set close to the width of the actual chromatographic/electropherographic peaks. During the run, the integrator updates the peak width as necessary to optimize the integration. The integrator calculates the updated peak width in different ways, depending on the instrument configuration: For LC/CE configurations, the default peak width calculation uses a composite calculation: 0.
4 Integration Peak Recognition When data is bunched, the data points are bunched as two raised to the bunching power, i.e. unbunched = 1x, bunched once = 2x, bunched twice = 4x etc. Bunching is based on the data rate and the peak width. The integrator uses these parameters to set the bunching factor to give the appropriate number of data points Table 8 on page 83. Bunching is performed in the powers of two based on the expected or experienced peak width.
4 Integration Peak Recognition HiVgije i' i& 7VhZa^cZ i( i* JehadeZ i) i&& i&% 9dlchadeZ i, i- i+ :migVXi EZV` i.
Integration Peak Recognition t6 4 • Peak valley found and upslope counter is greater than or equal to 2 or • Downslope sigma is greater than twice peak end sigma or • Baseline reset now or • Baseline reset next valley and peak valley found t7 Downslope criterion is no longer met t8 Downslope criterion is met again t9 • Peak valley found and upslope counter is greater than or equal to 2 or • Downslope counter equals zero or • Downslope sigma is greater than peak end sigma or • Baseline reset now or •
4 Integration Peak Recognition Peak End In Table 10 on page 86the expected peak width determines which filter’s slope and curvature values are compared with the Slope Sensitivity. For example, when the expected peak width is small, Filter 1 numbers are added to the down-slope accumulator. If the expected peak width increases, then the numbers for Filter 2 and, eventually, Filter 3 are used. When the value of the down-slope accumulator is recognizes that a peak may be ending.
Integration Peak Recognition 4 KVaaZn ed^ci Figure 9 Merged Peaks The integrator processes merged peaks in the following way: 1 it sums the area of the first peak until the valley point. 2 at the valley point, area summation for the first peak ends and summation for the second peak begins. 3 when the integrator locates the end of the second peak, the area summation stops.
4 Integration Peak Recognition &*#**% V W &*#)&% &*#+.% Figure 10 Peak Shoulders Shoulders are detected from the curvature of the peak as given by the second derivative. When the curvature goes to zero, the integrator identifies a point of inflection, such as points a and b in Figure 10 on page 88. • A potential front shoulder exists when a second inflection point is detected before the peak apex.
Integration Baseline Allocation 4 Baseline Allocation After any peak cluster is complete, and the baseline is found, the integrator requests the baseline allocation algorithm to allocate the baseline using a pegs-and-thread technique. It uses trapezoidal area and proportional height corrections to normalize and maintain the lowest possible baseline.
4 Integration Baseline Allocation The Start of the Baseline If no baseline is found at the start of the run, the start of the baseline is established in one of the following ways: • from the start of the run to the first baseline point, if the start of run point is lower than the first baseline point, • from the start of the run to the first valley point, if the start of run point is lower than the first valley, • from the start of the run to the first valley point, if the first valley penetrates an imagi
Integration Baseline Allocation 4 Baseline Penetration A penetration occurs when the signal drops below the constructed baseline (point a in Figure 12 on page 91). If a baseline penetration occurs, that part of the baseline is generally reconstructed, as shown by points b in Figure 12 on page 91.
4 Integration Baseline Allocation HiVcYVgY WVhZa^cZ igVX`^c\ Figure 13 NOTE 7VhZa^cZ igVX`^c\ cd eZcZigVi^dc Standard baseline tracking and baseline tracking (no penetration) Baseline tracking (no penetration) is not available for solvent peaks, with their child peaks and shoulders.
Integration Baseline Allocation =& 4 =' =k Figure 14 Peak Valley Ratio The peak valley ratio is calculated using the following equations: H1 ¡ H2, Peak valley ratio = H2/Hv and H1 < H2, Peak valley ratio = H1/Hv Figure 15 on page 93 shows how the user-specified value of the peak valley ratio affects the baselines. &-#*'. &-#*'. &.#%+% EZV` kVaaZn gVi^d adlZg i]Vc jhZg"heZX^[^ZY kVajZ Figure 15 &.
4 Integration Baseline Allocation Tangent Skimming Tangent skimming is a form of baseline constructed for peaks found on the upslope or downslope of a peak. When tangent skimming is enabled, four models are available to calculate suitable peak areas: • exponential curve fitting, • new exponential skim • straight line skim, • combined exponential and straight line calculations for the best fit (standard skims).
Integration Baseline Allocation 4 peaks after the first child peak are separated by drop lines, beginning at the end of the first child peak, and are dropped only to the skim (see Figure 17 on page 95). EVgZci eZV` &)#'.+ &)#(*, 8]^aY eZV`h &)#&)) 9gde a^cZh :medcZci^Va h`^b XjgkZ 7VhZa^cZ d[ eVgZci eZV` Figure 17 New mode exponential skim Straight Line Skim This skim model draws a straight line through the start and end of a child peak.
4 Integration Baseline Allocation Standard Skims The appropriate calculation is chosen for a particular application; by default, the chosen method is a combination of exponential and straight line calculations for the best fit. The switch from an exponential to a linear calculation is performed in a way that eliminates abrupt discontinuities of heights or areas. • When the signal is well above the baseline, the tail-fitting calculation is exponential.
Integration Baseline Allocation 4 You can disable exponential skimming throughout the run by setting the value of the tail skim height ratio to a high value or to zero. Valley Height Ratio is the ratio of the height of the child peak above the baseline (Hc in Figure 19 on page 96) to the height of the valley above the baseline (Hv in same figure). This ratio must be smaller than the specified value for the child peak to be skimmed.
4 Integration Baseline Allocation The exponential model is fitted through the part of the tail of the parent peak immediately before the first child peak. Figure 21 on page 98 shows the corrected curve of a child peak after tangent skimming. 8dggZXiZY X]^aY eZV` XjgkZ Figure 21 Tail-corrected child peak Front Peak Skimming As for child peaks on the tail of a parent peak, special integration is required for some peaks on the front/upslope of a peak, see Figure 22 on page 98.
Integration Baseline Allocation 4 The valley height ratio takes the same value for both front peak skimming and tail peak skimming (see "Valley height ratio"); the front skim height ratio is calculated in the same way as the tail skim height ratio (see "Tail skim height ratio"), but can have a different value. Unassigned Peaks With some baseline constructions, there are small areas that are above the baseline and below the signal, but are not part of any recognized peaks.
4 Integration Baseline Allocation B The peak started or stopped on the baseline. V The peak started or stopped with a valley drop-line. P The peak started or stopped while the baseline was penetrated. H The peak started or stopped on a forced horizontal baseline. F The peak started or stopped on a forced point. M The peak was manually integrated. U The peak was unassigned. Additional flags may also be appended (in order of precedence): Character 3 D The peak was distorted.
Integration Baseline Allocation f Peak defined by a front shoulder tangent. b Peak defined by a rear shoulder tangent. F Peak defined by a front shoulder drop-line. B Peak defined by a rear shoulder drop-line. U The peak is unassigned.
4 Integration Peak Area Measurement Peak Area Measurement The final step in peak integration is determining the final area of the peak. Peak areas are calculated from the contents of the cardinal point file. Cardinal points are the points chosen by the integrator to define and quantify a peak (see “Identifying the Cardinal Points of a Peak” on page 76). These include baseline points, valley points, peak apex, points at peak half height.
Integration Peak Area Measurement 4 • for valley-to-valley (VV) peaks, the area above the baseline, segmented with vertical dropped lines from tick marks, as in Figure 25 on page 103, KK Figure 25 Area Measurement for Valley-to-Valley Peaks • for tangent (T) peaks, the area above the reset baseline, • for solvent (S) peaks, the area above the horizontal extension from the last-found baseline point and below the reset baseline given to tangent (T) peaks.
4 Integration Peak Area Measurement IdiVa odcZ VgZV " 6gZV WZadl WVhZa^cZ 2 7VhZa^cZ XdggZXiZY VgZV Figure 26 Area Measurement for Negative Peaks Units and Conversion Factors Externally, the data contains a set of data points; they can be either sampled data or integrated data. In the case of integrated data, each data point corresponds to an area, which is expressed as Height × Time. In the case of sampled data, each data point corresponds to a height.
4 Integration Integration Events Integration Events The integrator provides you with a number of initial and timed integrator events. Many events are on/off or start/stop pairs. Initial Events Initial Peak Width Initial peak width sets the integrator’s internal peak width to this value for the start of run. This initial peak width is used to scale the accumulator that detects peak up-slope, down-slope, and tailing.
4 Integration Integration Events Choosing Peak Width Choose the setting that provides just enough filtering to prevent noise being interpreted as peaks without distorting the information in the signal. • To choose a suitable initial peak width for a single peak of interest, use the peak’s time width as the base as a reference.
4 Integration Integration Events Tuning Integration It is often useful to change the values for the slope sensitivity, peak width, height reject, and area reject to customize integration. Figure 27 on page 107 shows how these parameters affect the integration of five peaks in a signal. =Z^\]i gZ_ZXi 7VhZa^cZ ( + & * EZV` l^Yi] , ' ) Figure 27 Using Initial Events A peak is integrated only when all of the four integration parameters are satisfied.
4 Integration Integration Events Table 11 Height and Area Reject Values Integration Parameter Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 7 Height reject Above Above Above Below Above Above Area reject Above Below Above Below Below Above Peak integrated Yes No Yes No No Yes Timed Events You can use timed events to customize signal baseline construction when default construction is not appropriate.
Integration Integration Events 4 0.2% for GC. The initial area reject is set to zero and a trial integration is performed. The trial is repeated several times if necessary, adjusting the parameters each time until at least 5 peaks are detected or integration is performed with an initial height reject of 0. The trial integration is terminated if the above conditions are not met after 10 trials.
4 Integration Manual Integration Manual Integration This type of integration allows you to integrate selected peaks or groups of peaks. Except for the initial area reject value, the software's event integration is ignored within the specified range of manual integration. If one or more of the peaks resulting from manual integration is below the area reject threshold, it is discarded. The manual integration events use absolute time values. They do not adjust for signal drift.
4 Integration Manual Integration Saving Manual Integration Events As manual integration events are typically very specific for a certain data files, in ChemStation B.04.01 these events can be stored directly in the data file instead of the method. Each time the data file is reviewed or reprocessed, the manual events in the data file are automatically applied. A run containing manual integration events is marked in the Navigation Table in the corresponding column.
4 Integration Manual Integration In case the Manual Events checkbox of the Integration Events Table of a method is enabled, the manual events of the method are always applied when loading a data file using this method. If the data file contains additional manual events, they are applied after the events of the method. When the Manual Events checkbox is enabled, the user is never asked to save the events to the data file.
Understanding Your Agilent ChemStation 5 Quantification What is Quantification? 114 Quantification Calculations 115 Correction Factors 116 Absolute Response Factor Multiplier 116 Dilution Factor 116 Sample Amount 117 116 Uncalibrated Calculation Procedures Area% and Height% 118 Calibrated Calculation Procedures ESTD Calculation Norm% Calculation 118 119 120 122 ISTD Calculation 123 Run 1: Calibration 124 Run 2: Unknown Sample 124 ISTD Calculation of Calibrated Peaks 125 ISTD Calculation of Uncalib
5 Quantification What is Quantification? What is Quantification? After the peaks have been integrated and identified, the next step in the analysis is quantification. Quantification uses peak area or height to determine the concentration of a compound in a sample. A quantitative analysis involves many steps which are briefly summarized as follows: • Know the compound you are analyzing. • Establish a method for analyzing samples containing this compound.
5 Quantification Quantification Calculations Quantification Calculations The ChemStation offers the following calculation procedures for determining the concentration of each component present in a mixture: • Percent • Normalization • External standard (ESTD) • ESTD% • Internal standard (ISTD) • ISTD% The calculations used to determine the concentration of a compound in an unknown sample depend on the type of quantification.
5 Quantification Correction Factors Correction Factors The quantification calculations use four correction factors, the absolute response factor, the multiplier, the dilution factor, and the sample amount. These factors are used in the calibration procedures to compensate for variations in detector response to different sample components, concentrations, sample dilutions, sample amounts, and for converting units.
5 Quantification Correction Factors Sample Amount If the ESTD% or ISTD% calculations are selected, the ESTD and ISTD reports give relative values rather than absolute values, that is, the amount of each component is expressed as a percentage of the sample amount. The sample amount is used in ESTD% and ISTD% reports to convert the absolute amount of the components analyzed to relative values by dividing by the value specified.
5 Quantification Uncalibrated Calculation Procedures Uncalibrated Calculation Procedures Uncalibrated calculation procedures do not require a calibration table. Area% and Height% The Area% calculation procedure reports the area of each peak in the run as a percentage of the total area of all peaks in the run. Area% does not require prior calibration and does not depend upon the amount of sample injected within the limits of the detector. No response factors are used.
5 Quantification Calibrated Calculation Procedures Calibrated Calculation Procedures The external standard (ESTD), normalization, and internal standard (ISTD) calculation procedures require response factors and therefore use a calibration table. The calibration table specifies conversion of responses into the units you choose by the procedure you select.
5 Quantification ESTD Calculation ESTD Calculation The ESTD procedure is the basic quantification procedure in which both calibration and unknown samples are analyzed under the same conditions. The results from the unknown sample are then compared with those of the calibration sample to calculate the amount in the unknown. The ESTD procedure uses absolute response factors unlike the ISTD procedure. The response factors are obtained from a calibration and then stored.
5 Quantification ESTD Calculation D is the dilution factor. GZhedchZ G; m GZhedchZ m 6bdjci m Figure 28 6bdjci Response Factor The multiplier and dilution factor are read either from theCalibration Settings or from theSample Information dialog box.
5 Quantification Norm% Calculation Norm% Calculation In the normalization method, response factors are applied to the peak areas (or heights) to compensate for changes that occur in detector sensitivity for the different sample components. The Norm% report is calculated in the same way as an ESTD report except that there is an additional step to calculate the relative rather than absolute amounts of compounds. The Norm% report has the same disadvantage as the Area% and Height% reports.
5 Quantification ISTD Calculation ISTD Calculation The ISTD procedure eliminates the disadvantages of the ESTD method by adding a known amount of a component which serves as a normalizing factor. This component, the internal standard, is added to both calibration and unknown samples. The software takes the appropriate response factors obtained from a previous calibration stored in the method.
5 Quantification ISTD Calculation Run 1: Calibration 1 The calibration points are constructed by calculating an amount ratio and a response ratio for each level of a particular peak in the calibration table. The amount ratio is the amount of the compound divided by the amount of the internal standard at this level. The response ratio is the area of the compound divided by the area or height of the internal standard at this level.
5 Quantification ISTD Calculation ISTD Calculation of Calibrated Peaks The equations used to calculate the actual amount of a calibrated component x for a single-level calibration are: where: RFx is the response factor for compound x; The actual amount (Actual Amt) of ISTD is the value that was entered in the Calibration Settings dialog box or the Sample Info dialog box for the internal standard added to the unknown sample; M is the multiplier. D is the dilution factor.
5 Quantification ISTD Calculation RFx is the Response Factor set in the Calibration Settings dialog box. You can see from these formulae that the variations in the ISTD response are used to correct the quantification of the unknown component. 2 Use a calibrated peak. This ensures that the same response factor is used for the quantification of all peaks. The response factor of the selected compound and the uncalibrated peaks is corrected during all recalibrations.
Understanding Your Agilent ChemStation 6 Peak Identification What is Peak Identification? Peak Matching Rules 128 129 Types of Peak Identification 130 Absolute Retention/Migration Time 130 Corrected Retention/Migration Time 130 Peak Qualifiers 130 Amount Limits 131 Absolute Retention/Migration Time Corrected Retention/Migration Times Single Reference Peaks 134 Multiple Reference Peaks 134 132 134 Peak Qualifiers 136 Signal Correlation 137 Qualifier Verification 137 Qualifier Ratio Calculation 137 The
6 Peak Identification What is Peak Identification? What is Peak Identification? Peak identification identifies the components in an unknown sample based on their chromatographic/electropherographic characteristics determined by the analysis of a well-defined calibration sample. The identification of these components is a necessary step in quantification if the analytical method requires quantification.
6 Peak Identification Peak Matching Rules Peak Matching Rules The following rules apply to the peak matching process: • if a sample peak falls within the peak matching window of a component peak from the calibration table, the peak is given the attributes of that component, • if more than one sample peak falls within the peak matching window, then, the peak closest to the expected retention/migration time is identified as that component, • if a peak is a time reference or internal standard, then the larg
6 Peak Identification Types of Peak Identification Types of Peak Identification There are different techniques that can be used to match sample peaks with those in the calibration table of the ChemStation software. Absolute Retention/Migration Time The retention/migration time of the sample peak is compared with the expected retention/migration time specified for each component in the calibration table.
Peak Identification Types of Peak Identification 6 Amount Limits The amount limits defined in the Compound Details dialog box are used to qualify the peak identification. If the amount of the identified compound is inside the amount limits the peak identification is indicated in the report.
6 Peak Identification Absolute Retention/Migration Time Absolute Retention/Migration Time A retention/migration time window is used in the peak matching process. The retention/migration time window is a window which is centered on the retention/migration time for an expected peak. Any sample peak that falls within this window may be considered as a candidate for component identification. Figure 30 on page 132 shows a retention/migration time window for peak 2 which is between 1.809 and 2.
6 Peak Identification Absolute Retention/Migration Time • specific window values for individual components which are set in the Compound Details dialog box. The default values for these windows are entered in the Calibration Settings dialog box. The width on either side of the retention/migration time that defines the peak matching window is the sum of the absolute and percentage windows. A window of 5 % means the peak must have a retention/migration time between less than 2.5 % and more than 2.
6 Peak Identification Corrected Retention/Migration Times Corrected Retention/Migration Times To match peaks by absolute retention/migration times may be simple but not always reliable. Individual retention/migration times may vary slightly due to a small change in conditions or technique. As a result peaks may occur outside the peak matching windows and therefore are not identified.
6 Peak Identification Corrected Retention/Migration Times progresses. Often during a long run the retention/migration time changes non-uniformly. In such cases better results are obtained using multiple reference peaks spaced at intervals across the run. This splits the signal into separate zones. Within each zone the deviation between retention/migration times is assumed to change linearly, but the rate of change is determined separately for each zone.
6 Peak Identification Peak Qualifiers Peak Qualifiers A component can be detected with more than one signal. Although applicable to all forms of chromatography using multiple detectors or detectors capable of producing multiple signals, multisignal detection is most commonly used in liquid chromatography with multiple wavelength or diode array detectors.
6 Peak Identification Peak Qualifiers Signal Correlation Signal correlation means that two peaks measured in different detector signals within a defined time window are assigned to the same compound. The signal correlation window can be controlled by the SignalCorrWin parameter in the QuantParm table of the _DaMethod register. Signal correlation is disabled when setting the signal correlation window to 0.0 minutes (see the Macro Programming Guide for more information).
6 Peak Identification Peak Qualifiers The tolerance can be set in the calibration table user interface (Identification Details) and is an absolute percentage. For multilevel calibrations, the ChemStation calculates a minimum qualifier tolerance based on the measured qualifier ratios at each calibration level. The minimum qualifier tolerance is calculated using the following equation: where qi is the measured qualifier ratio at level i.
6 Peak Identification The Identification Process The Identification Process When attempting to identify peaks, the software makes three passes through the integration data. Finding the Reference Peaks The first pass identifies the time reference peaks. The software searches peak retention/migration times from a run for matches within the retention/migration windows of the reference peaks in the calibration table.
6 Peak Identification The Identification Process Finding the Remaining Calibrated Peaks The third pass identifies all remaining peaks listed in the calibration table. The non-reference peaks in the calibration table are matched to the remaining run peaks by using their RT window. Each non-reference calibrated peak has its own retention/migration time in the calibration table. This is adjusted for the particular run based on the pre-identification of the time reference peaks.
Understanding Your Agilent ChemStation 7 Calibration Definition of Terms 142 Calibration Table 143 Calibration Curve 144 Unknown Samples 146 Types of Calibration 147 Single-Level Calibration 147 Multilevel Calibration 148 Calibration Ranges 150 Calibration Curve Fits 150 Origin Treatment 150 Group Calibration Peak Summing 153 154 Recalibration 155 What is Recalibration? 155 Why Recalibrate? 155 Manual Recalibration 155 Recalibrations with Peak Summing Recalibration Options 156 Ways to Recalibrate
7 Calibration Definition of Terms Definition of Terms Calibration Calibration is the process of determining response factors used to calculate absolute component concentrations by injecting specially prepared calibration samples. The calibration table is also used for identification. See “Peak Identification” on page 127. Compound A chemical compound can comprise several peaks, in a multiple signal calibration, typically one per signal. In a single signal calibration a compound refers to one peak.
Calibration Calibration Table 7 Calibration Table The calibration table specifies conversions of peak areas or heights into the units you choose according to the calculation procedure you select. It contains a list of retention/migration times from a calibration run. These retention/migration times are compared with retention/migration times of peaks from a sample run.
7 Calibration Calibration Curve Calibration Curve A calibration curve is a graphical presentation of the amount and response data for one compound obtained from one or more calibration samples. Normally an aliquot of the calibration sample is injected, a signal is obtained, and the response is determined by calculating the area or height of the peak, similar to Figure 32 on page 144.
Calibration Calibration Curve 7 where: relRES = relative residual in percent The calculated response represents the point on the calibration curve.
7 Calibration Unknown Samples Unknown Samples An unknown sample is a sample containing an unknown amount of the compound to be quantified.
7 Calibration Types of Calibration Types of Calibration The ChemStation offers two types of calibrations, single-level and multilevel calibrations. Single-Level Calibration The calibration curve shown in Figure 34 on page 147 contains one point, that is, one level. For the single-level calibration curve, the response of the detector is assumed to be linear over the working range of concentrations for the samples of interest.
7 Calibration Types of Calibration GZhedchZ &%%% 8Va^WgVi^dc XjgkZ &%% & 8dcXZcigVi^dc Pc\$¥aR Figure 35 &% Two-level calibration curve For example, if you want to quantify a compound, and the unknown samples are expected to range from 1 – 10 ng/µl, then a calibration curve should have at least the two levels as shown in Figure 35 on page 148. Amount Limits The ChemStation allows you to define the valid quantification ranges in terms of absolute amounts for each component.
7 Calibration Types of Calibration assumed to be linear. The difference between the two calibration types is that, with linear fit, the slope of the detector response can be determined by a best fit through a number of points, one for each level.
7 Calibration Types of Calibration Calibration Ranges Each multilevel calibration is valid over the range of concentrations used in the calibration samples. Extrapolation of a calibration curve, especially if it is non-linear, is at best an approximation. The valid calibration range for each compound may be defined in the Compound Details dialog box. Each entry for that compound can be expressed as lower and upper limits. If these limits are exceeded, the report is annotated.
7 Calibration Types of Calibration To force the origin to be included in the calibration curve the calibration points are mirrored about the origin from the first quadrant into the third quadrant. Using all points for the regression calculation ensures that the resulting calibration curve passes through the origin. This is also explained in Figure 37 on page 151.
7 Calibration Types of Calibration Weight Description Equal All calibration points have equal weight in the curve. Linear (Amnt) A calibration point with the amount x has the weighting 1/x normalized to the smallest amount so that the largest weight factor is 1. Normalization is done by multiplying the weight with the smallest amount.
7 Calibration Group Calibration Group Calibration Group calibration can be applied for compounds where the individual concentrations are not known but the sum of concentrations for a group of compounds is known. An example are isomers. Complete compound groups are calibrated.
7 Calibration Peak Summing Peak Summing The peak sum table is provided for certain applications in the petrochemical and pharmaceutical industries that can be performed more effectively with the following features: • Sum the areas of peaks that lie within a range specified by the user • Sum the areas of a range of peaks and perform the calculations using a single multiplier • Sum the areas of all peaks having the same name The peak sum table is similar to, but distinct from the standard calibration table
Calibration Recalibration 7 Recalibration What is Recalibration? Recalibration is the process used when you want to update a level on a calibration curve. When you recalibrate you run another sample that contains the same calibration compounds as the original, and most important, the same amount of these compounds. When you run the calibration sample, you obtain updated response factors and retention/migration times.
7 Calibration Recalibration where the same compounds have been analyzed for many years and the response factors for various compounds and detectors are readily available. You create the calibration table manually by entering peaks and their response factors into the calibration table, recalibrating the method using a standard that contains at least one response reference peak, and selecting Delta% update.
7 Calibration Recalibration Ways to Recalibrate Recalibration can be done in two ways using the ChemStation software. You can recalibrate interactively or automatically during a sequence of automated analyses. Interactive recalibration is when you directly go through the process of recalibration using the ChemStation software after injecting one or more calibration samples.
7 158 Calibration Recalibration Understanding Your Agilent ChemStation
Understanding Your Agilent ChemStation 8 Automation What is Automation? 161 What is a Sequence/Sequence Template? Preferences - Sequence Tab Sequence Parameters Sequence Table 162 163 165 166 Creating Sequences (Sequences and Sequence Templates) Using the Sequence Table Editor 167 Using the Insert Vial Range Button 167 Using the Append Line Button 167 Using the Custom Fields Button 168 167 Working with Sequences (Sequences and Sequence Templates) Priority Samples 169 Sequencing with Control Sample
8 Automation Recalibration Specifying Recalibrations 180 Recalibration Parameters in the Sequence Table Types of Sequences 180 183 Explicit Calibration Sequences 184 Cyclic Single-Level Calibration Sequences 185 Cyclic Multiple-Level Calibration Sequences Method A Analysis Order 187 Method B Analysis Order 188 Explicit and Cyclic Calibrations Together Example 190 SimpReg Analysis Order 191 186 190 Cyclic Calibration Sequences with Bracketing Example 192 Bracketed Sequence Operation 193 Example 19
Automation What is Automation? 8 What is Automation? Automation is the unattended analysis of more than one injection. The sequence part of the ChemStation software allows you to automate acquisition, data evaluation, and report generation.
8 Automation What is a Sequence/Sequence Template? What is a Sequence/Sequence Template? A sequence is a series of instructions that automates the analysis of samples. A sequence can be used to automatically inject each sample, and to acquire and analyze the data according to the method specified for that sample. Each sample vial in a sequence may be analyzed with a different analytical method and, thus, use different sets of chromatographic/electropherographic conditions and evaluation parameters.
Automation Preferences - Sequence Tab 8 Preferences - Sequence Tab On the Sequence tab, the user has the choice of two different data storage models. These modes define how the sequence data are stored in the ChemStation. Unique Folder Creation ON In this mode of data storage, there is a robust and permanent link between the raw data and the method.
8 Automation Preferences - Sequence Tab Unique Folder Creation OFF In this mode of data storage, the method name is the only link that exists between the data file and the method that was used to acquire and process it. No copies of the method are saved with the sequence or with the data file; if the method is changed, or a new method with that name is created, then the sequence cannot be exactly reproduced.
8 Automation Sequence Parameters Sequence Parameters The Sequence Parameters dialog box contains information common to all sample vials in a sequence. Use this dialog box to: • select the data directory using the Path combo box, and enter information about the operator name (the operator name entered in the access level dialog box is shown), and • specify how the sequence processing should be done by choosing particular part of Methods and Run parameters.
8 Automation Sequence Table Sequence Table The sequence table determines which methods are used to analyze the sample vials and the order in which the vials are analyzed. This table also contains information about each sample, including the name, quantification parameters, and recalibration parameters. The injector group box is displayed for instruments that support dual sampling (GC). Selecting Front or Back displays the lines in the sequence table and the currently running status for that injector.
Automation Creating Sequences (Sequences and Sequence Templates) 8 Creating Sequences (Sequences and Sequence Templates) Use the sequence table to specify the samples, methods, and vials to run in the sequence. The sequence table lists each sample in the sequence in the order it will be run and contains the necessary vial, method, and calibration information for each sample.
8 Automation Creating Sequences (Sequences and Sequence Templates) Using the Custom Fields Button If custom fields have been set up in the method(s) used in the sequence table, select the Custom Fields button in order to edit the custom fields values for each sample (sample related custom fields) or for each compound in the method of a sample (compound related custom fields).
8 Automation Working with Sequences (Sequences and Sequence Templates) Working with Sequences (Sequences and Sequence Templates) Sequences (Sequences and Sequence Templates) are accessed and created from the Sequence menu. Sequences may be created and saved in the same way as methods. When you save a sequence, a file is created with a .S extension. When you want to edit or use the sequence again, you access it by, for example, using the Load Sequence item in the Sequence menu.
8 Automation Working with Sequences (Sequences and Sequence Templates) Aborting a Sequence The abort function terminates a currently active sequence immediately. Pausing a Sequence During a sequence pause, the sequence table file name and the data file name cannot be changed. In the sequence table you can only change sequence lines that have not already been executed or change the vial number in the current sequence line. You can add, delete, and change sequence lines for future analyses.
8 Automation Working with Sequences (Sequences and Sequence Templates) Figure 38 Partial Sequence Dialog Box Partial Sequence in Preference Mode “Unique Folder Creation ON” The sequence data are stored in a sequence data container using a defined sequence container name. If you run a partial sequence the system will create a new sequence data container based on the Preference settings, each time a part of that sequence is executed. So it is possible to create e.g.
8 Automation Sequence Log File Sequence Log File A sequence log file is produced that indicates what has happened during the running of the sequence. It is useful for identifying when errors occurred if the sequence is running unattended or overnight. The name of the logbook file always has the .log extension. The logbook file is located in the directory where the data of the sequence is stored.
Automation What Happens When a Sequence is Run? 8 What Happens When a Sequence is Run? Starting a Sequence using “Unique Folder Creation ON” The system creates a sequence data container based on the path definition in the sequence parameters and the sequence preference settings. The sequence template *.s, all methods defined in the sequence table belonging to this particular sequence are copied to the sequence data container. The system continue to work with these files during acquisition.
8 Automation What Happens When a Sequence is Run? • During the entire process, the ChemStation tracks the sequence’s progress in real time and produces a sequence log file.
Automation Sequence Data File Structure (Unique Folder Creation ON) 8 Sequence Data File Structure (Unique Folder Creation ON) In the ChemStation revision B.02.01 and later, the link between the raw data and the method has been strengthened, as shown in Figure Sequence Data File Structure “Unique Folder Creation ON”.
8 Automation Data File Naming in a Sequence Data File Naming in a Sequence Data file naming in a sequence can be done in the following ways: • automatic, • manual, or • prefix/counter. Automatic Data File Naming During a Sequence Sample Vials For example 017-0103.D where: • The first three digits are the vial number, for example, 017.
Automation Data File Naming in a Sequence 8 Entering Data File Names Manually One of the columns in the sequence table is named Datafile. When it contains no entry, the data file naming scheme specified (automatic or prefix-counter) is used to create the data file name. If any text is entered into the Datafile column, the ChemStation uses that text as the data file name for the run.
8 Automation Postsequence Operation Postsequence Operation You can specify what happens after a sequence has finished during normal execution or when the ChemStation encounters an error during sequence operation.
8 Automation Automatic Recalibration Automatic Recalibration Calibration is often done after a change in operating conditions, for example, after changing a column or capillary. Automatic recalibration is usually done at the start of a sequence of analyses or at regular intervals during a sequence as part of a program to compensate for factors that affect the analytical performance.
8 Automation Specifying Recalibrations Specifying Recalibrations The recalibration parameters for the sequence are entered directly into the sequence table. These parameters define how the method is recalibrated during a sequence. Recalibration Parameters in the Sequence Table The response factor and retention/migration times can be updated in several ways.
Automation Specifying Recalibrations 8 The table shows the columns in the sequence table that contain the recalibration parameters and the values that can be entered. No Update Does not change the response factor or retention/migration time. Replace Replaces the previous retention/migration times and the response (areas or heights) with those from the current run only. The response is not changed for any peak that is not found in this recalibration run.
8 Automation Specifying Recalibrations Delta% The delta% calculation allows you to compare response factors from an analysis with response factors entered manually into a calibration table. The delta% is then applied to all of the calibrated peaks in the table. You can identify several internal standards, and their measured response factors are then used to calculate new response factors for the other peaks.
Automation Types of Sequences 8 Types of Sequences The following are types of sequences: • explicit calibration sequences, • explicit single-level calibration sequences, • cyclic multiple-level calibration sequences, • explicit and cyclic calibrations together in a sequence, and • cyclic calibration sequences with bracketed calibrations.
8 Automation Explicit Calibration Sequences Explicit Calibration Sequences This type of sequence recalibrates at defined intervals specified by you in the sequence table. For explicit calibration sequences, the calibration samples are entered in the sequence without an interval entry in the sequence table. A recalibration is done once for each calibration sample entry in the sequence table.
8 Automation Cyclic Single-Level Calibration Sequences Cyclic Single-Level Calibration Sequences This type of sequence uses the same vial, that is, the calibration sample at regular intervals in the sequence. The interval entry in the sequence table determines how the recalibration is done. For example, an interval value of 2 will recalibrate after every two sample vials in the sequence.
8 Automation Cyclic Multiple-Level Calibration Sequences Cyclic Multiple-Level Calibration Sequences This type of sequence uses different calibration samples to recalibrate a multiple-level calibrated method. The following example describes a two-method sequence comprising method A and method B to analyze two groups of samples. Both methods are multiple-level calibration methods that will recalibrate automatically at defined intervals.
8 Automation Cyclic Multiple-Level Calibration Sequences Table 16 Line Sequence Table for Method A and Method B Vial Method Name Inj/Vial Sample Type Cal Level Update RF Update RT 6 13 Method A 1 7 14 Method A 1 8 3 Method B 9 5 10 Interval 1 Calibration 1 Average No update 3 Method B 2 Calibration 2 Average No update 3 20 Method B 1 11 21 Method B 1 12 22 Method B 1 13 23 Method B 1 14 24 Method B 1 Method A Analysis Order This section describes the a
8 Automation Cyclic Multiple-Level Calibration Sequences Table 17 Method A Analysis Order 9 Method A 1 Calibration level 1 and report 10 Method A 2 Calibration level 2 and report 11 Method A 14 Sample analysis and report Method B Analysis Order This section describes the analysis order for Method B, which is the second part of the two-method sequence. Method B has the following differences compared with Method A: • There are two injections per vial for calibration level 2.
Automation Cyclic Multiple-Level Calibration Sequences 8 Note that the results shown in Table 17 on page 187and Table 18 on page 188can be obtained by using Partial Sequence to see a preview of the run order after setting up the Sequence Table.
8 Automation Explicit and Cyclic Calibrations Together Explicit and Cyclic Calibrations Together This type of sequence comprises explicit and cyclic calibrations in the same sequence. This feature allows you to recalibrate the method completely at the beginning of a sequence (explicit recalibration) and then update the calibration (cyclic recalibration) during the sequence. • Two calibration lines for each calibration level in the Sequence table must be specified.
8 Automation Explicit and Cyclic Calibrations Together • The first calibration line is for the same level but averages the calibration parameters. The interval entry specifies that the recalibration is done after every three samples. • The second entry replaces all the recalibration parameters, that is, a complete recalibration is done. It has no recalibration interval. Sequence Table The sequence table comprises seven lines. The first line specifies the cyclic recalibration sample.
8 Automation Cyclic Calibration Sequences with Bracketing Cyclic Calibration Sequences with Bracketing In a cyclic calibrated sequence with bracketing, the calibration table used to calculate the unknown quantitative results is generated by averaging the results of the current calibration with those of the previous calibration. This new calibration table is a more accurate representation of the instrument response at the time the sample was analyzed.
Automation Cyclic Calibration Sequences with Bracketing >chigjbZci Yg^[i I^bZ 8Va^WgVi^dc & HVbeaZ Figure 41 8 8Va^WgVi^dc ' Bracketing Bracketed Sequence Operation • The first calibration vials are analyzed. • The sample vials are analyzed. • The next calibration vials are analyzed. • The calibration table is produced by replacing the existing response factors by new ones and averaging the following calibration runs into a new calibration table.
8 Automation Cyclic Calibration Sequences with Bracketing Sequence Table The sequence table of Brack.M (next page) is truncated to simplify the example. It consists of seven lines. The first two lines define the recalibration conditions for each level. The remaining lines define the samples to be analyzed. More specifically, the sequence table of Brack.M method has: • A Bracket entry in the Update Response Factor column that specifies brakketing of samples with calibration samples.
Automation Cyclic Calibration Sequences with Bracketing 8 Bracketed Sequence Analysis Order Understanding Your Agilent ChemStation 195
8 Automation Cyclic Recalibration Sequences with Multiple Vials Containing the Same Dilution of a Standard Cyclic Recalibration Sequences with Multiple Vials Containing the Same Dilution of a Standard Cyclic Recalibration Sequence with “Round-Robin” Calibration Vial Usage When running a large sequence that performs cyclic recalibrations, that is, performs an automatic recalibration after a fixed number of sample injections, there is a potential risk of emptying the volume of calibration vial during the c
Automation Cyclic Recalibration Sequences with Multiple Vials Containing the Same Dilution of a Standard Table 22 8 Cyclic Recalibration Sequence with 3 Vials Defined for Each Level Vial No. Sample Name Sample Type Method Name No. of Inj.
8 Automation Cyclic Recalibration Sequences with Multiple Vials Containing the Same Dilution of a Standard Cyclic Recalibrations Where Each Calibration Uses a Different Vial To ensure that every calibration vial be injected only once, the sequence must define a sufficient number of different calibration vials, so that the round-robin order described in the previous example is not applied.
Automation Cyclic Recalibration Sequences with Multiple Vials Containing the Same Dilution of a Standard Table 23 Different Vials Used for Opening and Closing Brackets Vial No. Sample Name Sample Type Method Name No. of Inj.
8 200 Automation Cyclic Recalibration Sequences with Multiple Vials Containing the Same Dilution of a Standard Understanding Your Agilent ChemStation
Understanding Your Agilent ChemStation 9 Data Review, Reprocessing and Batch Review Navigation Table in Data Analysis 202 Navigation Table Configuration 202 Navigation Table Toolbar 203 Data Review Using the Navigation Table 204 Sequence Reprocessing Using the Navigation Table What is Batch Review? 205 207 Enabling Batch Review Functionality with ChemStation OpenLAB Option Installed 208 Batch Configuration 209 Batch Table 209 Compound Table 209 Batch Report 210 User Interface 210 Review Functions 211 Ca
9 Data Review, Reprocessing and Batch Review Navigation Table in Data Analysis Navigation Table in Data Analysis The Data Analysis view includes a Navigation Table that is designed to facilitate navigation through data files. The Navigation Table shows the runs contained in a selected data or sequence data subdirectory. You can use the Navigation Table to load access individual runs, or to automatically scroll through the loaded signals.
9 Data Review, Reprocessing and Batch Review Navigation Table in Data Analysis Table 24 Navigation Table Columns Single Runs Columns Sequence Runs Columns Dilution Multiplier --- Dilution --- Data File The Navigation table includes standard table configuration features, such as sorting and drag-and-drop options to move columns to different places. It is also possible to select the columns that are displayed in the Navigation Table.
9 Data Review, Reprocessing and Batch Review Navigation Table in Data Analysis Data Review Toolset The review functionality of the Navigation Table allows you to step automatically or manually through the loaded signals. Depending on the selection specified in the Preferences / Signal/Review Options, the system can automatically integrate the signal and print a report for each file as it is loaded. The method applied to the data file is shown in the top menu.
Data Review, Reprocessing and Batch Review Navigation Table in Data Analysis 9 is visible in the Status Bar; the system adds from data file in brackets (from data file) to indicate that the loaded method is the individual method for the data file. 2 Review your data using the sequence method: Use the option Sequence Method in the Preferences / Signal/Review Options to have the system load the sequence method corresponding to the current line of the Navigation Table.
9 Data Review, Reprocessing and Batch Review Navigation Table in Data Analysis • sequence-related batch (*.b) file • sequence-related logbook (*.log) file During reprocessing, the individual methods DA.M for the data files are updated as well as the batch file (*.b) file. With the Data Analysis reprocessing functions, it is possible to modify the sequence template (*.s) in the data container in order to change the multiplier, dilution etc., or to use a different method for reprocessing.
Data Review, Reprocessing and Batch Review What is Batch Review? 9 What is Batch Review? Batch review is a capability within data analysis designed to help an analyst perform a “first-pass” review of the results of a sequence or a selection of runs quickly and easily. It will save time especially when reprocessing large numbers of samples. Whenever a sequence is run, a batch file (with a .b extension) is automatically generated and placed in the data directory along with the data files.
9 Data Review, Reprocessing and Batch Review Enabling Batch Review Functionality with ChemStation OpenLAB Option Installed Enabling Batch Review Functionality with ChemStation OpenLAB Option Installed When ChemStation OpenLAB Option is installed, the Batch Review functionality is not available by default. In order to use Batch Review, this functionality has to be enabled by an entry in the [PCS] section of the ChemStation.ini file. The file is located in the windows directory c:\ WINDOWS.
9 Data Review, Reprocessing and Batch Review Batch Configuration Batch Configuration A batch is a user-selected series of data files that is processed using a user-defined method. All data files in the batch are processed using the same method. The processing steps carried out each time a new sample is loaded for review can be selected (integration, identification/quantitation, reporting).
9 Data Review, Reprocessing and Batch Review Batch Configuration • the compound list contains all compounds found in the method that was loaded for batch review. • if calibration samples only are displayed in the batch table (samples and control samples are hidden), the compound table shows additional columns for calibration-related information (expected amount, relative error and absolute error).
9 Data Review, Reprocessing and Batch Review Review Functions Review Functions Data files can be displayed in one of two ways: • manually, by selecting a run to display from the table, • automatically, with a predefined interval between each data file. During automatic display, only those sample types displayed in the table are displayed; the runs are displayed in the order in which they appear in the table. The automatic review can be paused and later resumed, or stopped.
9 Data Review, Reprocessing and Batch Review Batch Reporting Batch Reporting The user-configurable “Batch Table” on page 209, can be printed directly on the printer, displayed on the screen or printed to a file with a user-specified prefix in one of the following formats: • UNICODE text file with the extension .TXT • Data Interchange Format with the extension .DIF • Comma-Separated Values format with the extension .CSV • Microsoft Excel format with the extension .XLS.
Understanding Your Agilent ChemStation 10 Using the ChemStation Reports What is a Report? 214 Reporting Results 215 Uncalibrated Reports 215 Calibrated Reports 215 External Standard Report 215 Internal Standard Report 216 Control Charts Report 216 Quantitative Results 217 Reporting Custom Field Values 219 Report Styles 220 Adding a Customized Report to Report Styles Other Report Style Parameters 223 Summed Peaks Table 223 Report Layout for Uncalibrated Peaks 222 223 Report Destination 224 Report F
10 Using the ChemStation Reports What is a Report? What is a Report? A report can comprise quantitative and qualitative information of the sample you analyze. The report can be a hardcopy printout, a display on the screen or an electronic file. The report can include details of the peaks detected during the run and plots of the signals that were acquired.
10 Using the ChemStation Reports Reporting Results Reporting Results Two types of reports are available: • an uncalibrated report which does not correct for detector response, and • a calibrated report that shows results corrected for the difference in the detector response to various components of the sample. Uncalibrated Reports The uncalibrated reports include the Area% and Height% reports. These reports are mainly used in the preparation of calibrated reports.
10 Using the ChemStation Reports Reporting Results calibration and unknown samples must be known accurately. The reliability of the external standard report is limited by the reproducibility of the injection and any other factors that might change from sample to sample. Internal Standard Report The limitations of the external standard procedure can be overcome by using the internal standard approach.
10 Using the ChemStation Reports Quantitative Results Quantitative Results The report type is identified by the name of the calculation method used to prepare it, for example, an ISTD report. Each type is briefly described below. The calculations for each report are given in “Quantitative Results” on page 217. Area% provides the simplest report and requires no calibration data since no correction is made for the difference in detector response of sample components.
10 Using the ChemStation Reports Quantitative Results ISTD% produces a report of the relative amount of each substance as a percentage of the injected sample. The use of an internal standard in both the sample and the calibration mixture eliminates the need to know and control the volume of sample injected. This also corrects for any variation in instrument performance between runs.
Using the ChemStation Reports Reporting Custom Field Values 10 Reporting Custom Field Values The values of the custom fields attached to a certain sample according to its acquisition method can be added to the report. Sample custom fields are listed at the end of the report header that contains the general sample information. Compound custom fields appear at the end of the report.
10 Using the ChemStation Reports Report Styles Report Styles The following report styles are available: You choose to add a signal to any of the report styles by checking the appropriate box in the Specify Report dialog box. • None - No text will be reported. The chromatogram will be reported only if the Add Chromatogram Output option is selected.
10 Using the ChemStation Reports Report Styles For uncalibrated methods, the report parameters include the peak number, retention/migration time, peak area, peak height, signal description, true half-height peak-width (see also “True Peak Width Wx [min]” on page 246), symmetry, k’, efficiency (plates) and resolution for each peak.
10 Using the ChemStation Reports Report Styles Adding a Customized Report to Report Styles You can add a custom report template that you have created in the Report Layout view of the ChemStation to the list of available report styles. NOTE 222 All reports apart from the performance reports list the peakwidths calculated with a more complex formula by the integrator (for details on the peakWidth calculation please refer to “Peak Width” on page 80).
Using the ChemStation Reports Other Report Style Parameters 10 Other Report Style Parameters Summed Peaks Table The peak sum table is provided for certain applications in the petrochemical and pharmaceutical industries that can be performed more effectively with the following features: • Sum the areas of peaks that lie within a range specified by the user • Sum the areas of a range of peaks and perform the calculations using a single multiplier • Sum the areas of all peaks having the same name When the r
10 Using the ChemStation Reports Report Destination Report Destination The report can be sent to either: • Screen The report (including text and graphics) is displayed on the screen in the report preview window from which it can be printed. • Printer The report comprising text and graphics is printed on the currently selected printer. • File The report is saved to a file. If the data is saved to a file it is possible to reprocess the data with another program, for example, Microsoft Windows EXCEL.
Using the ChemStation Reports Report Destination .CSV 10 The report is in Comma Separated Values (CSV) format. This is a very simple format for tabular data that is accepted by many spreadsheet programs and databases. Independent from the report style selected, only the information contained in the report style “Short” will be saved. There can be several .DIF and .CSV files for a single report. For each report block, the first file, for example, REPORT00.CSV, contains the report header information.
10 Using the ChemStation Reports Sequence Summary Reporting Sequence Summary Reporting Overview The ChemStation can print a variety of standard reports for individual sample analyses. Sequence summary reporting is an additional way of reporting, that allows you to calculate and report parameters across a number of different analyses. It is useful, for example, to test the stability of an instrument or the robustness of a new method.
Using the ChemStation Reports Sequence Summary Reporting 10 One Page Header The GLP template prints GLP in large letters as a title page for the following report. It also includes the date and a place for a signature. Configuration Select Configuration if you want to include the instrument configuration and analytical column/capillary specifications in the report. Sequence Table Select Sequence table to include a list of the samples, sample quantification parameters and method names in the report.
10 Using the ChemStation Reports Sequence Summary Reporting prints the statistical trends of the analyses as graphs, whereas the Standard Statistics selection prints only text. The selections that you make in the Items and Limits for Extended Statistics dialog box are used only when you choose the Extended Statistic option(s) in the Sequence Summary Parameters dialog box.
Using the ChemStation Reports Sequence Summary Reporting 10 Sequence Output In the Sequence output dialog box you can also define where the sequence summary report should be printed. Selecting Report to file and entering a file name prints the report to the specified file. The default setting is that data are saved to the file GLPrprt.txt. In GC systems with dual injection the data are saved in GLPrptF.txt and GLPrptB.txt for the front injector and back injector respectively.
10 Using the ChemStation Reports Sequence Summary Reporting 230 Understanding Your Agilent ChemStation
Understanding Your Agilent ChemStation 11 Evaluating System Suitability Noise Determination 235 Noise Calculation Using Six Times the Standard Deviation Noise Calculation Using the Peak-to-Peak Formula 236 Noise Calculation by the ASTM Method 237 Signal-to-noise calculation 239 Drift and Wander 239 Calculation of Peak Symmetry 235 240 System Suitability Formulae and Calculations 242 General Definitions 243 Void Volume 243 Retention Time of Unretained Compound t (m) [min] 243 Performance Test Definit
11 Evaluating System Suitability Sequence Summary Reporting Regression Coefficient Standard Deviation (S) 253 254 Internally Stored Double Precision Number Access 255 This chapter describes what the ChemStation can do to evaluate the performance of both the analytical instrument before it is used for sample analysis and the analytical method before it is used routinely and to check the performance of analysis systems before, and during, routine analysis.
11 Evaluating System Suitability Sequence Summary Reporting • peak width at half height, • peak symmetry, • peak tailing, • capacity factor (k´), • plate numbers, • resolution between peaks, • selectivity relative to preceding peak, • skew, and • excess. The mean value, the standard deviation, the relative standard deviation and the confidence interval are calculated. You can set limits for either standard deviation, the relative standard deviation or the confidence interval for each of these parameters.
11 Evaluating System Suitability Sequence Summary Reporting • column/capillary details, • analytical method, • sample information, • acquisition information, • signal description and baseline noise determination, and • signal labeled with either retention/migration times, or compound names.
Evaluating System Suitability Noise Determination 11 Noise Determination Noise can be determined from the data point values from a selected time range of a signal. Noise is treated in three different ways: • as six times the standard deviation (sd) of the linear regression of the drift, • as peak-to-peak (drift corrected), and • as determined by the ASTM method (ASTM E 685-93).
11 Evaluating System Suitability Noise Determination Std is the standard deviation of the linear regression of all data points in the time range. Noise Calculation Using the Peak-to-Peak Formula cd^hZ 2 bVm# eZV` b^cjh b^c# eZV` [ i^bZ Figure 43 Noise as Maximum Peak to Minimum Peak (Distance) The drift is first calculated by determining the linear regression using all the data points in the time range (see “Regression Analysis” on page 253).
Evaluating System Suitability Noise Determination 11 Noise Calculation by the ASTM Method cd^hZ 2 bVm# eZV` b^cjh b^c# lVcYZg Yi Figure 44 i^bZ Noise Determined by the ASTM Method ASTM noise determination (ASTM E 685-93) is based on the standard practice for testing variable-wavelength photometric detectors used in liquid chromatography, as defined by the American Society for Testing and Materials. Based on the size of the time range, three different types of noise can be distinguished.
11 Evaluating System Suitability Noise Determination Very-short-term noise is determined for a selected time range between 1 and 10 minutes. The time range for each cycle (dt) is set to 0.1 minute which will give at least 10 cycles within the selected time range. Determination of the Number of Cycles, n where t is the cycle time and ttot is the total time over which the noise is calculated.
Evaluating System Suitability Noise Determination 11 Signal-to-noise calculation For the signal-to-noise calculation, the ChemStation uses the six times the standard deviation (sd) of the linear regression of the drift to calculate the noise. The range closest to the peak is selected from the ranges as specified in the system suitability settings. The signal-to-noise is calculated using the formula: The signal-to-noise is calculated for each peak in the signal.
11 Evaluating System Suitability Calculation of Peak Symmetry Calculation of Peak Symmetry The ChemStation does not determine the asymmetry ratio of a peak, usually done by comparing the peak half-widths at 10% of the peak height, or 5% as recommended by the FDA.
Evaluating System Suitability Calculation of Peak Symmetry 11 = =[ hiVgi d[ eZV` i& Figure 45 =g V& V' V( i' i( V) i) ZcY d[ eZV` 7VhZa^cZ I^bZ Calculation of the Peak Symmetry Factor where: ai = area of slice ti = time of slice Hf = height of front inflection point Hr = height of rear inflection point H = height at apex Understanding Your Agilent ChemStation 241
11 Evaluating System Suitability System Suitability Formulae and Calculations System Suitability Formulae and Calculations The ChemStation uses the following formulae to obtain the results for the various System Suitability tests. The results are reported using the Performance, Performance + Noise and Performance + Extended report styles. When ASTM or USP is specified for a given definition, then the definition conforms to those given in the corresponding reference.
Evaluating System Suitability General Definitions 11 General Definitions Void Volume where: d = diameter of column [cm] π = constant, ratio of circumference to diameter of a circle l = length of column [cm] f = fraction of column volume that is not taken up by stationary phase but available for mobile phase; default value for f = 0.
11 Evaluating System Suitability Performance Test Definitions Performance Test Definitions Statistical Moments where: N Number of area slices Ai Value of area slice indexed by i dt Time interval between adjacent area slices t0 Time of first area slice Sum of starting index 1 to final index N for discrete observations 244 Understanding Your Agilent ChemStation
Evaluating System Suitability Performance Test Definitions 11 Statistical Moments, Skew and Excess Statistical moments are calculated as an alternative to describe asymmetric peak shapes. There is a infinite number of peak moments, but only the first five are used in connection with chromatographic peaks. These are called 0th Moment, 1st Moment, … 4th Moment. The 0th Moment represents the peak area. The 1st Moment is the mean retention time, or retention time measured at the center of gravity of the peak.
11 Evaluating System Suitability Performance Test Definitions True Peak Width Wx [min] WB base width, 4 sigma, obtained by intersecting tangents through the inflection points with the baseline (tangent peak width) W4.4 width at 4.4% of height (5 sigma width) W5.0 width at 5% of height (tailing peak width), used for USP tailing factor W50.0 width at 50% of height (true half-height peak width or 2.35 sigma).
Evaluating System Suitability Performance Test Definitions 11 L = CBTFMJOF ) 53 UJNF UX 8 8# Figure 46 Performance Parameters Number of Theoretical Plates per Column (USP, ASTM) n Tangent method (USP, ASTM): where: WB = base width [min] Half-width method (ASTM): where: W50 = peak width at half-height [min] Understanding Your Agilent ChemStation 247
11 Evaluating System Suitability Performance Test Definitions 5 Sigma method: where: W4.4 = peak width at 4.
Evaluating System Suitability Performance Test Definitions 11 Resolution (USP, ASTM) R (Pertaining to peaks a and b, TR of peak a < TR of peak b; TR in min) Tangent method (USP, ASTM): 5 Sigma method: Half-width method (Resolution used in Performance Report): Statistical method: where: M1(x) = mean retention time for peak x (1st Statistical Moment) [min] WB(x) = base width for peak x [min] W4.4(x) = width at 4.
11 Evaluating System Suitability Definitions for Reproducibility Definitions for Reproducibility For the statistical review of analytical data in terms of reproducibility the sequence is considered as a small random sample taken out of an infinite number of possible experimental results. To accomplish a complete set of results, an unlimited amount of sample material as well as time would be required. Strictly statistical data does only apply to a complete self-contained set or population of data.
11 Evaluating System Suitability Definitions for Reproducibility The sample standard deviation S differs in two points from the standard deviation s for the whole population: • instead of the real mean value only the sample mean value M is used and • division by N-1 instead of N. Relative Standard Deviation RSD[%] (USP) The relative standard deviation is defined as Standard Deviation of the Mean SM Let M be the sample mean and S the sample [or (N-1)] standard deviation.
11 Evaluating System Suitability Definitions for Reproducibility Confidence Interval CI The confidence interval is calculated to give information on how good the estimation of a mean value is, when applying it to the whole population and not only to a sample.
Evaluating System Suitability Definitions for Reproducibility 11 Regression Analysis Let N = number of discrete observations Xi = independent variable, ith observation Yi = dependent variable, ith observation Linear function: Coefficients: where: Regression Coefficient where: Understanding Your Agilent ChemStation 253
11 Evaluating System Suitability Definitions for Reproducibility Standard Deviation (S) 254 Understanding Your Agilent ChemStation
11 Evaluating System Suitability Internally Stored Double Precision Number Access Internally Stored Double Precision Number Access For validation purposes, it might become necessary to manually recalculate the ChemStation results such as calibration curves, correlation coefficients, theoretical plates, etc. When doing so the number format used in the ChemStation has to be taken into account. For all numbers stored internally within the ChemStation, the “C” data type DOUBLE is used.
11 Evaluating System Suitability Internally Stored Double Precision Number Access • Area • Height • Width (integrator) • Symmetry • Peak Start Time • Peak End Time Use Command Line Entry: DUMPTABLE CHROMREG, INTRESULTS,”C:\CHEM32\1\TEMP\ INTRES.
Evaluating System Suitability Internally Stored Double Precision Number Access 11 • Resolution – 5-Sigma (Extended Performance) • Resolution - Statistical (Extended Performance) Use Command Line Entry: DUMPTABLE CHROMRES, PEAK,”C:\CHEM32\1\TEMP\PEAK.TXT” Processed Compound Information: • Calculated Amount Use Command Line Entry: DUMPTABLE CHROMRES, COMPOUND,”C:\CHEM32\1\TEMP\ COMPOUND.
11 Evaluating System Suitability Internally Stored Double Precision Number Access 258 Understanding Your Agilent ChemStation
Understanding Your Agilent ChemStation 12 System Verification Verification and Diagnosis Views System Verification 260 The GLPsave Register 260 263 DAD Test Function 265 Review DAD Test Function 265 This chapter describes the verification function and the GLP verification features of the ChemStation.
12 System Verification Verification and Diagnosis Views Verification and Diagnosis Views If supported by the configured instrument, for example, the Agilent 1100/1200 Series modules for LC, the ChemStation comprises two additional views to perform instrument verification and diagnosis tasks. For more information, see the online help system. System Verification System verification is a key component in the routine use of an analytical instrument in a regulated laboratory.
System Verification Verification and Diagnosis Views 12 Verification test results are saved in binary format to the default subdirectory: c:\CHEM32\1\Verify, together with the method and data files. The Verify subdirectory is at the same level as the sequence, methods and data subdirectories. You can send the results to a printer or to a file. The test results, including a combined verification test result, are rated as either pass or fail.
12 System Verification Verification and Diagnosis Views 262 Understanding Your Agilent ChemStation
System Verification The GLPsave Register 12 The GLPsave Register The GLPsave register is saved at the end of each analysis when selected in the run time checklist. It contains the following information: • signals, • logbook, • integration results table, • quantification results table, • instrument performance data, and • data analysis method. This register is a complete protected record, generated at the time of analysis. You can recall it at any time in the future as proof of your analytical methods.
12 System Verification The GLPsave Register • reload and reprint the data analysis part of the method used at the time of the sample analysis to prove that the data evaluation, presented as the results of the analysis has not been modified in any way, and • review without recalculating, the integration and quantification results to prove the authenticity of the report.
System Verification DAD Test Function 12 DAD Test Function Detector tests can be used as a step in the routine system validation of an analytical instrument in a regulated laboratory. The DAD test assesses the performance of your diode array detector. When you select the DAD test from the Instrument menu (for LC3D and CE only) it checks the instrument for intensity and wavelength calibration.
Index Index A abort sequence 170 absolute response factor 116 retention time 130 retention time 132 accuracy analysis 157 amount limits 131 amount limits 148 analog signal 60 analysis accuracy 157 apex 73 area reject 105 area% calculation 118 report 217 ASTM noise determination 237 automatic batch review 211 library search 54 recalibration 179 shutdown 178 automation 26, 159 what is? 161 B baseline allocation 89 baseline tracking 74 baseline allocation 75 baseline construction 89 baseline penetration 91
Index confidence interval 252 configuration 15 control chart reports 25 control limits 210 corrected retention time 130, 134 correction factors 116 curve calibration 144 fits 151 customization data analysis 55 customized reports 24 cyclic recalibration 196 cyclic calibration bracketing 192 D Da.
Index manuals 32 method information 43 method file instrument parameters 51 method create 48 directory 51 edit 49 GLPSave.
Index average 181 complete 157 interval 181 partial 157 retention time 157 unidentified peaks 157 what is? 157 why 157 reference peaks finding 139 multiple 136 single 134 using 134 reference window 133 regression analysis 253 regression regression coefficient 253 relative retention 248 replace 181 report area% 217 calibrated 215 control chart 25 customized 24 destination 224 ESTD 215, 217 file formats 224 height% 217 reporting results 215 report norm% 217 sequence summary 24 reports system suitability 23 r
Index signal-to-noise calculation 239 single reference peaks 134 single-level calibration cyclic sequences 185 skew 245 slope sensitivity 105 slope 78 software overview data model 16 system configuration 15 software overview methods and sequences 15 operating system 15 solvent peak 103 standard deviation definition 254 standard deviation of mean 251 relative 251 sample 250 standard external 120 internal 123 recalibration with multiple vials standby state 178 start time 73 statistical moments 245 status ins
www.agilent.com In This Book This handbook describes various concepts of the Agilent ChemStation. It is intended to increase your understanding of how the ChemStation works. For information on using the ChemStation please refer to the general help system and the Online help "Tutorial".
