Varian PolyView2000 Operation Manual

Varian Analytical Instruments

NOTICE: This document contains references to Varian. Please note that Varian, Inc. is now part of Agilent Technologies. For more information, go to

www.agilent.com/chem.

2700 Mitchell Drive Walnut Creek, CA 94598-1675/usa

PolyView2000™

©Varian, Inc. 2002 03-914774-00:Rev. 4

Operation Manual

Trademark Acknowledgment

Microsoft, Windows, Windows 95, Windows 98, Windows NT, and Windows 2000 are registered trademarks of Microsoft Corporation.

Other brand and product names are trademarks or registered trademarks of their respective holders.

9065 PolyView Users

Please see the PolyView Operator’s Manual, 03-914738-00.

Overview................................................................................................... 1

PolyView2000 Functions .....................................................................................................2

Plotting a Chromatogram ..............................................................................................2

Examining and Analyzing Spectra ................................................................................3

Building, Editing, and Searching Spectral Libraries......................................................3

Determining the Purity of a Chromatographic Peak .....................................................3

Purity Evaluation by Multicomponent Analysis .............................................................4

Preparing Reports of Diode Array Data ........................................................................4

Recalculating the Peak Sense Events in a Diode Array Raw Data File .......................4

Quantitative Analysis by Multicomponent Analysis.......................................................4

Creation of Reduced Data Files....................................................................................5

PolyView2000 Windows ......................................................................................................5

Plot Window ..................................................................................................................7

Spectra Manager Window.............................................................................................8

Library Manager Window ..............................................................................................9

Reports Window..........................................................................................................10

Peak Sense Window ...................................................................................................11

MCA Window ..............................................................................................................13

PolyView2000 and Other Star Workstation Applications...................................................14

Operation................................................................................................ 17

Configuring PolyView2000 Windows.................................................................................18

Tutorial: Configuring PolyView2000 Windows...................................................................19

Plotting a Chromatogram...................................................................................................25

Tutorial: Plotting a Chromatogram.....................................................................................26

Examining Spectra from a Chromatogram ........................................................................35

Tutorial: Spectral Plots ......................................................................................................36

Tutorial: Spectral Math Functions......................................................................................41

Performing Library Functions ............................................................................................47

Tutorial: Creating a Library ................................................................................................50

Tutorial: Building and Editing a Library..............................................................................52

Tutorial: Conducting a Spectral Library Search.................................................................57

Reporting Data...................................................................................................................61

Tutorial: Making a Report ..................................................................................................62

Tutorial: Viewing and Printing of an Existing Report .........................................................69

Determining Peak Purity....................................................................................................71

Purity Parameter .........................................................................................................71

PolyView2000

Tutorial: Purity Parameter Plots.........................................................................................73

Tutorial: Peak Purity Survey Report ..................................................................................76

Performing Peak Sensing..................................................................................................77

Tutorial: Building a Peak Sensing Method ........................................................................79

Tutorial: Conducting Peak Sensing ...................................................................................81

Tutorial: Troubleshooting Peak Sensing............................................................................85

Automating PolyView2000 Operations..............................................................................90

Using On-Line Help ...........................................................................................................90

Tutorial: Using On-Line Help .............................................................................................91

Quantitative Analysis by MCA ...........................................................................................92

Tutorial: Using the MCA Window for Quantitative Analysis...............................................93

Tutorial: Automating the Quantitative Analysis..................................................................98

Purity Evaluation by MCA............................................................................................... 100

Tutorial: Using the MCA Window to Evaluate Purity ...................................................... 100

Creating Workstation Channels...................................................................................... 102

Library Searches ................................................................................. 105

Peak Sensing ....................................................................................... 109

Aurora................................................................................................... 115

Appendix A........................................................................................... 117

Interactive Reports.......................................................................................................... 117

Appendix B........................................................................................... 133

Non-Interactive Reports.................................................................................................. 133

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Overview

The PolyView2000™ Spectral Processing application, in conjunction with the Varian Star Workstation, provides post-run analysis of data from the 330 and the 9065 Diode Array Detectors. Unlike other chromatographic UV absorbance detectors that provide data in two domains, time and absorbance, the Diode Array Detectors yields data in the additional domain of wavelength.

Complete Spectral Data

PolyView2000

Domains of 330 Diode Array Data

While the data from a diode array detector is obviously more complex than a typical UV absorbance detector, this complexity can provide the chromatographer with considerably more information as well, when it is presented in an easily understandable form.

The PolyView2000 application is designed to handle this more complex data structure, enabling the user to extract a spectrum from any point in time within a chromatogram, or to generate an absorbance chromatogram at any wavelength (or set of wavelengths) within the wavelength range of the Diode Array detector. In this way, PolyView2000 can provide the user with qualitative information regarding an eluting compound. When PolyView2000 is used to generate an absorbance chromatogram, the Diode Array detector functions in much the same way as any other UV absorbance detector, yielding a concentration profile that changes with time. When PolyView2000 is used to examine UV absorbance spectra, this information, in conjunction with the retention time of the eluting compound, provides qualitative information that can be used to identify the compound of interest. In addition, the MultiComponent Analysis process (MCA) combines accurate quantitative analysis and purity evaluation, even in cases of coelution.

PolyView2000 Functions

The principal functions of the PolyView2000 Spectral Processing application are described in the following paragraphs.

Plotting a Chromatogram

The PolyView2000 application can provide plots of absorbance as a function of time at any of the available wavelengths within the range of the Diode Array Detector. While the absorbance chromatogram is the mainstay of chromatographic analysis, and as such, is often the starting point within the PolyView2000 application, UV absorbance chromatograms are not the only plots provided within PolyView2000. Additional treatments of the 330 data are also available, including plots of Purity Parameter™, absorbance ratios, wavelength maxima, and absorbance maxima. Just as an absorbance chromatogram provides absorbance as a function of time, these other parameters are also generated as a function of time. The results are a series of time dependent profiles that provide qualitative information on the spectral characteristics of eluting peaks. All chromatogram plots can be obtained both interactively and under automation.

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VERVIEW

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UNCTIONS

Examining and Analyzing Spectra

As mentioned earlier, through the PolyView2000 application, all of the spectra that constitute a raw data file are available for examination and analysis. Spectra can be viewed individually, or up to seven spectra can be overlaid and viewed at the same time. This spectral plot can be expanded to visually show detailed information about spectra. In addition, these spectra can be operated on arithmetically, enabling you to create a composite spectrum that is the sum or difference of several spectra. Most spectral functions are performed interactively, but reports comparing sample spectra to reference spectra are produced as part of the library search and MCA automated commands.

Building, Editing, and Searching Spectral Libraries

A particularly useful feature of PolyView2000 is the ability to create and maintain libraries of spectra. Within a spectral library, you can archive those spectra that are relevant to a particular chromatographic application. PolyView2000 can then provide ordered searches of these libraries, comparing a spectrum of interest within a chromatogram to the spectra stored within one or more spectral libraries. Library building and editing are interactive tasks. Library searching can be performed interactively or under automation.

PolyView2000

Determining the Purity of a Chromatographic Peak

Co-elution is often a problem with samples composed of structurally similar constituents. PolyView2000 provides several means of determining the purity of a chromatographic peak by displaying the spectral characteristics across the width of the peak. As an example, the Purity Parameter of a UV spectrum uniquely distinguishes it from other similar spectra. By plotting the Purity Parameter as a function of time, slight differences in the UV spectra across a peak can be determined, indicating the presence of impurities. Similarly, absorbance ratio and wavelength maxima plots can be used to indicate spectral dissimilarities across the width of a peak.

Purity Evaluation by Multicomponent Analysis

Multicomponent Analysis also provides a powerful tool to assess the spectral homogeneity of a peak or the presence of unexpected impurities. All purity features are available both interactively and under automation.

Preparing Reports of Diode Array Data

An important function of PolyView2000 is the ability to generate reports that contain the results of the data analyses. These reports are available in a variety of formats and cover most of the operations that are possible with PolyView2000. You can generate reports while working at the Star Workstation itself (interactive reports) or at the end of a run and some time later in an automated fashion (non-interactive reports).

Recalculating the Peak Sense Events in a Diode Array Raw Data File

Associated with every UV absorbance chromatogram obtained with the Diode Array detector is a series of peak sense events. These events mark important points within a chromatogram, and include peak start, peak end, peak apex and peak upslope and downslope. At times, the peak sense events that are calculated and set at the time of acquisition of a diode array data file may need to be redetermined. PolyView2000, in conjunction with the Method Builder application of the Star Workstation, provides the ability to recalculate these peak sense events.

Quantitative Analysis by Multicomponent Analysis

By integrating a group of fused peaks at each wavelength, one obtains an Area Spectrum. This Area Spectrum is the baselinecorrected spectrum of an elution fraction. If the spectra of all components are known, the Area Spectrum can be decomposed by a simple mathematical process known as Multicomponent Analysis. This process is routinely used in spectrophotometers. In PolyView2000, this process is used to obtain precise quantitative results from overlapping peaks.

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Creation of Reduced Data Files

PolyView2000 can reduce a data file to a few channels of data corresponding to time-programmed wavelengths or wavelength ranges, while preserving full spectra for the events detected by peak sensing. These channels can also be created while preserving all spectral data. These files can be created both interactively and under automation.

PolyView2000 Windows

The PolyView2000 Spectral Processing application is organized into several distinct child windows within the PolyView2000 main application window. These include the Plot window, Spectra Manager window, Library Manager window, Reports window, Peak Sense window, and the MCA window. The following illustration shows the organization of the PolyView2000 windows and the transfer of data between the windows. Multiple copies of several of the windows are available as indicated.

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Manager Window

PolyView2000

Library

Poly View

Main Application

Window

Spectra

MCA

Window

Spectra

Manager

Window

Spectra

.LBR

Files

.LBR Files

.RUN Files

Plot

Window

.RUN

Files

.RUN, .MTH

Perm anent

Disk Storage

Organization of the PolyView2000 Windows

Peak

Sensing

Window

Files

.SRP,.MTH

Files

Reports

Window

The three windows used most in PolyView2000 functions are the Plot, Spectra Manager, and Library Manager Windows. When PolyView2000 is first started, the screen is split between these three windows.

You will find that many of the data analyses performed using PolyView2000 requires only these three windows and you will not need to alter the screen configuration or create additional windows. Of course, if your work requires the use of additional windows, they are easily created and the screen configuration can be easily altered. Following is a brief description of the six PolyView2000 windows. For more detail on the functions available in each and the operation of On-Line Help, refer to the operation section.

PolyView2000 Configuration Upon Start-Up

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VERVIEW

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Plot Window

The Plot window is used to plot chromatograms from diode array raw data files (.RUN files). The plots can display the absorbance, Purity Parameter, absorbance ratio, absorbance maxima or wavelength maxima, each as a function of run time. Once displayed, regions of the plot can be enlarged (zoomed) and a variety of spectral correction methods can be selected. Only one chromatogram at a time can be displayed in the window. However, at the same time for comparison of different .RUN files or to compare different plot conditions on the same .RUN file. Additional features in the plot window include the ability to display peak events (peak start, peak end, apex, etc.) and to transfer selected spectra from the displayed raw data file to the Spectra Manager window.

several plot windows can be displayed on the screen

PolyView2000

The Plot Window

Spectra Manager Window

The primary function of the Spectra Manager window is the display and analysis of UV spectra from selected points in a chromatogram. A single spectrum can be displayed or up to seven spectra can be overlaid. Information about the spectra, such as compound name, operator, and method name, can be added or edited in this window. This information becomes a part of that particular spectrum record and is transferred with it between the various PolyView2000 windows.

The Spectra Manager Window

A number of data analysis functions can be performed in the Spectra Manager window such as the calculation of Purity Parameters, the determination of their statistics, and the arithmetic combination of spectra. Also, the wavelength range over which the Purity Parameter is calculated can be optimized. Several different reports of the spectra and the spectral analyses can be prepared and printed directly from the Spectra Manager window. From this window, spectra can be transferred to the Library Manager window for use in spectral library functions.

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Library Manager Window

A variety of spectral library functions are performed within the Library Manager window. These include building and editing libraries, conducting library searches, and preparing reports of the search results. Any spectrum transferred into the Library Manager window from the Spectra Manager is held in a spectrum register. The Library Manager window has the ability to hold only one spectrum. Information about the spectrum (name, operator, etc.) can be added to this spectrum in the same manner as it was in the Spectra Manager window. In addition, the Library Manager provides for the management of disk libraries of up to 120 spectra each. Library management functions include adding, editing, and deleting spectra in libraries. Additionally, key word searches of the contents of a library can be made to quickly locate a selected spectrum. In addition to the library management functions, library searches are performed in the Library Manager window. To conduct a search, a spectrum is transferred from the Spectra Manager window into the Library Manager window. One or several disk libraries are specified and the search is conducted based on a number of retention time, and wavelength. The search results are ordered according to the similarity between the target spectrum and the match spectrum from the library. Reports can then be printed to document the results of the library search.

selectable criteria including Purity Parameter,

PolyView2000

The Library Manager Window

Reports Window

A wide variety of printed reports can be prepared in PolyView2000. Most operations within PolyView2000 windows can be documented through an “interactive” report obtained either by selecting Print... from the File menu or by selecting the Print pushbutton appearing in many of the dialog boxes. This is just one way to obtain a printed report. Reports can also be prepared in an automated, unattended fashion. In PolyView2000, a separate window, the Reports window, is used to prepare and manage these reports. The Reports window is not activated at the time of start-up and must be created using the New Reports Window command item under the Window menu. A variety of different reports can be prepared in this window including chromatograms, library searches, peak purity surveys, and tables of data characterizing the peak events in a data file. The contents of a report are first specified in the Method Builder application and then prepared in the Reports window. Once prepared, the report is saved as a disk file. It can then be loaded in the Report Window, viewed on the screen, edited, printed, or exported in a variety of formats to other windows applications.

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The Reports Window

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PolyView2000

Peak Sense Window

A number of functions within PolyView2000 require peak detection to determine the various peak events (peak start, peak end, inflection points, and apices). For instance, to provide efficient spectral correction to account for mobile phase absorbance, the peak start and ending points must be accurately determined. This ensures that the spectra obtained for an eluting peak accurately reflect the compound of interest, free from background interference. At the time of data collection, PolyView2000 peak sensing is automatically performed on diode array files, provided a PolyView2000 section is included in the Workstation method file (.MTH) under which the data is collected. However, if no PolyView2000 section was included or if the peak detection was not accurate (as indicated by misplaced peak events or baseline), then peak sensing may need to be repeated.

The Peak Sense window is used to perform a peak sense recalculation on a raw data file. Like the Reports window, the Peak Sense window must be created using the New Peak Sense Window menu item under the Windows menu. In the Peak Sense window, the absorbance chromatogram is displayed at the wavelength used for peak sensing. To perform peak sensing, the method to be used (wavelength, peak width, etc.) is specified in the Method Builder application and then

used in the Peak Sense window. Additional features include the ability to view and edit the noise data acquired by the Workstation during the diode array monitor period. The noise data is used in conjunction with the S/N ratio parameter to detect peaks, and editing of this data may be required in cases where there was excessive detector noise during the monitor period.

The Peak Sense Window

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VERVIEW

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MCA Window

The Multicomponent Analysis window contains all the functions required to perform MCA Quantitative Analysis and Purity Evaluation. The MCA window is composed of 3 areas. The bottom half displays a Chromatogram at a user selectable wavelength, which is used to select the integration limits. The upper right quarter contains 6 push buttons that implement the main commands, and the upper left quarter shows the spectrum of the integrated chromatogram region.

PolyView2000

The MCA Window

PolyView2000 and Other Star Workstation Applications

The PolyView2000 application software is integrated into the Star Workstation to enable the Star Workstation to conduct automated tasks that include PolyView2000 functions. For instance, at the end of a chromatographic run, the Star Workstation can automatically conduct PolyView2000 peak sensing on the .RUN data file and prepare selected PolyView2000 reports, all without your actually having to start the PolyView2000 application.

Upon installation, a PolyView2000 Icon is added to the Star Workstation’s Star Tool Bar. PolyView2000 interacts with two other Star Workstation applications, Method Builder and System Control/Automation. The .MTH file is used to specify the peak sensing conditions (wavelength, peak width, etc.) or the type of reports needed. Method Builder is used to build and manage the .MTH files that are used to perform these functions. In PolyView2000, the Method Builder application can be accessed directly, without going through the Star Tool Bar. When accessed in this manner, after the method editing session is completed and the Method Builder application is closed, control is returned to PolyView2000, right where you left off.

NOTE: See the Data Acquisition with LC Control Operation Manual for more

details on the functions and operation of the Method Builder application.

The other Star Workstation application that interacts with PolyView2000 is System Control/Automation. However, the interaction is not readily apparent to the user, as it involves internal processing within the Star Workstation via the AutoLink protocol. When a diode array detector is operating under the control of the System Control/Automation application, post-run PolyView2000 processing and reporting is performed on any diode array file based on the PolyView2000 section in the active method. If no PolyView2000 section exists in the method, default peak sensing conditions are used, and no reports are created.

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VERVIEW

OLYVIEW

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AND OTHER STAR WORKSTATION APPLICATIONS

The results of the peak sensing and/or reporting performed under System Control/ Automation are exactly the same as if these functions had been performed interactively in PolyView2000. System Control/Automation is also used if a series of .RUN files need to have peak sensing or reporting repeated on them. In this case, the recalculation capability of System Control/Automation is used to conduct

PolyView2000

peak sensing or reporting on a series of .RUN files.

NOTE: See the Data Acquisition with LC Control Operation Manual for more

details on the operation of the System Control/Automation application.

PolyView2000

Operation

The functions and operation of PolyView2000 are described in this section. PolyView2000 is a very powerful software program and often there are several different ways to accomplish the same task. It is not the purpose of this section to fully describe every operation available within PolyView2000. Rather, the primary functions will be described along with a series of short Examples to assist you in getting started using PolyView2000; in particular, learn to use the On-Line Help which contains both Reference and Procedural information. This may help you to accomplish a particular function in a different way than that described in the Tutorials. Use the commands that are best suited to your particular needs or application. The primary PolyView2000 functions covered in this section are:

PolyView2000

• Configuring PolyView2000 Windows

• Plotting A Chromatogram

• Examining Spectra from a Chromatogram

• Performing Library Functions

• Reporting Data

• Determining Peak Purity

• Purity Parameter

• Performing Peak Sensing

• Automating PolyView2000 Operations

• Using On-Line Help

• Quantitative Analysis by MCA

• Purity Evaluation by MCA

• Creating Workstation Channels

Following the description of each of these functions, one or more Tutorials are presented to help you get started. Read the functional description section, work through the Tutorials, and then refer to the section or On-Line Help if further information is needed on a particular window or menu item.

Configuring PolyView2000 Windows

PolyView2000 operates under Microsoft Windows Multiple Document Interface (MDI). The MDI tracks and reflects changes in the menus associated with each child window within a main application window. The menu at the top of the main application window changes as the nature of the active child window changes. For example, within the PolyView2000 main application window, if you move from a child window that is used to manage spectra, to a child window used to manage libraries, spectrumspecific menus will be replaced with library-specific menus. When a number of child windows are present in the main application window, a child window is made the active window by moving the mouse cursor to that window and clicking on its title bar. The title bar changes color (or contrast) to indicate that it is now the active window. The menu names and items reflecting the functions and operation of the active child window are then displayed within the menu bar of the main application window at the top of the screen. Organization of the child windows within the main application window can easily be accomplished using the commands under the Window menu. Individual windows can be created, hidden from view, recalled, and arranged in a variety of different configurations. In addition, all of the window sizing controls are available as they are in most Windows applications.

NOTE: If you require further explanation of the operation of the MDI within

Windows, or additional detail on Microsoft Windows itself, refer to the

Star Workstation Operation Manual, or the Microsoft Windows User's

Guide.

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UTORIAL

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ONFIGURING POLYVIEW

Tutorial: Configuring PolyView2000 Windows

On the Star Tool Bar, double-click on the PolyView2000 icon. The application opens with the screen split between the Spectra Manager, Library Manager and Plot windows with the focus on the Plot Window.

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PolyView2000

PolyView2000 Configuration Upon Start-Up

All of the windows can be sized using the border controls and the sizing control in the upper right corner of each child. In addition, the main application window can be sized using its borders and controls when it is not maximized. The one restriction is that all the child windows must always remain within the confines of the main application window. Practice some of these window sizing functions and make the screen appear as shown below.

PolyView2000 Window Configured to Increase

Spectra Manager Area

The number and type of windows displayed within the PolyView2000 main application window are controlled using the Window menu. This menu is visible and available at all times.

• Create a Reports window by selecting New Reports

Window under the Window menu.

• Repeat this process, but this time select a Peak Sense

window. These windows, like all the others, can be moved and sized to any configuration desired using standard Windows methods. But, don't do that now. There is a much easier way as you will see in a minute.

Any of the currently active windows within PolyView2000 can be instantly accessed and displayed full screen.

• Drop down the Window menu and note that the five

PolyView2000 windows are listed at the bottom of the menu.

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UTORIAL

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ONFIGURING POLYVIEW

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Windows Menu Listing the Currently Active Windows

• Click on Spectra Manager in the Window menu and note that

this window is brought forward.

• You may maximize any window to the full size of the

PolyView2000 window simply by double-clicking on the title bar of the window you wish to maximize. Try this now, to maximize the Spectra Manager window.

PERATION

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PolyView2000

• Any of the open windows can be accessed instantly in this

manner. Multiple copies of the Plot, Peak Sense, and Reports windows can be active simultaneously and all of these multiple copies will be listed under the Window menu. This allows you to rapidly select from a number of chromatograms that may be currently active within PolyView2000.

A rapid method is provided that allows you to view all the currently active windows.

• Drop down the Window menu and select “Arrange All”. The

screen is split between all the windows under the Window menu.

Screen Split Among Five PolyView2000 Windows

• Individual windows can be hidden from view also. Click on

the title bar of the Library Manager window to make it the active window, drop down the Window menu and select Hide. Notice that the Library Manager is hidden from view, but it is not removed from the list of active windows (i.e., it still appears in the list under the Window menu).

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UTORIAL

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ONFIGURING POLYVIEW

PolyView2000 with Library Manager Hidden from View

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PolyView2000

• Now, the remaining windows can be organized. Select

“Arrange Visible” under the Window menu and note that the screen is partitioned between the four remaining visible windows.

PolyView2000 Screen Split Between the Four Visible Windows

• In this way the screen can be rapidly configured to meet the

needs of the PolyView2000 function you are performing.

• Also try “Arrange Plots”.

Except for the Spectra Manager and Library Manager windows, the individual child windows within PolyView2000 can be closed.

• Drop down the Window menu and select Arrange All.

• Close the Reports, Plot, and Peak Sense windows by

double-clicking in their Control Menu boxes. Note that the Control Menu box does not appear in a window's title bar until that window has been selected as the active window by clicking within it. You may go ahead and double-click where the Control Menu box would be and the window will be both selected and closed at the same time.

• Drop down the Window menu and note that these three

windows are no longer listed. It is important to remember the difference between a window that is merely hidden and one

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that is closed. All data in a window is lost when it is closed, but simply hiding the window does not destroy the data.

• Close PolyView2000 by double-clicking on the Control Menu

box in the main application window.

Plotting a Chromatogram

With the PolyView2000 application, raw data files transmitted from the Diode Array detector and collected at the Star Workstation can be plotted, examined on the display screen of the Star Workstation, and then printed out. The Plot Window, within the PolyView2000 main application window, is used to perform these functions. There are a variety of different plot types available in PolyView2000, each expressing the raw data in a different format. See On-Line Help (Help:Plot Manager) for an explanation of the different types of plots available and their uses. In the following Tutorial, the basics of chromatogram plotting are covered. In subsequent Tutorials, some of the more functional aspects of the chromatogram plots, such as their use in the determination of peak purity, will be addressed.

PERATION

LOTTING A CHROMATOGRAM

NOTE: It is not the purpose of this Tutorial to cover every possible function in the

Plot window. However, the basic functions are covered. For information on those functions not covered, such as those under the Peak Events menu, refer to On-Line Help.

In the following Tutorial, some of the files in the EXAMPLES directory will

be modified and/or created. Prior to performing this Tutorial, you should ensure that you are working with original copies of the data files called for. If you are not sure whether these files have been modified by previous use of the Tutorial, reinstall the PolyView2000 software from the CD using your serial number.

PolyView2000

Tutorial: Plotting a Chromatogram

On the Star Tool Bar, double-click on the PolyView2000 Spectral Processing icon. The application starts with the screen split between the Spectra Manager, Library Manager and Plot windows with the focus on the latter. To plot a chromatogram:

• Drop down the File menu and select Open File... . A dialog

box is displayed that lists directories and files with the extension .RUN. Press the “Help” button for more information on the operation of file selection dialog boxes.

File Selection Dialog Box

• Select the EXAMPLES directory from the list and note that

the Path field should display: C:\Star\Examples.

• Click once on a .RUN file. Try BASECORR.RUN. Notice that

the fields at the bottom of the screen fill with information about this file. The Status field indicates that a valid file has been selected. If you had selected a non-diode array file, an error message in red would have been displayed instead.

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PERATION

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LOTTING A CHROMATOGRAM

• Click on OK to accept this file. The absorbance

chromatogram at 253.6 nm is plotted in the Plot window at the bottom of the screen. The plot is autoscaled on the Yaxis and is plotted from time 0.0 to the end of the run

• Use the Select Plot Parameters command in the Plot menu

to plot the absorbance at 210 nm instead. If the plot goes off scale, double click to the left of the absorbance scale when a double headed arrow appears.

PolyView2000

BASECORR.RUN in the Plot Window

Many PolyView2000 functions can be performed with the window in this configuration, but there are many ways to alter the viewing of the chromatogram. Maximize the Plot window (click on the box with a solid line above it in the upper right hand corner of the Plot window). This enlarges the chromatogram to full screen.

Full Chromatogram

More detail in the chromatogram can be seen by utilizing the zoom functions. Zooming in PolyView2000 is accomplished by the drag method, the same as in the Interactive Graphics application.

• Using the mouse, position the cursor below the baseline just

to the left of the peak at 7.2 minutes. Press the left mouse button and hold it down as you drag the mouse diagonally toward the upper-right portion of the peak. The portion to be zoomed will appear in reverse video and when the mouse button is released, the plot will be zoomed.

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UTORIAL

Plot Window Zoomed in on a Peak

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LOTTING A CHROMATOGRAM

PolyView2000

• The plot can be zoomed again to observe even more detail.

Try zooming the top portion of the peak.

• Pressing the Escape (ESC) key during the drag process

aborts the zoom process.

To restore the scaling in effect prior to the latest zoom, drop down the View menu and select Previous. The plot is returned to the full peak. Selecting Previous again restores the zoomed top portion of the peak and thus functions as a toggle to switch between two zoom selections.

The full time range can be restored by positioning the cursor just below the time axis and double-clicking with the left mouse button. Note that the cursor changes to a double-headed arrow indicating that the plot will be restored to its full X-axis scaling. Similarly, the full Y-axis scaling can be restored by doubleclicking the mouse with the cursor positioned to the left of the Yaxis. Both can be restored simultaneously by double-clicking in the lower left corner. Try these functions now and restore the

scaling to its original setting when the chromatogram was first displayed.

NOTE: Chromatogram zooming can also be controlled using the commands under

the View menu. See On-Line Help (Help:Commands) for an explanation of these menu items.

To change the plot configuration (type, wavelength, etc.), drop down the Plot menu and click on the Select Plot Parameters menu item. This displays a dialog box that is used to specify the type of plot and features. The X and Y scales can be set here, the peak events can be turned on and off, and the wavelength range can be selected. Many of these features will be covered in future Tutorials.

The Select Plot Parameters Dialog Box

For this exercise:

• Change the events list box in the lower right corner to Draw

Apex Events.

• Set the wavelength range to 220-366 nm using the

Wavelength Range scroll boxes by either scrolling to the proper number or typing the number. Type the left hand lower wavelength first and then the right hand higher wavelength.

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PERATION

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LOTTING A CHROMATOGRAM

NOTES: For many PolyView2000 functions where either a single wavelength or a

wavelength range can be set, setting the right hand scroll box with cause both boxes to have the same wavelength. Setting the right hand scroll box will not effect the left hand box. Therefore, to set a single wavelength you only need to set the right hand scroll box.

Checking the “Save as default parameters dialog” check box will save

this plot type and condition (except time and scale ranges) and make them the default conditions for any plot windows created later, even in other sessions of PolyView2000. Do not check it at this time.

• Click on the OK pushbutton. The chromatogram will be

plotted using the average absorbance in the range of

220.00-366.00 nm with only peak apex events drawn. Notice that the original absorbance scaling was maintained.

To autoscale, simply double-click in the left margin of the Plot window. To prepare for the next step, zoom in on the peak at

19.3 minutes.

PolyView2000

BASECORR.RUN Displayed at 220.00-366.00

When spectra are extracted from a chromatogram, PolyView2000 automatically corrects them for background absorbance. Several different types of correction are available and are set in the Correction menu.

The default is baseline but other types are available, including the selection of one or more spectra in the file to use as a reference to which all others are corrected. The current correction method is indicated below the time axis. Let's try that function now:

• Drop down the Correction menu and select Reference. A

dialog box appears that prompts you to select the reference spectra.

• Move the cursor to any point of the chromatogram left of the

peak at 19.3 minutes, and press the left mouse button once. A vertical line is drawn to indicate the reference spectrum. Define another reference spectrum right of the peak, then press OK to indicate that you are done defining the reference spectra. The baseline is redrawn to indicate the change. The correction is interpolated between reference spectra, and uses the outermost reference spectra to correct spectra extracted from the corresponding regions. The correction method can easily be changed to a different mode by making a

different selection in the Correction menu.

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LOTTING A CHROMATOGRAM

BASECORR.RUN with Reference Correction

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• Make the correction method Peak Start and note that the

red lines under each peak originate from the peak start event and project horizontally.

The effect of the baseline will

be demonstrated in the next Tutorial.

correction mode on extracted spectra

Associated with each .RUN file is information that describes the conditions under which it was collected (date, time, etc.). Within PolyView2000, some of this information can be viewed and edited.

• Under the File menu, select Edit File Information. A dialog

box will appear that allows you to edit selected fields. Fields that can be edited are enclosed in boxes and they are edited by standard Windows techniques.

The Edit File Information Dialog Box

• Type your name in the Operator field and add any comments

into the Notepad.

• Click on OK to write the new information into the disk file for

BASECORR.RUN.

To print a chromatogram from PolyView2000, select the Print Plot command under the File menu and the chromatogram will be printed on the current Star Workstation printer. See Appendix

A for an example of this interactive plot report.

Close PolyView2000 by double-clicking on the Control Menu box in the main application window.

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XAMINING SPECTRA FROM A CHROMATOGRAM

Examining Spectra from a Chromatogram

The primary advantage of a diode array detector such as 330 is the availability of spectra at all points in the chromatogram. These spectra provide valuable qualitative data on the nature of an eluting peak. Using the PolyView2000 advanced baseline correction routines, the spectra extracted from a chromatogram are free of errors due to any background solvent absorbance and reflect only the spectra of the eluting compound. Within PolyView2000, the primary spectra handling utilities are provided in the Spectra Manager window. This window provides a temporary storage location for up to seven UV spectra. The spectra that are held in these spectrum registers are available for a variety of spectral processing and reporting functions. In addition, the window serves as a temporary storage location for spectra before they are transferred to the Library Manager window for inclusion in a disk library or as the target of a library search.

The spectral processing functions available include the calculation of Purity Parameter statistics, the optimization of Purity Parameter spectral ranges and the arithmetic combination of spectra. Details on these functions can be found in On-Line Help (Help:Commands:Spectrum).

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The following two Tutorials cover the Spectra Manager functions. As you are going through these Tutorials, keep in mind that there may be more than one way to perform many of these functions. An attempt has been made to present the most straightforward method. With experience, you may find that an alternate way of accomplishing the function may be more suited to your application. Feel free to explore these alternate command paths.

Tutorial: Spectral Plots

On the Star Workstation Star Tool Bar, double-click on the PolyView2000 Spectral Processing icon. The application starts with the screen split between the Spectra Manager, Library Manager and Plot windows with the focus on the latter.

Configure the screen to display the Spectra Manager window and the Plot window.

PolyView2000 Screen without Library Manager

Plot out the data file BASECORR.RUN from the EXAMPLES directory in the Plot window at 210 nm with Apex and Inflections events displayed (see the Tutorial: Plotting a Chromatogram).

Spectra are extracted from the chromatogram and transferred to the Spectra Manager window by clicking the mouse at the desired point in the chromatogram. Try that now:

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• Zoom in on the peak at 7.3 minutes and extract three

spectra at the apex and the front and back inflection points. Notice that each time you click on the chromatogram, a spectrum for that peak appears in the Spectra Manager window and the point in the chromatogram where the spectrum was extracted is marked with a corresponding colored line. Move the cursor to the Spectra Manager display and zoom on the spectra until your screen should look similar to the figure below.

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Overlaid Spectra, and Markers on the Chromatogram

The graphics display in the Spectra Manager window is controlled through a set of commands in the Display menu. Try some of these now:

• First, click on the title bar of the Spectra Manager window to

make it the active window.

Normalized Spectra

• Drop down the Display menu and select Normalized. Notice

that the three spectra are now normalized over their entire wavelength range. You can compare them over a limited range by zooming on the range as shown. This allows you to compare their shapes.

• Drop down the Display menu again and select Single Plots.

In this mode, only one spectrum is displayed at the time. Use the numbered colored buttons at the bottom of the Spectrum Manager Window to choose which spectrum you want to display.

The wavelength scale for the Spectra Manager window is variable depending on the wavelength of data that was collected. For the 9065, this is 190 to 367 nm (the limits of the 9065 diode array detector). For the 330 Diode array detector, this is the range over which the data was taken. If spectra from different diode array runs are compared where the range of wavelengths for the data is different, the widest range needed to encompass all of the data is used. When the spectra is zoomed, the entire screen is considered the working range. The wavelength flags

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that appear at the top of the Spectra Manager display indicate this range. This is the range considered when normalizing plots and in performing some of the math functions addressed in the next Tutorial. The range can be quickly altered by zooming in the Plot window. Try that now:

• Zoom (using the click and drag method) the Spectra

Manager window in the region between 250 and 300 nm.

The seven numbered boxes at the bottom of the Spectra Manager window screen represent the seven spectra storage registers in this window. When a spectrum is in one of the spectrum registers, a colored rectangle appears in the box. A filled rectangle means that the spectrum is currently displayed, while an empty rectangle indicates that the register position contains a spectrum but it is not currently displayed. You can turn on and turn off which spectra are displayed by clicking in the numbered boxes containing spectra.

Try that now. Point to the second box and click the mouse. Notice that the first spectrum was turned off, since Single Plots was selected. If Multiple Plots had been currently selected, this action would simply turn on and off that particular spectrum. Double-clicking on a particular box calls up that spectrum for editing. This feature will be covered in the Tutorial:Building and

Editing a Library.

Spectra can be removed from the Spectra Manager window using the controls under the Spectrum menu.

Drop down the Spectrum menu and select Delete Displayed Spectra.

NOTE: All spectra in the display area are removed. A deleted spectrum is

actually removed from the window and cannot be recalled without going back to the Plot window and extracting it again.

• Drop down the Spectrum menu and select Delete All

Spectra. Note that all of the boxes at the bottom of the screen are empty, indicating that there are no spectra left in the window.

PolyView2000

Now let's utilize the display functions you have learned to address a practical problem: the determination of peak purity. Using these spectrum display controls, you can quickly compare different spectra from within one eluting peak for an indication of peak purity.

• In the Plot window, open PV_SAM01.RUN and zoom in on

the fourth peak in the chromatogram at 3.45 minutes.

• Transfer spectra from the front and back portions of this

peak to the Spectra Manager window.

• In the Spectra Manager window, configure the display for

Multiple plots, 190.00-366.60 nm and Normalized. (Remember to click on the title bar to activate the Spectra Manager window.)

• Turn on both spectra and note that they do not overlay

perfectly, indicating the presence of co-eluting compounds in what appeared to be a single homogeneous peak. While visual overlays like this are useful for peaks with substantial impurities, numerical approaches, detailed in the next Tutorial, are less subjective and can be statistically validated.

• To see the results of this exercise on a pure peak, repeat

this on the peak eluting at approximately 5.0 minutes and observe how well the spectra are overlaid.

To print reports of spectra extracted from a chromatogram, click on the Spectra Manager title bar, then drop down the File menu and select Print Spectra. The report will either be a single spectrum report (if the window currently is displaying one spectrum) or a spectral overlay, if multiple spectra are currently displayed on the screen. (See Appendix A for example of the two types of reports).

Do not close PolyView2000. Instead, go on to the next Tutorial.

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Tutorial: Spectral Math Functions

A variety of spectral processing functions are available in the Spectra Manager window that help in the evaluation of spectral

data

• Using the procedures from the previous Tutorial, transfer two

spectra from the upslope and downslope portions from the impure peak at 3.45 minutes in PV_SAM01.RUN to the Spectra Manager window.

• Configure the window to display multiple plots between 190

and 367 nm with the ‘Normalized’ setting. The vertical lines in the Spectra Manager window correspond to the Purity Parameter calculated for each spectrum.

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PolyView2000

Upslope and Downslope Spectra from the Peak at 3.45 Minutes in PV_SAM01.RUN

• Display the numerical value for these Purity Parameters by

dropping down the Spectrum menu and selecting View PuPs. In the dialog box, you can change the wavelength range for the Purity Parameter calculation and the values are instantly recalculated.

NOTE: In PolyView2000, Purity Parameter is often abbreviated PuP.

• Close the Purity Parameters dialog box by double-clicking on

its Control menu box.

The Purity Parameter in PolyView2000 is a primary means to distinguish between different spectra. However, the ability to discriminate between spectra is highly dependent on the wavelength range over which the calculation is made. PolyView2000 provides a means to optimize the wavelength range used in this calculation. Drop down the Spectrum menu and select Optimize PuP. Notice that the cursor is replaced with the hourglass, indicating that the Star Workstation is calculating. Following completion of the calculation, select Yes in the informational pop-up and the wavelength range in the Spectra Manager window is set to the values that result in the optimum separation of Purity Parameter values for the displayed spectra.

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Screen Display Following Optimization of the Purity Parameter

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The optimum range for your spectra may not exactly match that shown, since it is dependent on exactly where in the peak that the spectra were extracted.

The Optimize PuP function is particularly useful for determining the proper wavelength range to use in assessing peak purity using Purity Parameter plots (see Determining Peak Purity on

page 71).

For the next two exercises, replace the spectra in the Spectra Manager window with three spectra corresponding to the upslope, downslope, and apex of the pure peak at 5.0 minutes in PV_SAM01.RUN. Configure the Spectra Manager window to display Multiple Plots, Normalized in the range 190-367 nm. The screen should appear as shown below.

Screen Display of the Three Spectra

Extracted from the Peak at 5.0 Minutes in PV_SAM01.RUN

The statistical variation in the Purity Parameters for a number of spectra can be calculated.

• Drop down the Spectrum menu and select Do Statistics.

• A dialog box appears that lists all the spectra in the window,

their correlation values, average and standard deviation. Initially, the wavelength range is set to what is selected in the spectrum window. Statistics over alternate ranges can also be calculated.

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The Purity Parameter Statistics Dialog

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• Check the Accumulate Info box, change the wavelength

range to 220-366.6 nm, and click on the Calculate pushbutton. Data for the new range is added to the original data.

• Try several more ranges and note that the statistics report

lengthens. The entire report can be viewed using the scrolling controls to the right of the report.

• Select the Print pushbutton to obtain a printed report of the

calculations (see Appendix A for an example of the PuP statistics report) for an example of the PuP statistics report).

• The Purity Parameter statistics can be used to help in

deciding the significance of subtle differences in values among several different spectra and for establishing expected correlation values for library searches.

• By entering a Purity parameter target and/or a standard

deviation in the fields composing the PuP Target group, you can express the purity parameter statistics relative to a standard. If filled, the Name field will be added to the report.

• Close the Statistics dialog box by double-clicking on its

Control menu box.

Spectra can be combined arithmetically in the Spectra Manager window.

• With the screen configured as above, drop down the

Spectrum menu and select Do Arithmetic. The Spectra Arithmetic dialog box will appear with all current spectra displayed.

The Spectra Arithmetic Dialog Box

• Edit the factor fields just to the right of each spectrum by

replacing the 0.0 with 0.333.

• Set the wavelength range to 190-367 nm and select

Compute.

• The spectrum that appears in the large graphics display is

the average spectrum, since factors of 0.333 were used for each of the three source spectra. From here, the calculated spectrum can be sent to the Library Manager or the next storage location in the Spectra Manager.

Close the PolyView2000 application by double-clicking on the Control Menu box in the main application window.

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NOTE: Spectra arithmetic functions are particularly useful for preparing spectral

libraries which contain averaged spectra resulting from several different injections. To do this, transfer apex or area spectra from several different injections of a standard solution to the Spectra Manager window. Average the spectra using the spectra arithmetic functions and transfer the resulting spectrum to the Library Manager for inclusion in the library.

Performing Library Functions

PolyView2000 incorporates extensive Spectral Library functions. These capabilities allow the user to match spectra resulting from the injection of unknown samples with reference spectra archived in spectral libraries. The Library functions in PolyView2000 include the ability to build a library from a .RUN file, edit the library records, search the list of library entries for a specific name or key word associated with the record, and conduct spectral searches of the library. Spectral searches can either involve searching the library for a specific spectrum picked from a .RUN file or searching a .RUN file for a specific spectrum from the library. Initially, the library search can be narrowed down by restricting the retention time and Purity Parameter match windows. Then, the library spectra that meet these first criteria are considered and their spectral similarity with the target spectrum is quantified, providing “Dissimilarity” and “Similarity” indices. The library operations are some of the most complex in PolyView2000. They routinely involve the use of three different windows (Plot, Spectra Manager, and Library Manager) with movement of data (spectra) between the three windows.

PolyView2000

Like similar operations in other related disciplines (Mass Spectrometry, Infrared Spectrometry), the library functions in PolyView2000 provide qualitative information about an eluting peak by correlating an unknown “target” spectrum with a known reference spectrum. In UV spectrometry, this task is somewhat more difficult, since the information content of UV spectra is low (nearly identical spectra can be obtained from different compounds) and the spectra obtained can be influenced by a number of factors (pH, ionic strength, detector non-linearity, mobile phase absorbance, etc.). Consequently, care must be taken in the interpretation of data from a Diode Array Detector. At best, the spectral correlations obtained from library searches should be used as a means to confirm the identity of an eluting

peak after taking into account other criteria such as the retention time comparison with a known standard. In very few cases is it possible to draw conclusions about the identity of an unknown based on the UV spectrum alone without reference to a known standard. The quality of library matches will depend greatly on the conditions under which the library spectra were saved. It is important to use the same HPLC conditions when constructing a library as are to be used in the analysis of unknowns, particularly if the analytes are sensitive to minor solvent effects. Care must be taken to insure that the detector and other components of the system are in good working order to obtain the best library search results. If in doubt about the quality of the spectra contained in one of your libraries, it is probably best to reinject known reference standards and obtain new spectra.

NOTE: For a detailed discussion on how library searches are conducted in

PolyView2000 and how to interpret the results of a library search, see

Library Searches on page 105. for an example of the PuP statistics

report.

In the following Tutorials, the primary library functions will be covered. Initially, you must “Create” a library disk file for storage of spectra. A Tutorial will be presented on Building and Editing that covers the techniques available to place spectra in a library and to add information to each library record that is helpful in characterizing it. PolyView2000 provides extensive editing capabilities for management of your disk libraries. Only the primary capability of locating a particular library record and editing its information section will be covered in this Tutorial. In the last Tutorial, the spectral search capabilities will be covered. It is important to keep in mind that the results actually obtained in library searches depend heavily on the nature of the analytes and the analytical conditions. It is important for each user to develop criteria based on their particular application for the interpretation of library search results.

The Tutorials are based on hypothetical standard and sample chromatograms. The Standard (PV_STD01.RUN) contains several peaks for which spectra have been archived in the spectral library provided with PolyView2000 (PV_STD01.LBR). In the Tutorial, you will be asked to create your own library and to store spectra in it from PV_STD01.RUN. Your library should be nearly identical to the one provided (PV_STD01.LBR). Next,

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you will be asked to determine if the peaks in the Hypothetical sample (PV_SAM01.RUN) match the peaks in the standard. Both the library you build and the one provided will be searched.

PolyView2000 can have different types of libraries. Because of the way the spectra are stored in a library, spectra collected with different wavelength ranges and different bandwidths MUST be stored in different libraries. (Data collected at different data rates can be stored in the same library because data rates do not effect spectral characteristics.) For example, data collected with a wavelength range of 200 to 400 nm could not be stored in a library with data collected over a wavelength range 200 to 500 nm. Also, data collected from 2 different diode arrays, e.g. the 9065 and the 330, cannot be stored in the same library.

Even though data with different acquisition parameters must be kept separate in different libraries, any library can be used to with any spectrum. The wavelength ranged searched with the wavelength range of overlap between the particular library and the target spectrum. For example, if the library has a wavelength range from 250 to 400 nm and the spectrum was taken from 300 to 450 nm, the target spectrum will be reduced to a spectrum from 300 to 400 nm and the library search will occur.

PolyView2000

Therefore, it is desirable for most standards which will be used in a library to take data with the same wavelength and bandwidth.

Tutorial: Creating a Library

From the Star Tool Bar, double-click on the PolyView2000 icon. PolyView2000 will open with the three primary windows displayed.

• Click on the Library Manager title bar to make it the active

window and select Create Library under the Library menu.

• In the file selection dialog box that is displayed, select the

EXAMPLES directory(double-click on it) and then edit the File name field to assign a name for the library you are about to create. Name the library MYLIB.LBR and select OK. If the file already exists, you need to delete it first. This can be done by pressing the Cancel button, then executing Library: Open to select MYLIB.LBR, then Library: Delete to delete it.

After the name for the new library is selected, the required disk file is created and the library becomes the Current Library in the Library Manager window. A dialog box is displayed that allows you to add information to the library header. In the dialog box, add whatever information you like to the three fields and select OK.

The Edit Library Information Dialog

Don't close PolyView2000. Instead, go on to the next Tutorial.

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NOTE: It is important to distinguish how the Library Manger addresses different

libraries. All libraries are maintained as disk files just as any other DOS file in the Star Workstation. The Library Manager maintains a list of those .LBR files to address during searches, called the Search List. In addition, one library can be designated as the “Current Library” for the Library Manager window. The Current Library is the one that is currently open for adding, editing, and deleting spectra, and is listed in the title bar of the Library Manager window. Libraries are added to the search list using the Add to List command in the File Selection dialog box. Also, when a new library is created, it becomes the current library and is added to the search list. A shortcut to making one of the libraries in the search list into the Current Library is to double-click on its name in the search list.

PolyView2000

Tutorial: Building and Editing a Library

Be sure to complete the previous Tutorial on creating a library. The screen should now display the three primary PolyView2000 windows and MYLIB.LBR should be the only library in the Search List in the Library Manager window.

To place a spectrum into a library, you must first extract it from the .RUN file and transfer it to the Spectra Manager window.

• Click on the title bar of the Plot window to make it the active

window.

• Plot out the absorbance chromatogram of PV_STD01.RUN

at 253.6 nm (refer to Plotting a Chromatogram on page 2.

• Transfer a spectrum from the apex of each peak to the

Spectra Manager window Examining Spectra from a

Chromatogram on page 35.

Screen Configuration for Building a Library File

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• Click on the title bar of the Spectra Manager window to make

it the active window.

• Select Edit Spectra in the Spectrum menu.

A dialog box is displayed, that allows you to view and edit a variety of information that is attached to each spectrum in the Spectra Manager. This information, along with the spectrum itself constitutes the “Spectrum Record” that can be transferred between the various PolyView2000 windows and becomes a permanent part of the disk file when it is stored in the Library. The number in the upper left corner of the dialog box indicates which of the seven spectrum registers you are currently viewing and editing.

PolyView2000

The Edit Spectra Dialog Box

• Initially, change the Valid wavelength range to 200-366.6 nm

(select Edit in the Valid Wavelength range box and set the new range in the small dialog box that appears).

• Add or alter any information you would like in the five edit

fields (Name, Note, Operator, Method and Instrument). Name the peak Compound #1.

• Once this has been completed, you can obtain a printed

report of the spectrum and its associated information. Select the Print pushbutton and wait for the report to be printed.

Select Next to access the next spectrum in the Spectra Manager window. Note that you must verify the edited changes. Select Yes. Continue editing the spectrum information sections for each of the six spectra in the Spectra Manager window. At this point in the Tutorial, all of the spectra and their associated information sections are temporarily stored in the Spectra Manager window. They are not yet part of the library.

After the last spectrum in the Spectra Manager window has been edited, select Quit. You are now ready to begin adding these spectra to the library (MYLIB.LBR).

• In the Spectra Manager window, select Single Plots under

the Display menu. The spectrum in register position #1 is displayed.

• Next, select To Library Manager under the Spectrum menu.

The displayed spectrum in the Spectra Manager window is transferred to the single spectrum register in the Library Manager window (i.e., it becomes the Current Spectrum in the Library Manager). Add it to the Library by selecting Add Spectrum to Library in the Library menu.

NOTE: In PolyView2000, multiple spectra can be transferred from the Spectra

Manager window to the Library Manager window and added to the current library in a single step.

• To add the other spectra in the Spectra Manager window to

the library, go back to the Spectra Manager window and select Multiple Plots under the display menu. Then, click on the boxes under the spectra to de-select spectrum # 1 and select spectra #2 through #6.

• Next, select To Library Manager under the Spectrum menu.

You will be told that since multiple spectra are selected, you cannot change the Library Manager’s current spectrum, and

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asked if you want to add the 5 selected spectra to the current library. Answer Yes.

NOTE: After the transfer of multiple spectra, a small message window informs

you of the success of the operation and disappears after a few seconds. You can close this message window earlier by pressing the Enter key or clicking on the window.

At this point, the library should contain spectra for all the peaks in PV_STD01.RUN. In the Library Manager window, select Select/Edit Library Record under the Edit menu. Notice that all of the spectra added to the library are listed.

A library can be searched for key words (in the Name and Notepad fields of each record) as a rapid means of locating a desired record. Try that now:

• Select Change Parameters.

• Type a key word in the Search Text field that can be found in

the Name or Notepad fields of only one of the records.

PolyView2000

• Check the Search in Name and Search in Notepad boxes

and select Do Search.

Notice that the list of records is updated to include only those with the specific key word. The other records are still in the library, but are not displayed in the list.

Individual records in the library can be edited or deleted.

• First, list the entire library by doing a key word search with

only a space (use the space bar) in the Search Text field. Then, select one of the records from the list in the List Library Records dialog box by pointing and clicking with the mouse.

• Select Edit Record. A dialog box appears that is very similar

to the Edit Spectra dialog box in the Spectra Manager window. This is your interface to the actual data stored on disk in the library. You can alter the information stored for that particular spectrum or delete the record from the library altogether. For more information on the functions available in this dialog box, press the “Help” button.

MYLIB.LBR after addition of spectra

The Edit Library Records Dialog Box

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• Select Quit to close the dialog box.

Don't close PolyView2000. Instead, go on to the next Tutorial to learn how to conduct spectral library searches.

NOTE: In the above description on building a library, the information associated

with each spectrum (Name, Operator, etc.) was added to the spectrum in the Spectra Manager window prior to its transfer to the Library Manager. Alternately, this information could have been added after the spectrum was transferred to the Library Manager window. The spectrum information fields are accessible after the spectrum becomes a part of the disk library and this information could have been edited after all spectra were in the library (see the above Tutorial). Also, the information is accessible when the spectrum is the Current Spectrum in the Library Manager window (i.e., after it is transferred from the Spectra Manager window but before it is added to the library). To access the Current Spectrum in the Library Manager, select Edit Spectrum under the Spectrum menu. When the desired information has been added, the spectrum can be added to the library directly. In building libraries, use whatever method seems the most straightforward for editing the information associated with the spectra and adding them to the library.

Tutorial: Conducting a Spectral Library Search

The first step in conducting a library search is to extract a spectrum for the peak of interest from the .RUN file.

• In the Plot window, select Open File under the File menu.

• In the file select dialog box, locate PV_SAM01.RUN and

double-click on it. The file will be plotted in the window and for this Tutorial, the default plot conditions can be used.

• In the Spectrum Manager, select Delete All Spectra in the

Spectrum menu, then set the Display mode to Single Plot.

• The target spectrum for all library searches is the Current

spectrum in the Library Manager window. The spectrum is transferred to this window in the same manner as was used in the previous Tutorial (i.e., by transfer through the Spectra Manager window). Using the cursor, extract the apex spectrum from the peak at 5.0 minutes and transfer it to the Spectra Manager window. Activate the Spectra Manager

PolyView2000

window and select To Library Manager under the Spectrum menu. You will not be asked if you want to add the spectrum to the current library because the Spectrum Manager was in the 'Single Plot' Display mode.

In the Library Manager, a search list of disk libraries must be built. The library you created in the previous Tutorial should already be listed in the search list. Let's add the library provided with PolyView2000.

• Click on the Library Manager title bar to make it the active

window and select Open Library under the Library menu.

• Select PV_STD01.LBR from the list and activate the Add to

List pushbutton. Click on the Cancel pushbutton to close the dialog box. Note that it becomes the current library and is added to the Search List.

Screen Configuration for Library Search

To send a single spectrum to the Library Manager without being asked about adding it to the library, set the Spectra Manager to the 'Single Plot' mode.

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NOTE: Management of the contents of the Search List is accomplished with the

controls under the Search menu. For more information on the use of these controls, refer to On-Line Help (Help:Commands:Search). Also, any of the libraries in the list can be designated the Current library by double-clicking on its name in the list. That library is then opened for editing. Note that the Current library is always listed in the title bar of the Library Manager window.

Now that the target of the search is the Current Spectrum in the Library Manager, and the search list of libraries is built, you can conduct the search.

Select Search for Spectrum under the Search menu.

The Library Search dialog box is displayed that is used to set the search parameters and display the results. Refer to Library

Searches on page 105 for more information on the way in which

library searches are conducted and search parameters are set.

The Library Search Dialog Box

PolyView2000

For now, simply change the wavelength range to 200-366.6 nm and leave all the other controls at their default settings. Select the Search pushbutton. The search is conducted and, after a short period of time, the results are displayed on the screen.

Results of a Library Search

Following the search, a wide variety of additional functions are available. Using the controls in the list box at the bottom of the screen, view the entire library search text report. This report lists the best 5 library matches (sometimes fewer if there were not 5 library spectra that met the retention time and Purity Parameter criteria.) and the search conditions, followed by the information pertaining to the target and match spectra. This report can be printed (Print List) and an example is shown in Appendix A. Other controls in the dialog box enable you to access all of the matches individually (Next and Prev) and obtain a detailed report for each (see Appendix A as an example).

NOTE: The library search results are ranked in order of decreasing similarity.

For a discussion of the indices used to quantify the match between two spectra, see Library Searches on page 105, or refer to On-Line Help.

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Reporting Data

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EPORTING DATA

By default, the result of the search (if successful) is to display the target spectrum overlay with the best match. Since these two spectra are often very close, their difference is displayed as a spectrum in the lower portion of the spectrum plot. To not display this difference, click on the check box titled Difference to uncheck it. This provides you with a larger size spectral overlay.

When you are finished using this particular target spectrum, close the search dialog box by double-clicking on its Control Menu box. To search any of the other peaks in the chromatogram, simply transfer a spectrum from the peak to the Library Manager (through the Spectra Manager window) and select Search for Spectrum. Try that now. Determine the name of the compound eluting at 8 minutes in PV_SAM01.RUN by searching the two libraries.

Close PolyView2000 by double-clicking on the Control Menu box in the main application window.

PolyView2000

PolyView2000 has two modes of operation: the interactive mode where the user is actually sitting at the Star Workstation,

and the non-interactive mode, where functions and reports are generated automatically using a set of pre-defined conditions contained in a PolyView2000 reports method. Most of the windows and dialog boxes in PolyView2000 permit you to document your work through an interactive printed report (select either the Print command under the File menu or the Print pushbutton in dialog boxes). Examples of these reports are presented in Appendix A. Most of the functions (and reports) that can be performed interactively can also be conducted in the non-interactive mode. In Automating PolyView2000 Operations on page 90 you will learn how to automate these data processing and reporting functions. In PolyView2000, your primary control over these non-interactive reports is through the Reports window. Non-interactive reports are specified using the Method Builder application (see the LC Star Workstation Manual) which can be accessed directly from the Reports window in PolyView2000. When the Method Builder application is accessed in this manner, control is passed directly back to PolyView2000 when it is closed. Once a report method is specified, the report can be generated and stored as a disk file. Then, you can view

or print the report at some later time. The following Tutorial is broken into two parts. The first part covers the viewing and printing of an existing report file on the disk. The second part covers the specification and generation of a report file.

NOTE: In the following Tutorial, some of the files in the EXAMPLES directory will

Tutorial: Making a Report

The first step in making a report is to construct a report method in the Method Builder application.

Activate the Reports window (if not already active).

Drop down the Reports menu and select Edit Method.

The Method Selection dialog box allows you to create a new method or edit an existing method. Let's create a new method.

The Method Selection Dialog Box

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AKING A REPORT

Select the New Method pushbutton and accept the default bus address of 4, the address used in collecting the Example data files. Control is transferred to the Method Builder application and the window used to specify reports is displayed.

The PolyView2000 Reports Method Window

NOTE: For more details on the operation of the Method Builder application, refer

to the Data Acquisition with LC Control Operation Manual.

PolyView2000

The Report Method dialog box is used to manage the list of report types called for in the .MTH file. First, let's call for the generation of a Single Plot report.

Select Add and a list of the reports that can be generated is shown.

Reports that can be generated automatically

Select the Single plot report and press OK. Note that a dialog box is displayed that allows you to specify the parameters for the report.

Single Plot Report Parameters

Leave the Plot type at Chromatogram (the default), set the wavelength range to 220-311 nm, and leave the Time and Scale ranges at their default settings.

These parameters will generate a report of the entire data file using the average absorbance from 220 to 311 nm with the absorbance range autoscaled.

Click on Done at the bottom of the window. Notice that the Single Plot is included as the first report specified.

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AKING A REPORT

A Single Plot Report was added to the Report List

Now, let's include a library search in the report.

Click on Add and select Library Search.

Note that the Select Library File dialog box is displayed. You now specify the libraries that you want to use for a library search. Once you have specified the library, you can specify the parameters to use in conducting an automated library search. Let's duplicate the search that was conducted in the Tutorial, Conducting a Spectral Library Search.

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Leave the Report Type set to Short Graphics.

Set the wavelength range to 190-366.6` nm and leave all of the other fields at their default settings.

Now, build a list of libraries to search.

Select Add Library and the Library Select dialog box is displayed.

Select MYLIB.LBR. Select Open and the library will be added to the search list. Repeat this for the PV_STD01.LBR file.

Back in the Report Method window, select Add to append the Library Search report to the list of reports.

The Library Search Report, showing

the list of libraries to be searched

NOTE: Once a series of reports have been entered into the list, the controls in the

Report Method window are then used to manage the list. You can edit or delete selected reports and rearrange the order that the reports will be generated (and appear in the disk file). For details on the operation of these list management controls, see On-Line Help (search for Report Method).

Now that the types of reports have been specified, you need to assign the .MTH file a name and save it to the disk.

Drop down the File menu and select the Save As command.

In the dialog box, assign it the name MYMETH. You do not need to add the .MTH extension. It is automatically added to the method name.

Close the Method Builder application by clicking on the Control Menu box in the main application window.

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When the Method Builder application is exited, control is returned to PolyView2000, just where you left off (in the Reports Window). You can now generate a report using the .MTH file you just created. To generate the report, you must specify the .RUN file that is the source of the raw data, the .MTH file that will be used to process the data, and the .SRP file that becomes the disk file containing the report.

Drop down the Reports menu and select Create Report.

Report Creation Dialog Box

PolyView2000

Press Select Data File and choose PV_SAM01.RUN as the raw data file from which to construct the report.

Press Select Methods File and choose MYMETH.MTH as the method to construct the report.

Press Select Reports File, type MYREPORT into the File name field and select OK..

Press OK to start the report generation. A dialog box is displayed that informs you of the progress. After the report is completed, it is automatically displayed in the Reports window. It can now be accessed or printed just as any other existing report file.

MYREPORT.SRP displayed in Reports Window

Close PolyView2000 by clicking on the Control Menu box in the main application window.

NOTE: The above Tutorial covered the building of a new report generation

method. PolyView2000 also allows you to edit an existing report method by selecting Edit Method under the Report menu and specifying an existing .MTH file in the File Select dialog box. You can then add new report types to the file or edit the ones already in the .MTH file.

You may also extract the report method directly from an existing .SRP file even if the original .MTH file used to generate it doesn't exist on the disk. To do this, select Edit Current Method under the Report menu. The dialog box that appears allows you to generate a .MTH file with the same root as the .SRP file. You may choose another name if desired.

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IEWING AND PRINTING OF AN EXISTING REPORT

Tutorial: Viewing and Printing of an Existing Report

If you have not already executed the previous Tutorial, do so to create a report file.

If PolyView2000 is not already running, start PolyView2000 now.

• Select New Reports Window under the Windows menu.

To view a report file, select Open Report under the Reports menu and select MYREPORT.SRP from the EXAMPLES directory. Notice that as you select the disk file, the pages in the report are listed. This enables you to confirm the contents of the report before it is called into the window. Select OK to confirm your selection.

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Report window displaying an .SRP file

The first page of the specified report is displayed in the window. Initially, it is displayed in color, and the full page is reduced in size to fit within the window. Access the next page of the report by selecting Next Page under the Page menu. The commands under this menu can be used to access or delete sequentially the individual pages in the report. The Next Page and Previous Page commands can also be given through the Page Up and Page Down keys in the numeric keypad of your keyboard. The report can be viewed in monochrome by selecting that mode under the Page menu.

Library Search Report Zoomed in

Next, drop down the Report menu and select View Page List. This allows you to view the method that was used to generate the report and the resulting pages. Any of the individual pages can be accessed by selecting them from the page list and

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selecting the View Page pushbutton. Select a page of the Library Search Report.

Particular details in the report can be viewed by moving the cursor to the desired region of the report by a click and drag operation of the mouse. Zoom in on the region of the report dealing with a library match. Notice that the remainder of the report can still be accessed using the scrolling controls at the bottom and right of the screen. Clicking the left mouse button anywhere within the report un-zooms the display.

Drop down the File menu and select Print Page. The currently displayed page is printed on the Star Workstation printer. If desired, all the pages in the report can be printed using the Print File command in this menu.

Determining Peak Purity

A primary feature of a Diode Array Detector such as 330 is the ability to provide information on the purity of chromatographic peaks. These peak purity determinations are made by assessing the degree of difference in the UV spectra across the width of a peak. There are a number of mathematical parameters that can be used to compare spectra. The Purity Parameter used in PolyView2000 is perhaps the most sensitive to minor co-eluting impurities (contaminants) within a peak of interest (the analyte).

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PolyView2000

Purity Parameter

For a given wavelength range, the Purity Parameter is the average wavelength weighted by the square of the absorbance.

∑

PuP

(@λλ )

∑

Absorbance A wavelength

Consequently, by examining the Purity Parameter values across a peak, you can quickly determine the presence of contaminants. Rather than examining a table of Purity Parameter values, PolyView2000 provides the ability to plot the Purity Parameter as a function of time. If the peak is free of co-eluting compounds, the Purity Parameter will not change over the course of the peak and it will appear with a flat top. However, if impurities are present, the top of the peak will slope, indicating that the Purity

Parameter is not consistent across the width of the peak. In addition to the Purity Parameter, other qualitative parameters associated with UV spectra can be plotted and provide information on peak purity, including the wavelength of maximum absorbance and the ratio of absorbances at two different wavelengths. Like the Purity Parameter plot, a flat topped peak indicates that the parameter is not changing with time and provides a measure of peak homogeneity.

The accuracy of all the measures of peak purity are dependent on a number of factors. There must be some degree of separation between the analyte and contaminant to create a difference in the UV spectra across the width of the peak. If the two (or more) compounds exactly co-elute, then the UV spectra will not change across the peak and it will appear to be pure. Care must be taken to maximize the efficiency of your HPLC system. A Diode Array Detector cannot

make up for low efficiency columns and/or inadequate separation techniques. In addition, the degree of difference in the UV spectra between the analyte and the contaminant will have a profound effect on the peak purity determination. If spectral differences are small, it will be very difficult to determine impurities. However, the Purity Parameter is very sensitive to even slight spectral differences and this will not often be a problem for PolyView2000. A final consideration in peak purity determinations is the wavelength range used in specifying the plots. It is possible for the spectra of an analyte and a contaminant to be very similar over a certain portion of the UV spectrum and different over another portion. It is important to carefully consider the wavelength range used to assess peak homogeneity. To assist you in selecting the proper wavelength range for plotting the Purity Parameter, you can use the optimization routine in the Spectra Manager under the Spectrum menu.

PolyView2000 provides several ways to construct Purity Parameter plots. They can be plotted interactively in the plot window or generated automatically in a non-interactive report. In addition, the non-interactive Peak Purity Survey report presents the same information in a slightly different format. In this report, the Purity Parameters of all spectra within a peak are compared with the Purity Parameter of the apex spectrum and, if different, the peak is marked. With experience, you will find the technique that best suits your application.

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In the previous tutorials, you have already learned the techniques that are required to conduct these Peak Purity determinations. The basic technique for plotting a .RUN file was covered in Plotting a Chromatogram on page 25 and preparing non-interactive reports was covered in Reporting Data on page

61. Consequently, the details of these operations will not be covered here. Instead you will be asked to refer to these previous tutorials. The data file used in this tutorial, PV_SAM01.RUN, has several obvious as you work through this tutorial.

Note that Multicomponent Analysis can be also used to assess the purity of a peak, as detailed in Purity Evaluation by MCA on page 100.

Tutorial: Purity Parameter Plots

The first step is to configure your screen to compare two plots.

• At the Workstation Star Tool Bar, open PolyView2000 by

double-clicking on the PolyView2000 icon.

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PolyView2000

• Configure PolyView2000 with only the Plot window active,

hiding both the Spectra Manager and Library Manager windows.

• In the Plot window, plot out PV_SAM01.RUN at 254 nm.

• Drop down the Plot menu and select Duplicate Plot Window.

Wait for the plot to appear and then select Arrange Visible under the Window menu.

Your screen should display two identical plots of the file. This is the most convenient screen configuration for determining peak purity because you can view the absorbance chromatogram in one window and the purity information in another.

Duplicated Plot Window

You must now specify the Purity Parameter plot.

• With the focus on the bottom window, drop the Plot menu

and select Plot Parameters.

• Specify a Purity Parameter plot with a wavelength range of

200-366 nm. Accept the defaults for all of the other parameters.

The Purity Parameter is now plotted as a function of time between the upslope and downslope inflection points of each peak. Notice that the peak at 3.5 minutes has a sloping top, indicating that the Purity Parameter is changing across the width of the peak.

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Purity Parameter Plot indicates impure peak

at 3.5 minutes

You can easily verify the spectral dissimilarities in the impure peak by transferring the upslope and downslope spectra to the Spectra Manager window. In either Plot window, extract spectra corresponding to the upslope and downslope of the peak at 3.5 minutes. Activate the Spectra Manager window and observe the differences in the two spectra.

NOTE: The peak at 3.9 minutes in PV_SAM01.RUN also has a significant

impurity that can be clearly detected by plotting the Purity Parameter over the range of 224-278 nm.

Don't close PolyView2000. Instead, go on to the next tutorial.

PolyView2000

Tutorial: Peak Purity Survey Report

The Peak Purity Survey report is a non-interactive report and therefore must be specified in the Method Builder application.

• Call up the Reports window and create a new report method

(see Tutorial: Making a Report on page 62).

• In the Method Builder application, select Add and set the

type of report to Peak Purity Survey and Say OK.

In the dialog box, you can specify the conditions for report generation. For an example of a peak purity survey report, see

Appendix B.

• Set the PuP Interval field to 2 nm.

• Set the wavelength range to 200-367 nm and leave all other

parameters at their default conditions.

• Select Done and assign the report method the name of

PURITY.MTH. (Use the Save As command under the File menu.)

• Close the Method Builder application by clicking on the

Control menu in the main application window.

Now, let's use the report method to determine peak purity on a file. Back in the PolyView2000 Reports window, create a report of the data file PV_SAM01.RUN using the PURITY.MTH method. Call the report file PURITY.SRP (see Tutorial: Making a Report on page 62). The report displays the absorbance chromatogram (at the wavelength used for peak sensing) with impure peaks shaded. (Note that the peak at 3.5 minutes is indicated as being impure.

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Peak Purity Survey Report showing

impure peak at 3.5 minutes

Close PolyView2000 by double-clicking on the Control Menu box in the main application window.

Performing Peak Sensing

Several functions within PolyView2000 rely on an accurate determination of peaks in a chromatogram. To provide corrected spectra that accurately reflect eluting peaks, the baseline profile must be determined with precision. Also, many of PolyView2000's automated functions require the determination of peak placement within a chromatogram. It is the job of the peak sensing algorithm to accurately determine where a baseline segment starts and stops and where eluting peaks are located.

PolyView2000

There are a number of user-selectable parameters that must be set to insure accurate peak sensing. As with most data systems, including the Interactive Graphics application in the Star Workstation, you must set the peak width and S/N value (the threshold parameter) according to the peak width and size you expect. In addition, since you are dealing with multi-channel data, in PolyView2000 the wavelength used for peak sensing must be specified. A single wavelength or a wavelength range can be specified. All of the peak sensing parameters can be time programmed to accommodate changing conditions over the course of a chromatographic run.

Like the reports method, a separate portion of the Star Workstation .MTH file is set aside for the PolyView2000 peak sensing method. This enables peak sensing to be performed automatically under the control of the System Control/Automation application. Along with this, the Method Builder application is used to specify and manage the peak sensing method. Within PolyView2000, a separate window is provided in which to manage the peak sensing functions. In this Peak Sense window you can view a chromatogram at the peak sensing wavelength, view and alter the detector noise monitor data, access the Method Builder application, and conduct peak sensing on a file.

The following tutorials cover the basic operations of constructing a peak sensing method and performing peak sensing on a data file. In the last tutorial, the procedure for defining a new noise monitor will be covered.

NOTE: For more detailed information on peak sensing in PolyView2000,

including procedures to follow for troubleshooting peak sensing, refer to

Peak Sensing on page 109.

In the following Tutorial, some of the files in the EXAMPLES directory will be modified and/or created. Prior to performing this tutorial, you should ensure that you are working with original copies of the data files called for. If you are not sure whether these files have been modified by previous use of the Tutorial, reinstall the PolyView2000 software from the CD using your serial number.

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UILDING A PEAK SENSING METHOD

Tutorial: Building a Peak Sensing Method

From the Star Workstation Star Tool Bar, double-click on the PolyView2000 icon to activate the application.

Drop down the Windows menu, activate a New Peak Sense Window and maximize it.

You are now ready to build a new peak sensing method.

• Under the Peak Sensing menu, select Edit Method.

• To define a new .MTH file, select New Method in the dialog

box. Accept the default bus address (all methods sections are attached to an address, used under automated operation). Control will be transferred over to the Method Builder application and the Peaksense Time Program dialog box will appear. In this window, the default peak sensing time program is in the list box at the bottom of the window. The operation of the edit controls in this window is similar to other windows within the Method Builder application. Modify the Peak Sensing Method as follows.

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The Peak Sense Time Program Window

• Click on the first line 0.00 Peaksense..., and alter the

wavelength range fields to 273-273 nm. Note that, unlike previous versions of the Method Builder, the numbers change immediately in the Method listing and that there is no Change button. However, the parameters do not actually change in the method until you save the method.

• Click on the second line 0.00 S/N Threshold... and change

the S/N value to 6.

• Click on the third line 0.00 Inhibit Integrate... and change the

end time to 2.00 minutes. This ensures rejection of the region containing the void peak.

• Then, click on Add twice. This creates two new lines in the

method.

• Click on the second of the new lines and change the event to

Peaksense, the time to 16.0, and the wavelength range to 215-215.

• Click on the first of the new lines and change the event to

S/N Threshold, the time to 19.5 and the S/N value to 10. Your screen should look like the following.

The New Peak Sensing Time Program

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Next, assign a name to the method and save it to the disk.

• Drop down the File menu and select Save As.

• Save your method as PSENSE.MTH in the EXAMPLES

directory.

• Close the Method Builder application and note that control is

passed back to PolyView2000 right where you left off.

Don't close PolyView2000. Go on to the next tutorial to learn how to use the method you built to perform peak sensing.

NOTE: The above tutorial covered the building of a NEW peak sensing method.

PolyView2000 also allows you to edit an existing peak sensing method by selecting Edit Method under the Peak Sensing menu and specifying an existing .MTH file in the File Select dialog box. Control is transferred to the Method Builder application with the specified .MTH file already loaded. It can then be edited and saved for use later.

A similar feature allows you to extract the peak sensing method directly from an existing .RUN file even if the original .MTH file used to generate it doesn't exist on the disk. To do this, select Edit Current Method under the Peak Sensing menu. The dialog box that appears allows you to generate a .MTH file with the same root as the .RUN file. Simply select OK in the dialog box that appears and control will be transferred to the Method Builder application with the .MTH file just created loaded into the window. If a file by that name already exists on the disk in the current directory, you will be given the opportunity to verify your selection to prevent inadvertently overwriting it.

Tutorial: Conducting Peak Sensing

To begin this tutorial, your screen should be configured as in the last tutorial, with a single Peak Sense window. The task will be to perform peak sensing on BASECORR.RUN using the method you built in the last tutorial. Drop down the File menu and select Open File. Select BASECORR.RUN in the EXAMPLES directory and then OK. The file is plotted in the window.

PolyView2000

BASECORR.RUN in Peak Sense window

NOTE: The peak events are displayed in both the Peak Sense window and the

Plot window (optional). These events are color coded as follows: WhiteApex, Red-Peak Start, Yellow-Peak End, Purple-Inflection points, Dark Blue-Valley points, and Lt. Blue-Shoulder. The placement of peak events in a chromatogram indicates how well the peak sensing method was tailored to the actual chromatogram. For more information on how PolyView2000 conducts peak sensing and how to assess peak sensing results, see Peak Sensing on page 109.

Now, let's perform the peak sensing operation. Select Do Peak Sensing under the Peak Sensing menu. In the dialog box, select the file you created, PSENSE.MTH, in the EXAMPLES directory. Notice that the peak sensing method is listed on the screen. Select OK. Peak sensing is automatically performed.

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Often, the peak sensing method you specify cannot be executed exactly as it is called for in the .MTH file. For instance, timeprogrammed changes in peak sense parameters (wavelength, peak width, etc.) cannot be made within a peak. When this occurs, a dialog box appears that informs you of this situation. This is the case for the BASECORR.RUN file and the View Peak Sense Log dialog box is displayed on the screen. The Peak Sense Log shows the original method you specified, the resulting method (as executed), and the reasons why it was modified. After viewing the information, select Quit.

PolyView2000

Peak Sense Log showing changes to method

Following the peak sensing function, the display is updated to reflect the new peak sense results.

New Peak Sense Results

The chromatogram display in the Peak Sense window is very similar to the display in the Plot window, but it only displays the time-programmed absorbance specified in the Peak Sensing Time Program. Flags at the top part of the screen indicate where time-programmed peak sense events were executed. By default, the whole file is displayed and automatically scaled. You can use the mouse and the View menu to modify the time and amplitude settings.

Close PolyView2000 by double-clicking on the Control Menu box in the main application window.

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ROUBLESHOOTING PEAK SENSING

Tutorial: Troubleshooting Peak Sensing

Attached to all .RUN files is noise data transmitted to the Star Workstation from 330 when it is in the MONITOR state, just prior to the start of the run. During the peak sensing operation, PolyView2000 determines the level of chromatographic noise from this monitor data and differentiates peaks from noise using the S/N ratio you set in the peak sensing time program. Occasionally, the run monitor associated with the .RUN file may not be accurate, possibly due to the presence of a late eluting peak from a previous run or a gradient program that had not yet stabilized prior to the run monitor period. PolyView2000 provides the ability to redefine the data used as the noise monitor in these situations and this function will be illustrated here. Configure PolyView2000 with a single Peak Sense window and call up BADMON.RUN from the disk.

NOTE: See Peak Sensing on page 109 for more information on how PolyView2000

conducts peak sensing and how to set the peak sensing parameters.

PolyView2000

BADMON.RUN showing no detected peaks

If the file is in the original state, you will notice that the peak sensing function did not detect any peaks and the noise value at the upper left corner of the screen is very high. If this is not the case, this tutorial has already been executed on the file; restore the file to its original state by executing the Remove User Monitor command in the Peak Sensing menu.

You can examine the run monitor:

• Drop down the File menu and select View Run Monitor. You

will see that several high absorbance spikes occurred during the monitor.

The Run Monitor dialog box

• Using the wavelength control, you can view the run monitor

data at any of the diodes which were used to collect the data. Select Quit to close the dialog box.

If a file has a noise monitor that is corrupted or inappropriate, like BADMON.RUN, you can select a portion of the baseline in the chromatogram to be used for the noise calculation. This is called the User Monitor and after it is specified, peak sensing is automatically repeated.

• Drop down the Peak Sensing menu and select Define User

Monitor.

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The controls in the dialog box that appears are used to select a portion of the file as the noise monitor.

PolyView2000

The Define User Monitor dialog box

• Select a portion of the baseline between 0 and 1 minute

using the click and drag method. When you do this, the plot will zoom and a message will be displayed in the Number of Points field. If the piece of baseline is too large, Too many points will be displayed and you will need to select a smaller portion of the baseline until the proper number of points is selected. Use Select from Left Edge. Try for a number between 26 and 74. When assigning the User Monitor, be sure to use a flat portion of the baseline, free from eluting peaks. To autoscale the plot after selecting a new region, double-click left of the Absorbance axis, where the cursor becomes a double-headed vertical arrow.

Section of Baseline chosen as User Monitor

• After the number of points is refined, select OK and peak

sensing will automatically be performed using this new monitor.

When the plot is displayed, you will note that normal peak detection has occurred (see the following figure). Also, the new noise value is displayed in the upper left corner of the screen. The new monitor data can be viewed in the same manner as before. This time, select View User Monitor under the File menu. The dialog box that is displayed is very similar to the View Run Monitor dialog box.

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PolyView2000

BADMON.RUN with proper Peak Sensing

after definition of a User Monitor

Close PolyView2000 by double-clicking on the Control Menu box in the main application window.

Automating PolyView2000 Operations

One of the primary features of the PolyView2000 Spectral Processing Application is the ability to conduct automated analyses of the spectral data from the 330 or 9065 diode array detectors. Instead of having to process the data manually after it is collected, the Workstation has the ability to perform many of these functions automatically, immediately after data collection. The PolyView2000 functions that can be performed in this manner include peak sensing to locate the peaks in a chromatographic run and any of the non-interactive reports. The functions in the non-interactive reports allow you to perform sophisticated analyses of diode array spectral data in an automated fashion.

You can automatically add PolyView2000 functions to the processing of a diode array data file at the end of each chromatographic run by adding a PolyView2000 method associated with the detector (by means of the bus address) will be executed. During an automated sequence of injections, the PolyView2000 reports that are generated are written to a permanent disk file with a .SRP extension. These reports will also be printed automatically if the Automated Printing is enabled in System Control (at the bottom of the Automation menu). If you don't wish these reports to be printed automatically, simply disable the autoprint function. The .SRP files will still be generated and will be available for viewing in the Reports window. In addition to the generation of PolyView2000 report files during live runs, these .SRP files can be generated during a Recalculation sequence in System Control.

Using On-Line Help

There are three ways to access On-Line Help in PolyView2000: the F1 key, the Help menu, and the Help push button which is present in most dialog boxes.

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Tutorial: Using On-Line Help

Pull down the Help menu. Select Using Help to display the information file provided with Windows. Learn the basics, then close the Help window. Also try Index, Commands, Keyboard, Procedures.

Select the MCA Manager window, then press the F1 key. The Help window appears, displaying information relevant to the MCA Manager. Maximize the Help window. Note that the abbreviation MCA is underlined with a dotted line. This indicates a defined term. Move the cursor over the word MCA. Note that the cursor turns to an index. Click the mouse, and the definition is displayed as a pop-up definition. Click again, and the pop-up disappears. Now move down to terms with a solid underline. These represent Hypertext jumps, which means that selecting such an item by a single click will send you to another topic. Try Computing Area Spectra. You can come back to the previous topic using the Back pushbutton above the help text.

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SING ON-LINE HELP

PolyView2000

You can also print any topic by using the Print Topic command in the File menu of the Help window.

Display the Preferences dialog box by pulling down the File menu and selecting Preferences. Press the Help pushbutton. Maximize the Help window and scroll through the information. There is such a Help pushbutton in most dialog boxes in PolyView2000.

Try a keyword search. Press the Index button in the Help window, and start typing a keyword, for example Purity. Note that as you enter more characters, a list of keywords is scrolled to the point where it best matches what you requested.

The Keyword Search Dialog Box

Now select one of the keywords and press the Display button inside the dialog box (or press the Enter key).

Quantitative Analysis by MCA

Multicomponent Analysis is a proven technique, routinely used in spectrophotometry to determine the composition of a mix of known components in unknown amounts. In PolyView2000, an original adaptation of the technique provides easy quantitative analysis of groups of up to six overlapping peaks, regardless of the chromatographic resolution (J. Chrom, 631 (1993) 15-21).

This requires that the components have different enough spectra. If this is not the case, the report will let you know. The one-page MCA report provides amounts, peak profiles, and an evaluation of the quality of the decomposition. MCA can be invoked interactively or under automation.

In PolyView2000, you can also define a peak as an internal standard to refine the quantitation results. This process is documented in On-Line Help and is only available in the noninteractive MCA report. You can obtain such reports interactively by using the PolyView2000 Reports window to create and display a report including MCA commands specifying the use of the Internal Standard.

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PERATION

UTORIAL

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INDOW FOR QUANTITATIVE ANALYSIS

Tutorial: Using the MCA Window for Quantitative Analysis

Create an MCA window, using the New... command in the Windows menu. The MCA window will automatically fill up the

PolyView2000 workspace.

PolyView2000

The MCA Window

Pull down the File menu and select Open File. Select DIPA3020.RUN in the EXAMPLES directory. A chromatogram is displayed on the bottom half of the window. It contains one major peak of DIQUAT, which we will use as a reference.

To integrate this peak, press the Define Spectrum pushbutton. Click in front of the peak, on the peak start marker at about 1.8 minutes, then double-click on the peak end marker at about 3.3 minutes. This defines integration limits and baseline correction spectra for this peak (for more details, see On-line help on computing Area Spectra). A dialog box requests you to provide information to be saved along with this spectrum. Enter Diquat in the peak name field, 10 for the Amount and ug for the unit (Note that the flow rate was read from the file). Press OK after all the fields have been updated. The MCA window now shows the area spectrum and the integrated region is highlighted.

The Amounts Dialog

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PolyView2000

The MCA window, showing the

integrated region and the Area Spectrum

Create a new library, using the Create Library command in the Library menu. Name it PQDQ.LBR. We will now compute area

spectra and add them to this library.

Add the spectrum to the library by pressing Add To Library.

Now repeat the process with DIPA3037.RUN. The compound name is Paraquat, the amount 10 and the unit ug. Add the spectrum to the library. Create a new library, PQ.LBR, and press Add to Library again. At this point, you have created 2 libraries: PQDQ.LBR contains Paraquat and Diquat spectra, while PQ.LBR contains Paraquat only.

Now repeat the integration process described in step 3 with DIPA3053.RUN. This is an unknown mix, so do not enter any values in the Amount and Unit fields. Instead, just press OK.

Pull down the Library menu and select Open Library. Select PQDQ.LBR.

Press the MCA button. Press OK in the dialog box requesting additional parameters. This displays an MCA Report in a Preview window. This report has 3 main components: the Spectrum Analysis, the Plot Analysis and the Error Analysis. You can use the mouse to zoom in on a particular region of the report. To do this click on the left mouse button and drag to highlight the area of interest. Double-click to restore. Note that the right half of the Spectrum Analysis provides quantitative results, while the right half of the Plot Analysis provides qualitative results such as retention times and resolution. The Error Analysis compares the residual error in each point of the chromatogram to the noise. See On-Line Help, Interpreting MCA reports for more details. Press Print Page if you want to obtain a hardcopy, then Delete Page to terminate the Preview.

The MCA Report Parameters

NOTE: The MCA process uses up to six spectra from the library which match

the specified time range. If more than six spectra fit in the range, only the closest to the center of the range are used. As all component spectra must be independent, avoid placing multiple copies of the same spectrum in a library.

When only one component is specified, the best match in the time range is determined based on spectral comparison, not retention time.

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Varian PolyView2000 Operation Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Table of Contents

Overview

PolyView2000 Functions

Plotting a Chromatogram

Examining and Analyzing Spectra

Building, Editing, and Searching Spectral Libraries

Determining the Purity of a Chromatographic Peak

Purity Evaluation by Multicomponent Analysis

Preparing Reports of Diode Array Data

Recalculating the Peak Sense Events in a Diode Array Raw Data File

Quantitative Analysis by Multicomponent Analysis

Creation of Reduced Data Files

PolyView2000 Windows

Plot Window

Spectra Manager Window

Library Manager Window

Reports Window

Peak Sense Window

MCA Window

PolyView2000 and Other Star Workstation Applications

Operation

Configuring PolyView2000 Windows

Tutorial: Configuring PolyView2000 Windows

Plotting a Chromatogram

Tutorial: Plotting a Chromatogram

Examining Spectra from a Chromatogram

Tutorial: Spectral Plots

Tutorial: Spectral Math Functions

Performing Library Functions

Tutorial: Creating a Library

Tutorial: Building and Editing a Library

Tutorial: Conducting a Spectral Library Search

Reporting Data

Tutorial: Making a Report

Tutorial: Viewing and Printing of an Existing Report

Determining Peak Purity

Purity Parameter

Tutorial: Purity Parameter Plots

Tutorial: Peak Purity Survey Report

Performing Peak Sensing

Tutorial: Building a Peak Sensing Method

Tutorial: Conducting Peak Sensing

Tutorial: Troubleshooting Peak Sensing

Automating PolyView2000 Operations

Using On-Line Help

Tutorial: Using On-Line Help

Quantitative Analysis by MCA

Tutorial: Using the MCA Window for Quantitative Analysis