UniPoint System Software User s Guide

Transcription

1 UniPoint System Software User s Guide LT Gilson, Inc. All rights reserved

2 Exercise 3-Creating operations list Create and name operations list Set up steps Identify sample descriptions Save operations list and close its window Exercise 4-Viewing calibrations Exercise 5-Viewing data file Exercise 6-Viewing report file Exercise 7-Re-Analyzing data Common Procedures Using the navigator File maintenance Creating a directory Creating a file Opening an existing file Saving an untitled file Saving a named file Making a copy of a file Deleting files and directories Specifying default font for document windows Working in the table pane Displaying the table if it is not shown Sizing the table pane Changing column width using the mouse Setting column attributes Manipulating table rows Changing the table font Finding and replacing information Working in the graph pane Displaying the graph pane if it is not visible Sizing the graph pane Sizing and moving the graph Sizing and moving a summary graph Changing the font Setting graph attributes Removing items from display Displaying/removing gridlines Legend Annotation Manipulating the toolbar Using the notes or log file text editor Using the browse feature within dialogs

3 Customizing the UniPoint workspace Arranging elements in a workspace Saving a workspace Opening a workspace file Printing Specifying printer setup options Selecting what to print and starting the print Transferring information between files Gettng software version number Control Methods Control Method window Checking configuration file Assigning descriptions Additional device control parameters Mobile phase settings Data channel parameters Injector options Detector options Fraction collector options Interface only and contact event option High and low temperature limits Scheduling timed events Constructing a mobile phase profile Device commands Pumps Data channels Contacts Detector Injector and its syringe pump or dilutor Fraction collector System interface System (computer) control Valve actuator Temperature regulator (or column oven) Rack temperature regulator Pressure regulator (controller) GSIOC-to-RS-232 converter GSIOC command strings Variables Error handling

4 7 Analysis Methods Analysis Method window Setting analysis conditions Integration events Channel scales Peak table Naming peaks Entering level information for peaks Indicating internal standard peak(s) Grouping peaks Additional peak options Background removal Producing reports Report type Report output Report format review and modification Report customization Variables Error handling Operations 9 Programmed Run Operations window Setting up an operations list Using express entry dialog for list setup Using step entry dialog for list setup and modification Using the tray file to generate the operations list Setting up operations list for fraction collection Generating operations list when re-injecting collected fractions Modifying the operations list using edit menu Reviewing where run-generated files are stored Method Executor Starting a run During the run Manipulating run-time graph Interrupting a run Resuming a run after termination After the run Status of UniPoint Shutting down the HPLC system Viewing log file

5 10 Manual HPLC System Control Manual control method Control of mobile phase pumps and manometric module Strip chart control Contact control Control of injector, detector, collector, computer, and other devices GSIOC command strings Results 12 Calibrations Results window Opening report and data files Selecting report file information to view Selecting data file information to view Locating and opening a data file using sample description Overlaying (or merging) additional chromatogram data Analyzing data or analyzing it in a different way Linking to an analysis method Linking to a calibration file Linking to a control method Manipulating graph and table Viewing and scaling techniques in graph pane Peak integration techniques Generating new report file Comparison commands Calibration window Building calibrations Viewing calibrations Merging calibrations Error messages Batch Re-Analysis Processing Re-Analysis window Setting up a re-analysis list Using express entry dialog for list setup Using step entry dialog for list setup and modification Modifying the re-analysis list using edit menu

6 Indicating baseline adjustment Reviewing where files are stored Previewing the re-analysis Executing the re-analysis Resuming a re-analysis after termination Protocols 15 Routines 16 Tray Editor Protocol windows Overview of creating a protocol Assigning descriptions Issuing commands Variables Saving the protocol file Synchronizing injector protocol and control method Gilson-supplied protocols Updating protocol configuration Checking protocol Routines windows Overview of creating routines Assigning descriptions Issuing commands Viewing and modifying routines via the edit menu Variables Saving a routine and the routines file Gilson-supplied routines file Updating routines configuration Starting the tray editor Tray editor window Setting up a tray file Choosing a template Defining racks Setting up and modifying zone names Assigning zone and number to tubes Viewing techniques Managing tray files Printing tray files Identifying custom racks and tubes Quitting the tray editor

7 17 Extract, Import, and Export Features Extracting archived information Importing data Viewing Gilson 160 chromatogram Viewing chromatogram from non-gilson detector 17-5 Exporting data and reports Exporting chromatogram data Exporting report information Exporting calibration information Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Appendix G System Configuration Working with configuration files... A-2 Reviewing configuration file... A-2 Modifying the configuration file... A-3 Searching for configuration file... A-4 Scanning the GSIOC for devices... A-5 Specifying system units... A-6 Saving changes to the configuration file... A-7 Configuration checks... A-7 Sample Tracking Before the run... B-2 After the run... B-6 File Extensions Password Protection Setting up password protection... D-2 Indicating new password... D-3 Removing password protection... D-3 Unlocking columns in operations or re-analysis list... D-4 Merging Security Keys Calculations Peak Table Retention time windows... G-1 Description... G-1 Setting time window parameters... G-2 Resolving overlapping windows... G-3

8 Using the peak table during data analysis... G-4 Locating reference peaks... G-5 Locating non-reference peaks... G-6 Updating peak retention times... G-7 Generating a calibration table... G-8 Appendix H Appendix I Appendix J Appendix K Appendix L Appendix M 715 Data Translator GSIOC Utility Using the GSIOC utility within Windows 95/NT... I-1 Using the GSIOC utility within Windows I-3 GILSON.SYS Device Driver File GSIOC Configuration Utility Gilson Executable (.GEX) File UniPoint Software History Index

9 1 Software Installation and Startup This section describes how to install the Gilson UniPoint System Software and its supporting files and how to start the software. before you begin UniPoint requires a set of programs called Microsoft Windows (Version 95/NT or Version 3.1 ). For information on installing and using Windows, refer to the Microsoft Windows User s Guide. Note: The window and dialog box examples in this user's guide are from UniPoint running under Windows 95. Verify that the UniPoint security key is in place. The key must be installed in the Key port of a Gilson 506C System Interface. Refer to the UniPoint System Installation Guide if necessary. Note: If your system has a Gilson 506B System Interface or 621 Data Master, contact Gilson. Your system must include a 506C System Interface, and the key must be installed in its Key port, to run UniPoint. 1-1

10 unipoint user's guide Installing UniPoint software package After Microsoft Windows is installed, you can install UniPoint. The procedures for installing UniPoint vary depending on whether your computer has Microsoft Windows 95/NT or Windows 3.1. Installation also varies depending on whether this is the first time you are installing UniPoint. Refer to the appropriate instructions below. You can install UniPoint on a computer s fixed disk or on a network drive so multiple computers can access it. Installing UniPoint under Windows 95/NT if you are installing UniPoint for the first time Follow these procedures: 1 Turn off then on your computer and start Windows. 2 Insert UniPoint Disk 1 into a floppy drive of the computer. 3 On the Windows desktop, click on Start. 4 From the Start menu, click on Run. 5 When the Run box appears, type a:\setup.exe (or b:\setup.exe if the disk is in drive B). Then click on Ok. 6 Follow the on-screen instructions to install UniPoint and its supporting files. 7 When the Setup Complete box appears, ensure that the Yes, I will restart my computer now option button is selected. Remove the UniPoint installation disk, then click on Finish to complete installation and restart the computer. if UniPoint was previously Follow these procedures: installed and you are updating it to a newer 1 Turn off then on your computer and start Windows. version Note: If you do not restart the computer before installing UniPoint, you may see the following error message during installation: An error occurred during the move data process: -115 If this message appears, remove installation disk from floppy drive and restart the computer. 1-2

11 software installation and startup 2 On the Windows desktop, delete the icons with the following labels. To delete each icon, click on its picture with the mouse pointer and then press the Delete key on the computer s keyboard. UniPoint v1.xx UniPoint v1.xx Update Info GSIOC Utility Key Utility 715 Data Translator (if present) This step deletes the icons and prevents duplicates from being displayed after installation. It does not delete the files associated with the icons. The files are replaced during UniPoint installation and new icons are displayed. 3 Insert UniPoint Disk 1 into a floppy drive of the computer. 4 On the Windows desktop, click on Start. 5 From the Start menu, click on Run. 6 When the Run box appears, type a:\setup.exe (or b:\setup.exe if the disk is in drive B). Then click on Ok. 7 Follow the on-screen instructions to install UniPoint and its supporting files. 8 When the Setup Complete box appears, ensure that the Yes, I will restart my computer now option button is selected. Remove the UniPoint installation disk, then click on Finish to complete installation and restart the computer. Installing UniPoint under Windows 3.1 if you are installing UniPoint for the first time Follow these procedures: 1 Turn off then on your computer and start Windows. 2 Insert UniPoint Disk 1 into a floppy drive of the computer. 3 In the Program Manager, choose Run from the File menu. 4 When the Run box appears, type a:\setup.exe (or b:\setup.exe if the disk is in drive B). Then click on Ok. 5 Follow the on-screen instructions to install UniPoint and its supporting files. 1-3

12 unipoint user's guide 6 If your computer does not require Win32S installation, click on the Yes, I want to restart my computer now option button when the Setup Complete box appears. Remove the UniPoint installation disk, then click on Finish to complete installation and restart the computer. If your computer requires Win32S installation, do the following: a) When the Setup Complete box appears, click on the No, I will restart my computer later option button, remove the UniPoint installation disk, and click on Finish. b) Insert the Win32S Disk 1 into a floppy drive of the computer. c) In the Program Manager, choose Run from the File menu. d) When the Run box appears, type a:\setup32.exe (or b:\setup32.exe if the disk is in drive B). Then click on Ok. e) Follow the on-screen instructions. f) When installation completes, remove the Win32S installation disk and restart the computer. if UniPoint was previously Follow these procedures: installed and you are updating it to a newer 1 Turn off then on your computer and start Windows. version 2 In the Program Manager, delete the icons with the following labels. To delete each icon, click on its picture with the mouse pointer and then press the Delete key on the computer s keyboard. UniPoint v1.xx UniPoint v1.xx Update Info GSIOC Utility Key Utility 715 Data Translator (if present) This step deletes the icons and prevents duplicates from being displayed after installation. It does not delete the files associated with the icons. The files are replaced during UniPoint installation and new icons are displayed. 3 Insert UniPoint Disk 1 into a floppy drive of the computer. 4 In the Program Manager, choose Run from the File menu. 5 When the Run box appears, type a:\setup.exe (or b:\setup.exe if the disk is in drive B). Then click on Ok. 6 Follow the on-screen instructions to install UniPoint and its supporting files. 7 When installation completes, remove the UniPoint installation disk and restart the computer. 1-4

13 software installation and startup Starting UniPoint startup To start UniPoint, do the following: 1 Start Microsoft Windows. 2 Double-click on the UniPoint icon in the Gilson Applications box. You see the UniPoint startup screen. The startup screen displays for approximately 30 seconds while the software is loading. Note: The startup screen will display the text UniPoint System Software with SF Pressure Control if you purchased that software package. after the startup screen If this is the first time the software has been started, you see a message box when the startup screen disappears. This box tells you that the software is going to scan the Gilson Serial Input/Output Channel (GSIOC). See Setting up configuration file(s) on page 1-7. If UniPoint has been opened previously, you see the UniPoint window with the Navigator window open within it (see figure on next page). The Navigator enables you to quickly select the part of UniPoint to use. For more information on the Navigator, see page

14 unipoint user's guide if the software fails to start Any of several situations can prevent UniPoint from starting as expected. If you see an error message at startup, follow the instructions that appear. If any of the device drivers are missing or outdated, try re-installing the software as described in this section. If the security key is missing, install it as described in Section 3 of the UniPoint System Installation Guide. Then restart the computer. If the key for a different Gilson software product is in the Key port of the 506C System Interface, either install the proper key and restart the computer or merge the codes from both keys. Refer to Appendix E in this manual. 1-6

15 software installation and startup Setting up configuration file(s) After installing UniPoint and starting it for the first time, you see a message box telling you that the software is going to scan the Gilson Serial Input/Output Channel (GSIOC). 1 Do one of the following: If you are controlling one HPLC system with UniPoint, ensure that Gilson devices are connected along the GSIOC and powered on. Then click on OK and proceed to step 2. If you are running multiple HPLC systems, click on Cancel. Then click on Cancel again to exit the Default System Configuration dialog and proceed with setting configurations for multiple HPLC systems on page If the scan locates any Gilson devices that require additional setup information, a dialog box appears. Indicate the requested information and click on OK. When the scan completes, the configuration file (DEFAULT.CFG) is created and its information appears in the Default System Configuration dialog. The computer and Gilson 811 Mixer (if two or more pumps are found) are automatically added to the list in addition to any other Gilson devices that were found. 3 Check that all installed Gilson devices are named in the Unit ID/ Installed Devices list box. If not, you need to add the device by choosing its description in the Known Devices list box, indicating its unit ID, and choosing Add. Refer to the Note on the next page. 1-7

16 unipoint user's guide Note: When adding a 402 Syringe Pump, select the description specifying how the pump is configured: 402 Dual Syringe Pump, 402 Dual Syringe Pump w/ Tee, 402 Left Syringe Pump, or 402 Right Syringe Pump. When adding a 122 Fluorometer, choose the 122 Fluorometer/ 606 Converter description. Do not include the converter as a separate device when used with the fluorometer. If you have a 133 RI Detector connected to a 606 GSIOC to RS- 232 Converter, the software-selected description of 606 GSIOC to RS-232 Converter is appropriate. If you are using a 233 XL or 215 for both injection and collection, the Unit ID/Installed Devices list box must include the device as an Auto Injector and a Fraction Collector. If you scanned the GSIOC, the scan identifies the device as an Auto Injector. You must add the device as a Fraction Collector to the Unit ID/Installed Devices list box. When you add the device as a Fraction Collector, use the same unit ID as the Auto Injector device. For a 232 XL or 233 XL Injector, the setup options request the location of the right rinse station. If the rinse station is located above the right valve, choose Right. If the rinse station is located to the left of the right valve, choose Center. 4 Choose OK to exit the Default System Configuration dialog. If you made changes to the file, the software prompts you to save the changes. Save them to the same configuration file. The UniPoint application window appears, with the Navigator open within it (see page 1-6). setting configurations for multiple HPLC systems If you are running more than one HPLC system from UniPoint, you must create a configuration file for each of those systems. Store each configuration file in its own directory and store any files that use the configuration there also. GSIOC unit IDs for all Gilson devices in the systems must be unique. Plus, all configurations share the computer and the 506C System Interface. On the next page is an example Gilson HPLC onfiguration whose modules have been separated into two systems, identified as System 1 and System 2. All Gilson GSIOC modules are connected along the GSIOC. 1-8

17 software installation and startup 1 Create a directory for each configuration using the File Manager, accessed via UniPoint's File menu. If you need help using the File Manager, refer to its on-line help. 2 When finished creating directories, exit the File Manager using its File menu. The UniPoint application window re-appears, with the Navigator open within it (see page 1-6). 3 Choose Default System Configuration from the File menu to display the Default System Configuration dialog. 4 Click on Install. 5 When the additional options appear in the dialog box: a) Choose the device in the Known Devices list box. b) Verify or enter its unit ID in the Unit ID text box. c) Choose Add. d) If the device requires additional setup information, the Setup dialog appears. Indicate the requested information and choose OK. e) Repeat the above for each additional device. 1-9

18 unipoint user's guide 6 When finished adding devices, choose Save. When the Save As dialog appears, use its options to indicate a name for the configuration file and where the configuration file will be stored. Then choose OK to save the configuration file. 7 When the Default System Configuration dialog re-appears, use the procedures in steps 5 and 6 to create configuration files for each additional HPLC system. 8 When finished creating configuration files, choose OK to exit the Default System Configuration dialog. The UniPoint application window appears, with the Navigator open within it (see page 1-6). Following are Default System Configuration dialogs for the example systems, System 1 and System 2, shown on page 1-9. Notice that the system interface and computer system are identified in the configurations for both systems. An 811 Mixer is installed in both systems; however, since mixers do not have a unit ID, they are identified by an * in the configurations. Also, notice the files are stored in different directories. 1-10

19 2 Background Information Getting help On-line help has been included with UniPoint and can be accessed any time you are using UniPoint. It describes commands and dialogs and discusses procedures used to perform tasks. Technical notes The following topic-specific UniPoint Technical Notes are available upon request from your local Gilson representative. 122 Fluorometer Control 133 Refractive Index Detector Control 170 Diode Array Detector Control Autopreparative System Control Fraction Collector Control Multi-System Setup Sample Tracking Supercritical Fluid System Control Using the 215/819 for Injection Using the 215/819 for Injection and Collection Using the 233 XL for Injection and Collection 2-1

20 unipoint user's guide Overview of UniPoint UniPoint is a multi-document software package that runs under Microsoft Windows. UniPoint is divided into these function-specific sections: control methods analysis methods operations lists results calibrations re-analysis lists protocols and routines Each of these sections has its own window for specifying run-time or post-run options or for viewing chromatographic results. More than one window of the same type or of different types can be open at the same time. Note: If you are not familiar with the Windows user interface, please refer to the Microsoft Windows User s Guide. Even though the software is separated into several sections, the parts are interdependent. Information set in one window may be used in or required by another window for proper operation. You use different windows depending on what you want to do. 2-2

21 background information As the diagram below shows, before a run, you set up information in Control Method, Analysis Method, and Operations windows, and optionally in the Protocol and Routines windows. To start a run and view run-time information, you use the Operations window. After the run, use the Results and Calibration windows to view acquired data and analysis report information. To batch analyze collected data to generate reports and calibration plots, use the Re-Analysis window. 2-3

22 unipoint user's guide Default directory and file organization The following diagram shows the default locations to which UniPoint and files created by it are stored, unless changed by you during installation or when operating the software. 2-4

23 3 Getting Started After installing UniPoint, you can ensure that it is communicating with Gilson GSIOC (Gilson Serial Input/Output Channel) devices in your HPLC system. To check communication, you create a manual control method that assigns descriptions to GSIOC devices, contact connections, and data channels. After the control method is created, you use manual control to talk to devices. This section takes you through the procedures for setting up a manual control method. The control methods that you create for your applications can be based on this method. Multi-system users. If UniPoint controls multiple systems, you must choose the correct configuration file (File menu/default System Configuration command); see page A-2 if necessary. Then repeat the steps in Setting up manual control method and Checking and controlling devices for each HPLC system. 3-1

24 unipoint user's guide Setting up manual control method The manual control method assigns names to devices, contact connections, and data channels. 1 If necessary, display the Navigator by choosing its command from the File menu. On the Navigator, choose the Control Method square and then choose New. The Control Method window appears. review pump descriptions and set parameters To check the default descriptions assigned to installed Gilson pumps and assign refill speed and compressibility parameters: 1 Choose Mobile Phase from the Device menu. 2 When the Mobile Phase dialog box appears, review the default description, based on unit ID, that UniPoint assigned to each pump. 3-2

25 getting started 3 (Optional) To assign a different description to a pump based on the liquid it pumps: a) Click on the first pump in the list box. b) Type a description in the Description text box, for example, Water. c) Choose Change. d) Repeat for each pump. 4 Click on Settings to display the Pump Settings dialog. a) Click on the first pump in the list box. b) Indicate a compressibility and refill speed for the liquid being pumped. If necessary, refer to the on-line help for more information on compressibility and refill speed. c) Repeat for each additional pump. d) Choose OK to accept the settings. Note: If you are controlling a Gilson Supercritical Fluid System, use the SF Setting button to reveal the Inlet Pressure P0 text box. Click on the description assigned to the supercritical fluid pump. In the Inlet Pressure P0 box, type the pressure at the pump's inlet, typically 6 MPa (60 bar or 870 psi) at 22 C. A table of inlet pressures at other temperatures is shown in the on-line help. 5 Choose OK to exit the Mobile Phase dialog. assign data channel descriptions For each data channel to acquire, assign a description. 1 Choose Data Channels from the Device menu. 2 When the Data Channel dialog appears: a) Choose a data channel in the Unit ID/Name list box. The list box includes the data channels for each installed Gilson model 116 to 119 Detector. It also lists the names of any analog inputs located on Gilson detectors or interface devices connected via the GSIOC. b) In the Description box, type a label for the data channel. The description can identify the wavelength and sensitivity for the data channel, for example, 254 nm at 0.1 sens. c) Click on the data channel in the Unit ID/Name list box to enter its description. d) Repeat the above for each data channel. e) Choose OK to exit the dialog box. 3-3

26 unipoint user's guide assign contact descriptions If contact connections exist between a Gilson device (usually the 506C System Interface) and another HPLC device, assign a description to each connection. Use the Contact command in the Device menu. To assign descriptions to contact connections, refer to assign data channel descriptions, above. The description could identify the action performed by the contact, the device being controlled by the contact, or a combination of those, for example, Start fraction collector. review descriptions for additional Gilson devices Use the remaining commands in the Device menu to review UniPoint descriptions assigned to each installed Gilson detector, injector and its syringe pump or dilutor, fraction collector, and so on. save and close control method When finished reviewing and assigning device descriptions: 1 Choose Save As from the File menu or the Save tool ( ) from the toolbar. If a Gilson injector is installed, you see a message asking for a tray file name, choose No at this time. If a Gilson fraction collector is installed, you see a similar message box, choose No. 2 When the Save As dialog appears: a) Type a valid name in the File Name box. As a suggestion, MANL_CTL.GCT. b) As necessary, use the Directories and Drives list boxes to choose where to store the file. The current path is listed above the Directories list box. c) Choose OK. 3-4

27 getting started 3 Choose Close from the File menu. Checking and controlling devices Using the Operations window, you can issue commands to Gilson devices and verify they can communicate with UniPoint. 1 Ensure that all Gilson devices are connected along the GSIOC and powered on. 2 If necessary, display the Navigator by choosing its command from the File menu. On the Navigator, choose the Operations square and then choose New. The Operations window appears. When you see the message System Status in the status box, UniPoint can communicate with the HPLC system. 3-5

28 unipoint user's guide check control method To verify that the manual control method that you created is used for device identification: 1 Choose Control Method from the Manual menu. 2 Check the method name shown in the Browse Control Method dialog. 3 If the method s name is shown, choose Cancel. If not, use the options in the Browse Control Method dialog to select the method. Then choose OK. UniPoint reads the information in the file and uses it for manual control. check communication To quickly check if UniPoint and installed devices are communicating: 1 Choose Generic from the Manual menu. 2 When the Generic Device Control dialog appears, choose the Custom> button. If the Custom> button is already active, its label is List>. Therefore, if you see a List> button, proceed to the next step because the options you need are already shown. 3-6

29 getting started 3 Choose the first device in the Device list box. 4 Type its unit ID in the Unit text box. The unit ID is shown at the left of the device name in the Devices list box. 5 Type % in the Command text box. 6 Choose the Immediate button. 7 In the Response field, read the response from the device. If you see the device s model and version number, the device and UniPoint are communicating. If you see an error in the Response field or an error message box, the device and software are not communicating. Check each of the following: Check that the device s power switch is ON. Check the physical GSIOC connections to the device as described in the UniPoint System Installation Guide. Check that the correct Unit ID was entered. If UniPoint still cannot communicate with the device, contact Gilson. optionally start flow To start the pumps if they are not currently pumping liquid: 1 Choose Mobile Phase from the Manual menu. 2 When the Mobile Phase Control dialog appears, type 1 in the Ramp Time box. 3 In the Total Flow box, type the desired flow rate. You can set a flow resolution of times the maximum flow of the pump head. At or below 10% of the maximum flow, settable resolution is times the maximum flow. 3-7

30 unipoint user's guide 4 In the mobile phase composition box for each pump except the first one, type the percent composition from that pump. The software calculates the percent composition from the first pump for you. 5 Choose Begin to ramp the pumps to the specified flow and composition conditions. 6 Choose Done to remove the Mobile Phase Control dialog from the screen. When you close the dialog, the conditions indicated remain in effect. autozero detector To autozero the detector: 1 Choose Detectors from the Manual menu. 2 Choose a detector in the Detector/Protocol File list box. 3 Choose Autozero Channels in the Command list box. 4 Choose the Send button. 5 Choose Done to remove the Manual Detector Control dialog. start strip chart To start a manual strip chart: 1 Choose Strip Chart from the Manual menu. 2 When the Manual Strip Chart dialog appears, click on Start to begin viewing data from the data channel(s). The traces for the channels appear in the Operations window. The Operations window's legend identifies each channel being collected. 3 To get a better view of the data, choose Done to remove the dialog. 3-8

31 getting started 4 To stop collection, display the Manual Strip Chart dialog again and click on Stop. 5 When data collection is halted, the following Manual Strip Chart dialog appears. Click on Done to exit the dialog without saving or printing the strip chart. exit operations window To close the Operations window: 1 Choose Close from the File menu. UniPoint continues monitoring the HPLC system until you exit the software. 3-9

32 4 Hands-On Look at the Software UniPoint is an application that runs under the Microsoft Windows operating environment. Therefore, it takes advantage of the Windows graphic interface. To use the software, you must understand how to use a mouse and how to use Windows icons and drop-down menus. For more information, refer to the Microsoft Mouse User s Guide and Microsoft Windows User s Guide. This section includes exercises that give you hands-on experience with the various document windows of UniPoint. Exercises 1-3 show you how to set up pre-run conditions (control method, analysis method, and operations list). Exercises 5-7 show you how to access and manipulate run output (data, reports, and calibrations) that Gilson generated from a chromatographic separation. Unless the default installation path was changed, the output files were copied to C:\GILSON\EXAMPLES\HPLC when UniPoint was installed. Exercise 8 shows you how to re-analyze data to generate new reports. 4-1

33 unipoint user's guide Exercise 1 Setting control method conditions In this exercise, you set and modify control method conditions. For the resources controlled by a method, you need to: assign a description, or use the default description assigned by Gilson, indicate any additional settings required for device control, and send timed events to or wait for input from the device. The control method for this exercise sets the mobile phase profile, starts an injector protocol, waits for the injector to inject the sample, and starts and stops data collection. Checking configuration file Before creating the control method, locate the configuration file that identifies the components of our example HPLC system. This file is EXAMPLE.CFG and is stored in C:\GILSON\EXAMPLES\HPLC, unless the default installation path was changed when UniPoint was installed. UniPoint uses the configuration to identify available system components for the control method. 1 Close any open document windows by choosing Close from the File menu. (The Navigator, if it appears, can remain on-screen.) 2 Choose Default System Configuration from the File menu. The Default System Configuration dialog appears. 3 Check the name of the configuration file listed at the bottom of the dialog. 4 If the EXAMPLE.CFG file is named, review the devices in the example system and click on OK. If the configuration for your HPLC system is named: a) Click on Browse. b) Use the options in the Browse System Configuration dialog to select the EXAMPLE.CFG file and place its name in the File name box. Then click on OK. c) When the Default System Configuration dialog re-appears, review the devices identified for the example system and click on OK. 4-2

34 hands-on look Create method To display the Control Method window: 1 If necessary, display the Navigator by choosing its command in the File menu. On the Navigator, click on the Control Method square and then click on New. The Control Method window appears. 2 Choose Show Graph from the Graph menu to display the graph pane. 3 Display each of the menus to become familiar with its commands. Set device descriptions and parameters For all installed resources, except data channels and contacts, UniPoint assigns a default description when the control method is created. Below you review and set descriptions for installed devices. You also identify additional control parameters for the pumps and injector. Whenever UniPoint refers to a device, it lists the device s description. 4-3

35 unipoint user's guide set pump descriptions and parameters To assign descriptions and indicate options for the pumps: 1 Choose Mobile Phase from the Device menu. 2 When the Mobile Phase dialog appears, notice that UniPoint assigned default descriptions to installed pumps, based on the pump s unit ID. 3 Click on Pump 21 in the Unit ID/Name list box. Then type 0.1% acetic acid in the Description box and click on Change. 4 Since Pump 22 is not needed for this method, click on its name in the Unit ID/Name list box. Press the DELETE or BACKSPACE key to remove its description. Then click on its name in the Unit ID/Name list box to update the list box. 5 Click on Pump 23 in the Unit ID/Name list box. Then type acetonitrile for its description and click on Change. 6 Click on Settings to set refill speed and compressibility parameters for the pumps. 7 When the Mobile Phase Settings dialog appears, type 46 in the Compressibility box and 125 in the Refill Speed box for the 0.1% acetic acid pump. 4-4

36 hands-on look 8 Click on acetonitrile in the list box. Then type 99 in the Compressibility box and 125 in the Refill box. 9 Click on OK to accept the settings and exit the Mobile Phase Settings dialog. 10 When the Mobile Phase dialog appears, click on OK. set data channel description and scaling To assign a description for the data channel to acquire and set run-time scaling parameters: 1 Choose Data Channel from the Device menu. 2 When the Data Channel dialog appears, click on 119 Channel 1 in the list box. Type 254nm, 0.01 sensitivity in the text box. Then click on 119 Channel 1 in the list box to enter its description. 3 Click on the Options button. 4 When the Strip Chart Options dialog appears, type 7 in the Display Time box. 5 Click on the User Units option button. 6 Type 10 in the mvolt box, 0.01 in the User Units box, and aufs in the Label box. 4-5

37 unipoint user's guide 7 Click on OK to accept the settings. 8 Click on OK again to exit the Data Channel dialog. check injector descriptions and choose protocol and tray files To check descriptions and select the protocol and tray files for the injector and its syringe pump or dilutor: 1 Choose Injector from the Device menu. 2 When the Injector dialog appears, notice that the injector and dilutor are assigned the same description: Injector. This combines their command sets and makes setting timed events for them easier. 4-6

38 hands-on look 3 To select an existing protocol file that contains commands for controlling the injector and dilutor during a run: a) Click on the Browse button. b) Use the options in the Browse dialog to choose C:\GILSON\EXAMPLES\HPLC\INJTOTL.GIP and then click on OK. When the Injector dialog appears again, it lists the protocol s file description: injection = total loop fill with initial rinses. 4 To select the tray file that identifies sample locations during the run: a) If necessary, click on the 231 injector description in the Unit ID/ Name list box so it is highlighted. b) Click on the Tray Editor button. c) If a Hint box appears on the Tray Editor window, click on OK to remove the box. d) Choose Open from the File menu of the Tray Editor window. e) Use the options in the Open Tray File dialog to select the C:\GILSON\EXAMPLES\HPLC\CODE_0.GTY file and click on OK. 4-7

39 unipoint user's guide f) Examine the example tray. It shows tube locations designated for sample tubes. g) Choose TUBES from the Zone menu to display numbers in the tubes. h) Choose Exit from the File menu to remove the Tray Editor window. When the Injector dialog appears again, it displays the tray file name. 5 Click on OK to accept the settings and exit the Injector dialog. remove fraction collector description Since the fraction collector is not used for this method, remove its default description. 1 Choose Fraction Collector from the Device menu. 2 When the dialog appears, click on the fraction collector s name in the Unit ID/Name list box. Press the DELETE or BACKSPACE key to remove its description. Then click on the fraction collector s name in the Unit ID/Name list box to update the list. 3 Click on OK. 4-8

40 hands-on look Save method You are done setting descriptions for the devices in the HPLC system. Before continuing, save the current conditions set in the control method: 1 Choose Save from the File menu or its tool ( ) from the toolbar. When the Save As dialog appears: a) Type a valid name in the File name box. As a suggestion, indicate your initials. The software automatically assigns the.gct extension. b) If necessary, use the dialog's other options to store the file to C:\GILSON\EXAMPLES\HPLC. c) Click on OK. 4-9

41 unipoint user's guide Setting events In this part of the exercise, set the timed events to issue during a chromatography run. specify mobile phase profile To set the mobile phase profile: 1 Choose Mobile Phase from the Event menu or click on its tool ( ) in the toolbar. 2 When the Mobile Phase Event dialog appears: a) Type 0.00 in the Time box. b) Type 0.30 in the Flow box. c) Type in the acetonitrile box. d) Click on Insert. The software automatically calculates for 0.1% acetic acid. e) Click on Done. 3 Notice the addition of the mobile phase event to the table and the gradient profile in the graph. set protocol event To set an event to start the protocol: 1 Choose Injector from the Event menu or click on its tool ( ) in the toolbar. 2 When the Injector Event dialog appears: a) In the Time box, type b) If necessary, choose the injector protocol s description in the Injector/Protocol File Description list box. c) Notice the TUBE and LOOP_VOLUME text boxes that appear. These are values requested by the protocol. d) Leave the variable name in the TUBE box. You will indicate a value for the variable when you set up the operations list (the steps executed during a run) in Exercise 3. e) Type 20 in the LOOP_VOLUME box. f) Click on Insert. g) Click on Done. 4-10

42 hands-on look 3 Notice the addition of the event to the table and graph. set synchronize event To synchronize the time of sample injection with the start of data collection, indicate a synchronize event: 1 Choose System from the Event menu or click on its tool ( ) in the toolbar. 2 When the System dialog appears: a) Type 0.06 in the Time box. b) Choose System Controller in the System Controller list box. c) Choose Synchronize in the Command list box. d) Click on Insert. e) Click on Done. 3 Notice the addition of the event to the table and graph. 4-11

43 unipoint user's guide start and stop data collection To set events for starting and stopping collection via the data channel: 1 Choose Data Channels from the Event menu or click on its tool ( ) in the toolbar. 2 When the Data Channel Event dialog appears: a) Type 0.07 in the Time box. b) Click on the Start All Channels option button. c) Click on Insert. d) Set the step to stop collection by typing 5.30 in the Time box, clicking on the Stop All Channels option button, and clicking on Insert. e) Click on Done. 3 Notice the addition of the events to the table and graph. Viewing protocol To view the steps in the protocol that you identified in the control method: 1 Move the mouse pointer so the arrow is positioned over the text injection = total loop fill with initial rinses. Then click to highlight the row and the cell. 2 Click the right mouse button to display the menu shown at the left. 3 Click on the Open Referenced File command. The Protocol window appears, listing the steps in the protocol file. 4-12

44 hands-on look 4 When you are finished looking at the protocol, choose Close from the File menu to return to the Control Method window. Save method and close its window You are done setting descriptions and events for the devices to be used during a programmed run. The Control Method window should look similar to the one below. 1 Choose Save from the File menu or its tool ( ) from the toolbar. 2 Choose Close from the File menu. 4-13

45 unipoint user's guide Select your system configuration So UniPoint uses the correct configuration information the next time you set up a control method, select the configuration file created for your HPLC system. 1 Choose Default System Configuration from the File menu. 2 When the Default System Configuration dialog appears, click on the Browse button. 3 Use the options in the Browse dialog to select the configuration file set for your system and then choose OK. 4 When the Default System Configuration dialog appears again, click on OK. 4-14

46 hands-on look Exercise 2 Setting analysis method conditions In the last exercise, you identified devices and set timed events for controlling them during a run. In this exercise, you specify conditions to analyze peaks in collected data and report on integrated peaks. Create method To display the Analysis Method window and name the method: 1 If necessary, display the Navigator by choosing its command in the File menu. On the Navigator, click on the Analysis Method square and then click on New. The Analysis Method window appears. Notice that the software automatically sets these default analysis conditions at 0.00 min: Default Baseline Peak Width: 0.20 min Peak Sensitivity: 2% Disable Negative Peak Integration 2 Choose Show Graph from the Graph menu to display the graph pane. 3 Display each of the menus to become familiar with its commands. 4-15

47 unipoint user's guide Modify integration events Below you modify the default values for peak width and sensitivity. 1 To change the default peak width: a) Double-click on its event in the analysis method table. b) In the Integration Events dialog appears, change 0.2 to 0.15 in the Width box and click on Change. 2 To change the default peak sensitivity: a) In the Integration Events dialog, click on the down arrow to display the peak sensitivity event. b) Type 5 in the Sensitivity box and click on Change. 3 To add an event to modify the peak sensitivity: a) In the Integration Events dialog, click on New. b) Click on Peak Sensitivity in the Command list box. c) Type 3.75 in the Time box and type 2 in the Sensitivity box. d) Click on Insert. e) Click on Done to exit the dialog. 4-16

48 hands-on look Select the analysis channel The analysis channel identifies the data channel whose information is reported on by the analysis method. 1 Choose the Channel Scales command from the Analysis menu. The Data Channel Scales dialog appears. 2 To display a data channel in the list box, click on Browse. When the Browse Control Method dialog appears, use its options to select the name of the control method created in Exercise 1 and choose OK. 3 When the Data Channels dialog re-appears, notice that the channel description and scaling settings entered into the control method appear. 4 If necessary, click on the channel description in the list box to highlight it. 5 Click on the Analysis Channel check box. 6 Click on OK to accept the settings. 4-17

49 unipoint user's guide Identify peaks in the peak table Next, you indicate peak information for the three peaks to be integrated and analyzed. They are 4-acetamidophenol, caffeine, and 2- acetamidophenol. 1 Choose Peak Table from the Analysis menu. The Peak Table dialog appears. 2 To identify the peaks and set their retention times: a) Type 2.70 in the Time box and 4-acetamidophenol in the Name box. Click on Insert. b) Repeat the above to set the following retention times for the other two peaks. Time Peak Name 3.55 caffeine acetamidophenol 3 To set up level information for calibrators, for the specified peaks, injected during the run: a) Click on 4-acetamidophenol in the Time/Name list box. b) Click on the Levels option button. c) In the Peak Levels area that appears, type 5 in the Amount box and click on Insert. d) Indicate two more levels of 25 and 45 by typing the amount and clicking on Insert. e) Click on caffeine in the Time/Name list box and indicate levels of 10, 40, and

50 hands-on look 4 To set 2-acetamidophenol as an internal standard: a) Click on its name in the Time/Name list box. b) Click on the Internal Standard check box. c) In the Amount box, type 150. d) For the Start Time and End Time, type 0 and 7. 5 Click on OK to save the settings and exit the Peak Table dialog. 6 Notice that a retention time window appears, on the graph, for each peak. Save method Before continuing, save the current conditions set in the analysis method: 1 Choose Save from the File menu or its tool ( ) from the toolbar. When the Save As dialog appears: a) Type a valid name in the File name box. As a suggestion, indicate your initials. The software automatically assigns the.gan extension. b) If necessary, use the dialog's other options to store the file to C:\GILSON\EXAMPLES\HPLC. c) Click on OK. 4-19

51 unipoint user's guide Selecting report parameters indicating report type To select the kind of report generated: 1 Choose Type from the Report menu. The Report Type dialog appears. 2 Choose the Internal Standard option button in the Report Type area. 3 In the Min. Area box, type Choose Linear in the Curve Fit drop-down list. 5 Leave all other options at their default settings. 6 Choose OK to accept the settings and exit. checking output To see which reports are saved to disk, printed, or exported at run time: 1 Choose Output from the Report menu. 2 Review the options available for saving, printing, and exporting reports. 4-20

52 hands-on look 3 Choose OK to exit. Save method and close its window You are done setting conditions for the analysis method. The Analysis Method window should look similar to the one below. 1 Choose Save from the File menu or its tool ( ) from the toolbar. 2 Choose Close from the File menu. 4-21

53 unipoint user's guide Exercise 3 Creating operations list In this exercise, set up an operations list that uses the control and analysis methods created in the previous exercises. An operations list identifies the steps the software performs when the run is started. These steps describe the samples to be injected and the methods that the software uses to acquire and analyze the sample s data. Create and name operations list 1 If necessary, display the Navigator by choosing its command in the File menu. On the Navigator, click on the Operations square and then click on New. 2 If you see any error messages, choose OK to remove their boxes. UniPoint starts communicating with HPLC devices when an Operations window is opened. Since your HPLC system is probably not the same as the example system, you may see messages indicating that some devices cannot be located or initialized. 3 Choose Save from the File menu or its tool ( ) from the toolbar. When the Save As dialog appears: a) Type a valid name in the File name box. Once again, you can use your initials since the software assigns the.gop extension. b) If necessary, use the dialog's other options to store the file to C:\GILSON\EXAMPLES\HPLC. c) Choose OK. 4-22

54 hands-on look 4 Display each of the menus to become familiar with its commands. Set up steps For our example, you will create steps that identify a group of calibrators followed by a group of unknowns. Those steps are followed by a second set of calibrators then unknowns. 1 Choose Express from the List Entry menu. The Express Entry dialog appears. 2 Indicate information in the text boxes of the Cycle Generation area. Text box Value Calib Levels 3 Repeats/Level 3 Unknowns 5 Repeats/Unknown 1 Number of Cycles 2 3 Click in the Control Method box and then click the Browse button to select the control method set up in Exercise 1. (Or, double-click in the Control Method box.) 4 Click in the Analysis Method box and then click the Browse button to select the analysis method set up in Exercise 2. (Or, double-click in the Analysis Method box.) 4-23

55 unipoint user's guide When you selected the control method, an additional text box, TUBE, appeared at the bottom of the Express Entry dialog. It requests information for the tube location in which a sample is located. 5 In the TUBE text box, indicate the location of the first sample, as follows. a) Click in the text box and click on Browse. (Or, double-click in the text box.) The Browse Tray Editor window appears, displaying information for the tray editor file specified in the control method. b) Double-click in the tube located in the upper left corner to select it. The tube s color changes to cyan and then red. c) Choose OK to exit the window. The Express Entry dialog re-appears and displays TUBES:1 in its TUBE text box. 6 Choose Generate then choose Done. 7 Review the list of steps in the Operations window. Notice how the software assigned tube numbers. For each calibrator level, the tube number remained the same. For each unknown, the tube number was incremented by one. 4-24

56 hands-on look Identify sample descriptions To assign sample descriptions: 1 Choose Paste Special from the Edit menu. 2 When the Paste Special dialog appears, click on Editor. 3 When the Notepad window appears, type standard and press the ENTER key. 4 Repeat step 3 eight times so the word standard is listed nine times on the screen. Each sample description must be on its own line. 5 For the next five lines, type unknown one, unknown two, unknown three, unknown four, unknown five. 6 Type standard and press ENTER nine more times. 7 For the next five lines, type unknown six, unknown seven, unknown eight, unknown nine, unknown ten. 8 Choose Select All from the Edit menu and then choose Copy from the Edit menu. 9 Choose Exit from the File menu. Indicate No when prompted to save the file. 10 When the Paste Special dialog re-appears, click on Description in the list box and click on Paste. The sample descriptions appear in the Operations window. 4-25

57 unipoint user's guide Save operations list and close its window You are done setting conditions for an operations list. If this list had been created for your HPLC system, you could start the run using the Run menu. However, since you created it for our example system, save the list and close the Operations window. 1 Choose Save from the File menu or its tool ( ) from the toolbar. 2 Choose Close from the File menu. 4-26

58 hands-on look Exercise 4 Viewing calibrations In this exercise, you view and manipulate information in a calibration file generated for an example chromatography run. 1 If necessary, display the Navigator by choosing its command in the File menu. On the Navigator, click on the Calibration square and then click on Open. 2 When the Open dialog box appears: a) Use its options to choose the C:\GILSON\EXAMPLES\HPLC \INT_STD.001 directory. b) Choose the INT_SEQ.GB1 file in the File Name list box. c) Click on OK. The Calibration window displays the summary report and calibration plot generated for the first standard peak of the selected calibration. 3 Use the vertical and horizontal scroll bars, if necessary, to view hidden information. 4-27

59 unipoint user's guide 4 To enlarge part of the graph: a) Click on the magnifying glass tool ( ). b) On the graph, click and drag to draw a rectangle around the repeats on one of the levels. Then release the mouse button. 5 To learn about the injected sample associated with a repeat: a) Click on the arrow tool ( ) and move the mouse pointer to a repeat s square. b) Click on the square using the right mouse button. c) When the menu appears, click on Info. A dialog giving information on the repeat appears. d) Choose OK to remove the dialog. 6 Click on the full-scale tool ( ) to return the X and Y axes to minimum and maximum values for the displayed calibration curve. 7 To view the curve for another standard peak: a) Choose Peaks from the View menu. b) When the dialog appears, double-click on another peak name. c) Watch as the Calibration window updates. d) Double-click on another peak name to view its calibration curve. 8 To view another calibration table for which calibration curves exist: a) Choose Calibrations from the View menu. b) When the dialog appears, double-click on the second calibration description in the list box. c) Watch as the Calibration window updates. 9 Use the Peaks dialog to view the curves for the peaks in the selected calibration. 10 Remove the Calibrations dialog and Peaks dialog by clicking on Done in each dialog. 11 Choose Close from the File menu to exit the window. 4-28

60 hands-on look Exercise 5 Viewing data file In this exercise, you open a data file and view traces acquired for injected samples. 1 If necessary, display the Navigator by choosing its command in the File menu. On the Navigator, click on the Results square and then click on Open. 2 When the Open dialog box appears: a) Use its options to choose the C:\GILSON\EXAMPLES\HPLC\INT_STD.001 directory. b) Choose the INT_STD.GDT file in the File Name list box. c) Click on OK. The Results window appears, displaying the trace for the first sample. The trace is displayed at full scale. Notice that the title bar identifies the name of the data file and the injection number of the sample whose information is displayed. 4-29

61 unipoint user's guide 3 To enlarge part of the chromatogram: a) Click on the magnifying glass tool ( ). b) On the graph, click and drag to draw a box around a peak. Release the mouse button. 4 Click on the full-scale tool ( ) to return the X and Y axes to the minimum and maximum limits for the displayed trace. 5 To view the trace for another sample: a) Choose Samples from the View menu. b) When the Sample's dialog appears, double-click on a sample description, for example, unknown five. c) Watch as the Results window updates. d) Double-click on another sample to view its trace. e) To close the dialog, double-click on its control-menu box in the upper left corner. integrating peaks To integrate peaks and view reporting information: 1 Choose Analysis Method from the Linking menu. The Analysis Method Link dialog appears. 2 To select the analysis method: a) Click on Browse. b) When the Browse Analysis Method dialog appears, locate the method you created in Exercise 2, or choose INT_SEQ.GAN, and click on OK. c) When the Analysis Method Link dialog appears again, choose the method in the list box and click on OK. 4-30

62 hands-on look The software uses the conditions in the method to scale the chromatogram plot and to generate reporting information. In the legend, the trace with a > sign in front of its description is the one being reported in the table pane. The table pane reports areas (or heights) for the channel. adjusting baseline To adjust the baseline beneath a peak: 1 Zoom on a peak using the magnifying glass tool ( ). 2 Click on the adjust baseline tool ( ). Begin points (up triangle) and end points (down triangle) appear on the baselines under each integrated peak. 3 Point and drag the begin or end point for the peak s baseline. Peak information in the table pane automatically updates. 4 Click on the arrow tool ( ) to remove peak begin and end points from being displayed. 4-31

63 unipoint user's guide reverting to method baselines If you made modifications to baselines and want to return to the conditions as set in the linked analysis method. 1 Click on the adjust baseline tool ( ). 2 Move the mouse pointer onto the chromatogram plot. 3 Click on the right mouse button. When the menu appears, click on Reset Baselines. 4 Click on the arrow tool ( ) to remove peak begin and end points from being displayed. 5 Click on the full-scale tool ( ) to return the X and Y axes to minimum and maximum values for the displayed trace. closing window 1 Choose Close from the File menu. 4-32

64 hands-on look Exercise 6 Viewing report file In this exercise, you open a report file and view reports within it. 1 If necessary, display the Navigator by choosing its command in the File menu. On the Navigator, click on the Results square and then click on Open. 2 When the Open dialog appears: a) Use its options to choose the C:\GILSON\EXAMPLES\HPLC\INT_STD.001 directory. b) Choose the.gr? extension in the List Files of Type list box. c) Choose the INT_SEQ.GR1 file in the File Name list box. d) Click on OK. The Results window appears, displaying the report information and chromatogram plot for the selected sample. The trace is scaled to the settings specified in the analysis method. The title bar identifies the name of the report file and the injection number of the sample whose information is being displayed. The table pane contains information for the analysis channel indicated in the analysis method. 4-33

65 unipoint user's guide 3 To view the trace for another report: a) Choose Samples from the View menu. b) When the Reports dialog appears, double-click on a report description, for example, calibration summary. c) Watch as the Results window updates. d) Double-click on another report, for example, unknown 8. e) Choose Show Graph from the Graph menu, if necessary to see the sample s trace. f) To close the Reports dialog, double-click on its control-menu box located in the upper left corner. 4 Choose Close from the File menu to exit the Results window. 4-34

66 hands-on look Exercise 7 Re-Analyzing data In this exercise, you generate new reports using the method that you created in Exercise 2. modify re-analysis file The quickest way to set up a re-analysis list is to open and modify a previously created re-analysis list. At run time, UniPoint automatically generates a re-analysis file (.GAR extension) for the samples being processed and stores it in the data/report directory. 1 If necessary, display the Navigator by choosing its command in the File menu. On the Navigator, click on the Re-Analysis square and then click on Open. 2 When the Open dialog box appears: a) Use its options to choose the C:\GILSON\EXAMPLES\HPLC\INT_STD.001 directory. b) Choose the INT_STD.GAR file in the File Name list box. c) Choose OK. 3 Review the steps in the re-analysis list. 4-35

67 unipoint user's guide 4 To replace the listed analysis method with the method you created: a) From the Edit menu, choose Replace. b) When the Replace dialog appears, type INT_SEQ.GAN in the Find What box. c) In the Replace With box, type the name plus the.gan extension of the analysis method created in Exercise 2. d) Choose Analysis Method in the Search Column list box. e) Choose the Replace All command button. f) If you see a message box asking to continue the search from the top of the table, click on Yes. When you see the message box indicating the search is complete, choose OK. g) To close the Replace dialog, choose Done. re-analyze data To start the re-analysis: 1 Choose Start from the Analyze menu or the Go tool ( ) from the toolbar. The software begins processing the first sample using the conditions in the analysis method. The status box and status line in the document window identifies step and method information. When the re-analysis completes, you can view the reports as described in Exercise 6. The report file is named after the analysis method used to create it and has the.gr1 extension. It is stored in the C:\GILSON\EXAMPLES\HPLC\INT_STD.001 directory. 4-36

68 5 Common Procedures This section deals with procedures that, for the most part, are the same in the various UniPoint document windows. Throughout the manual, you are referred to these procedures. The procedures in this section assume you know how to start UniPoint; see Section 1. It also assumes you can use the mouse and keyboard when working within windows and dialog boxes. For information on using the mouse and keyboard and sizing the application window and document windows, refer to the Microsoft Windows User s Guide. 5-1

69 unipoint user's guide Using the navigator When you start UniPoint, you may see the Navigator window. The Navigator provides for quickly creating and opening files in UniPoint. It also lets you view example files supplied with the software. To display the Navigator if it is not on-screen: 1 Choose Navigator from the File menu. To use the Navigator: 1 Click in the square next to the document type to access. The text in the different areas of the window helps you make your selection. (A description of the various windows is also given on page 5-5.) 2 If you do not see the document type you need, choose Advanced to reveal additional selections for protocols and routines. 3 Choose the New, Open, or Example command button. New. Displays the document window for the file type. The New button is not available for Results and Calibrations because those files are generated during a run or re-analysis processing or by saving an existing file to a new name. Open. Displays the Open dialog for choosing a file or information in a file. If you need help using the dialog, see page 5-6. Example. Displays a list box for selecting example information supplied with UniPoint and stored to disk during installation. 5-2

70 common procedures turning navigator off/on When the Show Navigator option is active, the Navigator appears each time you close all open document windows in UniPoint. You can turn off this option so the Navigator does not appear automatically. To turn off the Show Navigator option, choose Options in the File menu. When the Options dialog appears, click on the Actions tab and then remove the check mark from the Show Navigator box. redisplaying navigator at To display the Navigator while a document window is on-screen, any time choose its tool ( ) from the toolbar. Or, choose its command in the File menu. File maintenance You can create and open files in UniPoint using the Navigator, as described on the previous page, or using the New and Open commands in the File menu. On the following pages, you learn how to create, open, save, and delete files and how to create and delete directories. Note: In Windows 95/NT, directories are called folders. 5-3

71 unipoint user's guide Creating a directory To speed access to files, store similar files in directories. Gilson recommends that you create a directory for each experiment or user. Note: During a run or re-analysis processing, UniPoint automatically generates an output directory for data and reports. To create a directory: 1 Choose File Manager from UniPoint s File menu. The Windows File Manager appears. For details about the File Manager, see its on-line help. 2 Choose the disk drive and/or directory in which to create the new directory. 3 From the File menu, choose Create Directory. 4 In the Create Directory dialog, verify that the correct path to the directory is listed and then specify the name of the new directory. 5 Choose OK. 6 From the File menu, choose Exit to return to UniPoint. 5-4

72 common procedures Creating a file UniPoint can create several types of files for controlling HPLC devices and analyzing collected data. To create a file: 1 From the File menu, choose New. 2 When the dialog appears, select the type of file to create. (See the table below for a description of the files that you can create.) 3 If you do not see the needed file type, choose Advanced to reveal additional selections. For system-generated files, such as data, reports, and calibrations, you use the Open command to view the appropriate document window. Select: Control Method Analysis Method Operations Re-Analysis Injector Protocol Injector Routines Generic Protocol Generic Routines To do the following: Indicate device control events to issue when the control method is executed during a run. See page 6-1 for additional information. Identify how data is analyzed and reported. See page 7-1. Set the step-by-step actions to perform during a run. See page 8-1. Analyze data in a different way than it was originally analyzed. See page Create a set of untimed sequential instructions executed by the auto-injector and its dilutor. See page Identify named groups of commands that make building injector protocols easier. See page Create a set of untimed sequential instructions executed by one or more Gilson devices. See page Identify named groups of commands that make building generic protocols easier. See page Choose OK when the appropriate file type is selected. The document window that lists menu items for setting options for the file appears. Refer to the appropriate section in this manual, listed in the table above, for more information on setting parameters for the selected file type. 5-5

73 unipoint user's guide Opening an existing file To display the contents of an existing file in a document window: 1 From the File menu, choose Open. 2 In the File Name box, type or select the file to open. If the file is not listed in the box, do one or more of the following: In the Drives box, select a different drive. In the Directories box, select a different directory. Note: With data, report, calibration and routines files, you can choose among the individual items stored in those files. Consult Section 11, Results, Section 12, Calibrations or Section 15, Routines for details. 3 Choose OK when the desired name appears in the File Name box. The document window for the file type appears. For information on: See page: Control method 6-1 Analysis method 7-1 Operations 8-1 Results 11-1 Calibrations 12-1 Re-Analysis 13-1 Protocols 14-1 Routines 15-1 if you selected a log file Choosing an operations log file displays a text editor. See page 5-34 for more information on using the text editor. if you selected a workspace Choosing a workspace file displays the document windows, and their associated files on the computer screen. Any windows currently displayed are not affected by the workspace being opened. See page 5-38 for information on setting up a workspace. 5-6

74 common procedures shortcut The bottom of the File menu lists the most recently worked on and saved files. If the file is listed there, just choose its name to open it. Saving an untitled file To store the contents of a new unnamed (untitled) file to disk for future retrieval: 1 Choose Save or Save As from the File menu or click on in the toolbar. 2 When the Save As dialog appears, use its options to name and select a storage location for the file. 3 Choose OK to save the file. 5-7

75 unipoint user's guide Saving a named file To quickly save changes made to a file that is already named: 1 Choose Save in the File menu or click on in the toolbar. If this is the first time you are saving the file, the Save As dialog appears. Use the dialog to indicate a name for the file. See Saving an untitled file, above. Making a copy of a file To store the changes made to a file to a new name while still keeping a previous version, do the following: 1 From the File menu, choose Save As. 2 When the Save As dialog appears, use its options to name and select a storage location for the file. 3 Choose OK to create the new version of the file and exit the dialog. 5-8

76 common procedures Deleting files and directories If a file or directory is no longer needed, you can remove it from disk: 1 Choose File Manager from UniPoint s File menu. The Windows File Manager appears. For details about the File Manager, see its on-line help. 2 Locate and select the files and/or directories to delete. 3 From the File menu, choose Delete. Or, press the Delete key. 4 When the Delete dialog appears, verify that the correct names are listed. 5 Choose OK. 6 If a confirmation box appears, choose Yes or Yes to All to complete the deletion. 7 Choose Exit from the File menu to return to UniPoint. 5-9

77 unipoint user's guide Specifying default font for document windows You can select the font to apply to text in a new document window. When UniPoint is installed, the default font is Arial. Once in a document window, there are additional menu commands that allow you to change the font displayed in the table or graph. The default font is always used to display the text shown in the message line at the bottom of a document window. 1 To access the command for selecting the default font: a) Close all open document windows using the Close All command in the Window menu. (If it appears, the Navigator can remain onscreen.) b) From the File menu, choose Default Font. 2 When the Font dialog appears, use the drop-down list boxes to indicate font information. Font. Lists available screen and printer fonts. You can select the font in the list box or type its name in the text box. If there is a printer icon in front of a font name, the font is a printer font. The closest matching Windows font is used to display text onscreen. If there is a T T icon, the font is a True Type font. The same font is used on both the printer and on-screen. If there is no icon, the font is a screen font. The closest matching font is used when on-screen text is printed. Font Style. Lists available styles for the selected font, such as regular, italic, bold, bold italic. You can select the style in the list box or type its name in the text box. 5-10

78 common procedures Size. Lists available sizes for the selected font. You can select the size in the list box or type it in the text box. Sample. Shows a sample of text using the selected font, font style, and size. Script. Lists the available language scripts for the specified font. Pick the one appropriate for the language your computer is set up for. 3 Choose OK to accept the setting and exit the Font dialog. 5-11

79 unipoint user's guide Working in the table pane All document windows in UniPoint display a table in their window. UniPoint places information into the table when you set parameters in certain dialog boxes available within the document window. You can customize the table by adding or removing columns from display, changing the font, and specifying how text is aligned within columns. Displaying the table if it is not shown The table pane may not be displayed when you open a document window. To reveal the table pane: 1 From the Table menu, choose Show Table. Choosing the Show Table command again removes the table from display. 5-12

80 common procedures Sizing the table pane You can modify the size of the table pane so it takes up more or less of the document window s area. 1 Place the mouse pointer on the divider between the table and graph panes. The mouse pointer changes shape and becomes a two-headed arrow. 2 Click and drag the pointer up or down to manipulate the height of the table pane. 5-13

81 unipoint user's guide Changing column width using the mouse You can easily adjust the width of a column so all text within it can be seen: 1 Place the mouse pointer on the vertical line located on the right side of the column header whose column width is to be adjusted. The mouse pointer changes shape and becomes a two-headed arrow. 2 Click and drag the mouse pointer to the left to make the column narrower or to the right to make it wider. UniPoint saves column width information with the file so columns appear as you set them the next time you open the file. Setting column attributes In the table pane of a document window, you can set attributes for, rename, add, and remove columns. To display the Format Columns dialog: 1 From the Table menu, choose Format Columns. Or, double-click on one of the column headings of the table. 5-14

82 common procedures viewing column attributes To see the options set for a column: 1 Select the column in the Column Contents list box. The current attributes for the column appear in the text and drop-down list boxes, described below. Contents. The information found in the selected column. The types of contents that can appear in each column are set by Gilson. Heading. The descriptive text that appears at the top of a column and identifies the type of information that appears within the column. Width. The width of the column. Since it is not easy to know what width is required for columns containing text, you can use the mouse to adjust columns if the width is too large or small. The width set using the mouse inserts in this test box. See Changing column width using the mouse on page Factor. A number by which each value in a column is multiplied. Justify. How information in the column is aligned: left justified, right justified, or centered. Format. How numbers are displayed: decimal, with or without commas, or scientific notation. Decimals. The number of positions displayed after the decimal point. Password Protect (available in Operations and Re-Analysis windows only). Turns off/on password requirement for a column. This enables a user that does not have password authority to modify information in an Operations or Re-Analysis window without entering a password. The user can save the changes to a new file name, but not to the current file name. See the Password Protection appendix. changing an attribute To assign a new attribute to a column: 1 Select the column in the Column Contents list box. The current attributes for the column appear in the text and drop-down list boxes. 2 Use the text and drop-down list boxes to make the necessary changes. 3 Choose Change. 4 If you are done making modifications, choose Done to exit the dialog. Information in the column is formatted using the new attributes. 5-15

83 unipoint user's guide renaming a column To change a column heading: 1 Select the column in the Column Contents list box. 2 Type a new heading in the Heading box. 3 Choose Change. 4 If you are done making modifications, choose Done to exit the dialog. The table displays the new heading. inserting a column In the table, columns are inserted to the left of the column selected in the Column Contents list box. You can place the highlight after the last column to insert a column there. To add a column: 1 Select the appropriate column in the Column Contents list box. Or, click beneath the last column to add a column there. 2 Use the text and drop-down list boxes to set the attributes for the new column. 3 Choose Insert. 4 If you are done making modifications, choose Done to exit the dialog. The table displays the new column. removing a column To remove a column: 1 Select the column in the Column Contents list box. 2 Choose Delete. 3 If you are done making modifications, choose Done to exit the dialog. The table no longer displays the column. If you need the column later, use the inserting a column procedures. changing the position of a column To move a column: 1 Select the column in the Column Contents list box. 2 Click on the up and down buttons to change the position of the column in the report. 5-16

84 common procedures Changing the table font The size and appearance of the text shown in the table can be changed. To do this, use the Font dialog. 1 From the Table menu, choose Font. 2 When the Font dialog appears, use the drop-down list boxes to set font information. If necessary, refer to page 5-10 for a description of the areas of the Font dialog. Be aware that column and row headers are always shown in bold (darker) type. 3 Choose OK to exit the dialog. Manipulating table rows You can modify the location of rows in the table by deleting, copying, and pasting rows. You can also copy rows from the table of one document window to a different location in the same table or into the table of another document window of the same type. For example, you can copy the events from a control method into another control method. And, you can copy rows in an operations list and then duplicate them later in the same list. Note: Some table modifications via the Edit menu, such as deleting, are not available in all windows. UniPoint checks the information copied between windows and makes sure that the necessary parameters are set in the destination window. You see a message if UniPoint cannot place the information. For example, if you are copying events for a detector between two control methods, UniPoint verifies that the detector is named in the destination method. If the detector is not named, you must assign a description before UniPoint can paste the events. 5-17

85 unipoint user's guide deleting rows To remove one or more rows and place their contents in the Windows Clipboard: 1 Select the rows by clicking then dragging the mouse pointer to highlight them. Or, click on a row then choose Select All from the Edit menu. 2 Choose Cut in the Edit menu or press the CTRL+X key. This action not only deletes the rows but copies their information into the Clipboard. 3 To place the rows elsewhere, see pasting rows. To remove one or more rows without placing their contents in the Clipboard. 1 Click on a row in the table. 2 Choose Clear All from the Edit menu. copying rows To copy the information in one or more rows to the Clipboard: 1 Select the rows by clicking then dragging the mouse pointer to highlight them in the table. Or, click on a row and choose Select All from the Edit menu. 2 Choose Copy from the Edit menu. This copies the information into the Clipboard. 3 To place the rows elsewhere, see pasting rows. pasting rows Rows are inserted before the highlighted row. You can insert rows that were cut or copied from one document into the same window or into another window of the same type. To insert rows: 1 Click on the row before which the information stored in the Clipboard is inserted. 2 Choose Paste from the Edit menu. 5-18

86 common procedures Finding and replacing information You can locate and replace text in a table. For example, you could change each occurrence of an analysis method to another method in an operations list. finding text To locate text: 1 From the Edit menu, choose Find. 2 When the Find dialog appears, type the text to locate in the Find What box. 3 To further identify the text to locate, choose the Match the Whole Word Only box, the Match Case box, or both boxes. 4 To search an individual column, select it in the list box. If all columns shown in the list box are searched, select the Search All Columns check box. 5 Choose Find Next to begin the search. If UniPoint locates the text, it highlights the text. If UniPoint cannot find the text or has located the last occurrence of the text, it displays a message box telling you the text cannot be found. Remove the message box by choosing OK. 6 Repeatedly choose Find Next to locate any other occurrences of the text. 7 To close the dialog, choose Done. 5-19

87 unipoint user's guide replacing text To change text: 1 From the Edit menu, choose Replace. 2 When the Replace dialog appears, type the text to locate in the Find What box. 3 Type the replacement text in the Replace With box. 4 To further identify the text to locate, choose the Match Whole Word Only box, Match Case box, or both. Note: Not all columns shown in the table may be listed in the Search Column list box. The information in those columns is set by UniPoint and cannot be replaced with user-defined text. 5 Choose one of the command buttons. Command button Find Next Replace Replace All Function Searches for the word or text string. Changes the currently selected search text with the replacement text. Changes all occurrences of the search text with the replacement text. If UniPoint locates the text in the table, it highlights the text. If UniPoint cannot find the text or has located the last occurrence of the text, it displays a message box telling you the text cannot be found. Remove the message box by choosing OK. 6 To close the dialog, choose Done. 5-20

88 common procedures Working in the graph pane Most of the document windows available in UniPoint can display a graph in the lower pane of the window. The type of graph varies depending on the document window. Control and Analysis Method windows display a time line. Operations, Results, and Re-Analysis windows display a chromatogram plot. Calibration windows show calibration plots. Protocol and Routines windows do not have a graph pane. Document window for Control Method Following are procedures for displaying the graph pane and for sizing and moving the graph and if available summary graph within the pane. For further details on the graph shown in a particular document window, refer to the following: For information on: See page: Control method 6-1 Analysis method 7-1 Operations 8-1 Results 11-1 Calibrations 12-1 Re-Analysis

89 unipoint user's guide Displaying the graph pane if it is not visible The graph pane may not always be displayed when you open a document window. To show the graph pane: 1 From the Graph menu, choose Show Graph. Choosing the Show Graph command again removes the graph from display. Sizing the graph pane You can change the size of the graph pane. 1 Move the mouse pointer to the divider between the table and graph panes. 2 Move the pointer up or down to manipulate the height of the graph pane. 5-22

90 common procedures Sizing and moving the graph You can manipulate the size and change the location of the graph. sizing graph To enlarge or reduce the size of the graph: 1 Click within the graph. When selected, you see a sizing handle in the lower right corner. Note: If the legend is concealing the sizing handle, remove the legend by choosing Legend in the Graph menu. Or, move the legend, by clicking within it and dragging it to a new location. 2 Drag the sizing handle to the right, left, or diagonally to enlarge or reduce the size of the graph. moving graph To move the location of the graph: 1 Click within the graph to select it. 2 Drag the graph to the new location. Sizing and moving a summary graph In the graph pane of a Results or Re-Analysis window, you can view and manipulate a summary graph. The summary graph always shows the entire chromatogram plot even if you enlarge part of the graph. viewing summary graph To view and remove the summary graph: 1 Choose a Summary Graph command from the Graph menu. 2 Choose the command again to remove the summary graph from display. sizing summary graph To better view the traces shown in the summary graph, you can make it larger. 1 If the summary graph is not currently selected, click within its box. 2 Position the mouse pointer at the side or corner of the box. The mouse pointer changes to a two-headed arrow. 3 Click and drag the box to the desired size. 5-23

91 unipoint user's guide moving summary graph To change the on-screen location of the summary graph: 1 Click and drag the box to move it to the new location. Changing the font The size and appearance of text shown in the graph area, including the summary graph and legend can be changed. 1 From the Graph menu, choose Font (or Graph Font in some document windows). 2 When the Font dialog appears, use the drop-down list boxes to set font information. If necessary, refer to page 5-10 for a description of the areas of the Font dialog. 3 Choose OK to exit the Font dialog. 5-24

92 common procedures changing peak font Choosing the Peak Font command, available in the Graph menu of some document windows, enables you to change the size and appearance of peak names. In the Font dialog, two additional options are available. Rotation. Indicates the angle (0 to 360 ) at which peak names appear in relation to the X axis. Color. Identifies available color choices for displaying peak names. 5-25

93 unipoint user's guide Setting graph attributes The Attributes dialog provides for changing the appearance of lines or symbols shown in the graph. To display and set options in the Attributes dialog: 1 From the Graph menu, choose Attributes. Or, double-click on one of items shown in the legend, if it is displayed. 2 When the Attributes dialog appears, review and if necessary make changes to the attributes for a line or symbol. The options available vary depending on the document window. Legend. Identifies the lines and symbols currently shown in the graph. The types of lines and symbols vary between document windows. To change the attributes for a line or symbol select it from this list box. Display or Display Symbol/Line. If selected, indicates that the line or symbol is currently displayed in the graph. Size. Identifies the width of the line or size of the symbol in pixels. Color. Lists available color choices for the line or symbol. Display Peak Names. If selected, indicates that peak names will be displayed in the graph. Display Events. If selected, UniPoint displays run-time events (such as detector autozero) in the graph. Following are the symbols and text for run-time events. Text is black and displayed at a 45 angle. 5-26

94 common procedures Event Symbol Color/Fill Text Gradient* Detector Event light gray Displays Event Detector Autozero light gray Displays Zero Device Front Panel Query light gray Displays text retrieved from front panel Fraction Collection: Start green Displays tube designation, for example, TUBES:1 Fraction Collection: End red Displays tube designation, for example, FRACTIONS:1 User Pause: Start light red User Pause: End light red Synchronization: Start light gray Synchronization: End light gray *Note: A gradient line appears for each of the pumps used during the run. The color of the line is the same as that indicated for the pump in the control method. Fraction Sites. If selected, indicates that labels will be displayed in the graph for collected fractions. 3 Choose OK to accept any modifications and exit the dialog. Any change to a line or symbol is reflected in the graph and the legend. 5-27

95 unipoint user's guide Removing items from display To remove an item from display in the graph: 1 From the Graph menu, choose Attributes. Or, double-click on one of the items shown in the legend, if it is displayed. 2 When the Attributes dialog appears, select the description for the item in the Legend list box. 3 Remove the X from the Display check box. 4 Repeat steps 2 and 3 for any additional items to remove. 5 To disable display of peak names, remove the X from the Display Peak Names check box. 6 To disable display of run-time events (such as detector autozero), remove the X from the Display Event check box. For a description of run-time event symbols and lines, see page Choose OK to accept any modifications and exit the dialog. The items are no longer displayed. To redisplay the items, access the Attributes dialog and replace the X in the Display check box for each item. Displaying/removing gridlines You can turn on and off the display of gridlines in the graphs of Results, Calibration, Operations, and Re-Analysis windows. 1 In the Graph menu of the document window, choose one of the axis commands. For the Calibration window, the commands are Amount Axis (for the Y axis) and Area/Height (for the X axis). For the other three windows, the commands are Signal Axis (for the Y axis) and Time Axis (for the X axis). 2 When the dialog box appears, remove or insert an X in the Major or Minor check boxes and choose OK. 3 Repeat the above for the other axis. 5-28

96 common procedures Legend The legend identifies what the lines and symbols represent in the graph. UniPoint automatically sizes the legend box so all items are displayed. Note: For a list of run-time events whose symbols are not identified in the legend but may be shown in the graph of Operations, Results, and Re-Analysis windows, see page displaying legend To see the legend if it is not displayed: 1 From the Graph menu, choose Legend. Choosing the Legend command again removes the legend. moving legend To change the on-screen location of the legend: 1 Click on a white area within the legend box. 2 Drag the box to the new location. changing attributes To modify the attributes for an element shown in the legend, see page

97 unipoint user's guide Annotation All document windows can display an annotation box that lists information for the file being viewed. For example, in the annotation for a control method, you see configuration information for and parameters set in the method file. In all windows except Results and Calibration, you can add comments to the annotation for a file; see Using the notes or log file text editor on page 5-34 for more information. The annotations for UniPoint files used during an HPLC run are placed in and can be printed with the analysis report. displaying and removing annotation To see the annotation: 1 From the Window menu, choose Annotation. Choosing the Annotation command again removes the annotation. sizing annotation box To better view text in the annotation, you can make the box larger. 1 If the annotation is not currently selected, click within it. 2 Position the mouse pointer at the side or corner of the box. The mouse pointer changes to a two-headed arrow. 3 Click and drag the box to the desired size. moving annotation box To change the on-screen location of the annotation box: 1 Click and drag the colored bar at the top of the box to move the entire box to the new location. 5-30

98 common procedures changing font To select a different font for annotation text: 1 Position the mouse pointer in the annotation box. 2 Click the right mouse button. 3 When the menu appears, click on Set Font. 4 When the Font dialog appears, use its options to indicate font information. If necessary, refer to page 5-10 for a description of the areas in the Font dialog. 5 Choose OK to exit. copying text To copy text in the annotation to the Windows Clipboard. 1 To select a portion of text, move the mouse pointer to the left of the text to select. Click and drag to highlight the desired text. 2 Click the right mouse button. 3 When the menu appears, choose Copy or Copy All. 5-31

99 unipoint user's guide Manipulating the toolbar Each document window has a toolbar that provides quick access to some options available in the document window. selecting defaults You can choose whether or not to automatically display the toolbar when a document window is created and how many columns it should have. 1 Choose Options from the File menu. 2 If necessary, click on the Toolbar tab. 3 Use the options under the Toolbar tab to set the appearance of the toolbar when a document window is created or opened. Tool Arrangement. If you select None, no tools are displayed when a document window is created or opened. (You can still display the tools in a document window by selecting the Toolbar command from the Window menu.) If you select Floating Palette, the tools appear within a sizeable and moveable box. If you select Fixed Ribbon, the tools appear in a horizontal bar beneath the menu bar. Number of Columns (for Floating Palette arrangement). A value of 1 displays a vertical arrangement on-screen for any document window that is created or opened. Selecting a large number (for example, >15) displays a horizontal arrangement. 4 Choose OK to save the settings and exit the dialog box. The next time you open or create a document window, UniPoint uses the conditions set in the dialog box to display the toolbar. 5-32

100 common procedures removing toolbar To remove the toolbar when viewing a document window, choose Toolbar from the Window menu. (Or double-click on the control menu box in the upper left corner of a floating palette toolbar). Choose the Toolbar command again to restore the toolbar. sizing toolbar While in a document window, you can manipulate the size of a floating palette toolbar. 1 Position the mouse pointer at the side or corner of the box. 2 Click and drag the box to modify its size. UniPoint displays all of the available icons no matter how small you make the box. It can display the icons in a vertical or horizontal arrangement or in multiple columns. moving toolbar To change the on-screen location of a floating palette toolbar: 1 Click and drag the colored bar at the top of the box to move the box. displaying tool hints When the Display Tool Tips check box is selected under the Actions tab of the Options dialog (page 5-32), you see a tips box when you move the mouse pointer onto a tool icon in the tool bar. The picture at the left shows the Navigator tool and its tips box. 5-33

101 unipoint user's guide Using the notes or log file text editor A text editor like the one below appears when you do one of the following: Choose Notes from the Edit menu. (This command is not available in the Results and Calibration windows.) Any comments specified in the text editor appear at the bottom of the annotation portion of analysis reports. Open a log file (.GLG extension) generated from a run. (Modifications made to the log file in the editor are not saved to the file.) adding text To add information: 1 Point and click to position the insertion bar in the text editor box. 2 Type the text. Use the BACKSPACE key if you make a mistake and retype the correct text. editing text To modify existing text: 1 Point and click to position the insertion bar to the right of the text to change. Press BACKSPACE to remove text. Newly typed text is added at the insertion bar. Or, click and drag to highlight the text to change. Newly typed text automatically replaces the highlighted text. copying text To copy existing text: 1 Select the line(s) to copy. Select all text. Choose the Select All button. (Or, press CTRL+A.) Select portion of text. Move the mouse pointer to the left of the text to select. Click and hold the mouse button. Drag the mouse to highlight the desired text. 5-34

102 common procedures 2 Choose Copy. (Or, press CTRL+C.) The selected text is copied to the clipboard and can be inserted elsewhere in the annotation (see inserting text, below) or in another Windows document. inserting text To insert text: 1 Point and click to position the insertion bar at the end of the line after which you want a line inserted. You may need to press the right arrow key to move the cursor after the last character in the line. 2 Press ENTER. 3 Type the new line of text. Or, if you have copied or cut text to the Clipboard, choose Paste. (Or, press CTRL+V.) deleting text You can delete all or some of the text in the editor. To do this: 1 Select the line(s) to delete. Select all text. Choose the Select All button. (Or, press CTRL+A.) Select portion of text. Move the mouse pointer to the left of the text to select. Click and hold the mouse button. Drag the mouse to highlight the desired text. 2 Press the DELETE key. (Or, press CTRL+X to remove the text and copy it to the Clipboard.) changing font To change the font shown in the annotation: 1 Choose the Select All button. (Or, press CTRL+A.) 2 Choose the Font button. 3 When the Font dialog appears, modify the font selections and choose OK. (See page 5-10 for a description of the Font dialog.) printing To send the text to the printer: 1 Choose the Print button. 2 When the Print dialog appears, verify the selected options and make any necessary changes. 3 Choose OK to print. 5-35

103 unipoint user's guide sizing text editor You can enlarge or reduce the size of the text editor box. 1 Position the mouse pointer at the side or corner of the box. The mouse pointer changes to a two-headed arrow. 2 Click and drag the box to the desired size. pasting information into windows program One of the useful benefits of the Microsoft Windows user interface is the ability to share text among Windows programs. Information is copied (or clipped) from one program and pasted into another using an intermediary program called the Clipboard. To transfer text: 1 While viewing the editor, select the information to copy. Select all text. Choose the Select All button. (Or, press CTRL+A.) Select portion of text. Move the mouse pointer to the left of the text to select. Click and hold the mouse button. Drag the mouse to highlight the desired text. 2 Choose Copy. (Or, press CTRL+C.) 3 Choose Done to close the text editor. 4 Choose Minimize from the Control menu (or click on the Minimize box) to iconize UniPoint. 5 Open the Windows program. 6 Position the cursor within the window and choose Paste from the Edit menu. (Or, press CTRL+V.) 5-36

104 common procedures Using the browse feature within dialogs In many dialog boxes, there is a Browse button. This button enables you to select a file name to enter in one of the text boxes or to link to that file so the software can get information from it. Browse allows you to search the computer or a network disk to locate the appropriate file or information in a file. browsing for file To use the browse feature to select a file: 1 Click within the text or list box that requires a file name or a link to another file and then click on Browse. (Or, double-click in the text box.) Below is an example Step Entry dialog used to set up the operations list for a run. 2 When the Browse dialog appears, use options in the box to choose the appropriate file. An example dialog is shown below. 3 After you select the file(s), choose OK. The first dialog re-appears and the path and file name for the file(s) appear. Note: When browsing for protocol files, you can select multiple files in the Browse dialog and place the file descriptions in the control method. To select more than one file, press the CTRL key while clicking on each file name. To select a range of files, click on the first file; then while pressing the SHIFT key, click on the last file name. 5-37

105 unipoint user's guide Customizing the UniPoint workspace The workspace is the main work area used by UniPoint. It displays the document windows, or their icons, that are currently open. When you have multiple document windows, you can size the windows and arrange them to suit your needs. Once the workspace is organized, the software can automatically save the space when you exit or you can save the arrangement as a workspace file. When the workspace is saved, UniPoint not only saves the settings for the workspace but the name of the file that was open within a document window. The next time you open the workspace file, both the document window and the associated file are displayed. Any windows that are open when you restore a workspace have no effect on the workspace being recalled. If your workspace has multiple document windows open, it is important that you know which window is active. The items in UniPoint s menu bar change depending on which type of document window is active. In other words, a Control Method window has a different menu bar than an Analysis Method window or any other type of document window. Arranging elements in a workspace arranging document windows You can quickly arrange all open document windows so that all or part of each one is visible. 1 From the Windows menu, choose Cascade or Tile Horizontal or Tile Vertical. Cascade staggers the document windows so you can see each one s title bar. Tile Horizontal sizes and stacks the windows. Tile Vertical sizes and places the windows side-by-side. 5-38

106 common procedures arranging icons UniPoint can arrange icons for opened document windows, or you can position them yourself. To arrange icons automatically: 1 From the Windows menu, choose Arrange Icons. UniPoint aligns the icons for any open document windows along the bottom of its workspace. To arrange icons manually: 1 Drag each icon to its new location. Saving a workspace You can have UniPoint store the appearance of the workspace when the software is exited and recall it the next time the software is started. You can also save a workspace in a file so it can be retrieved by you at any time. automatically saving and recalling workspace The Save and Restore Workspace feature turns on or off the ability to recall the last workspace when the software is started. When the feature is active and you choose Exit in the File menu, the location of any open document windows and icons, and their files, is stored by UniPoint. The next time you start the software, it displays the windows and icons in the same location as before. This feature is turned off when you install the software. To turn on the Save and Restore Workspace feature: 1 Choose Options from the File menu. 2 When the Options dialog appears, click on the Actions tab. 3 Choose the Save and Restore Workspace check box. 4 Choose OK. 5-39

107 unipoint user's guide saving workspace file To save the appearance of the current workspace to file: 1 From the File menu, choose Save Workspace. 2 When the Workspace Save dialog appears, use its options to specify a name and storage location for the file. 3 Choose OK. Opening a workspace file To display a previously saved workspace: 1 From the File menu, choose Open. 2 When the Open dialog appears, use its options to locate and select the file. 3 Choose OK when the desired name appears in the File Name box. When you open the workspace file, the document windows appear in the same place they were located when the workspace file was saved, and any associated files are also opened. Any windows on-screen when you open a workspace remain open but may be hidden by the workspace. 5-40

108 common procedures Printing UniPoint supports printers with Windows drivers. You indicate printer information when installing Microsoft Windows onto the computer. Refer to the Microsoft Windows User s Guide for information on setting printer information. Before printing a document: Check that the columns in the table pane are wide enough so that the all of the information within them can be seen. Make sure the items in the graph pane are arranged and sized the way you want them. Verify that connections exist between the printer and computer. Check that the printer is turned on and has paper in it. Also, check the printer ON LINE (or equivalent) selection to make sure it is active. Specifying printer setup options To select and if necessary set options for the printer to use when generating printouts: 1 From the File menu, choose Print Setup. 2 When the dialog appears, indicate the printer options. You install printers and indicate the default printer using Microsoft Windows. See Microsoft Windows User s Guide for details. If you click on Options, another dialog displays so you can control default printer settings for the selected printer. The options in the setup box vary depending on the printer. Refer to your printer documentation or click on Help, if that button is available. 3 Choose OK. 5-41

109 unipoint user's guide Selecting what to print and starting the print To print information from a document window: 1 Choose the Print command from the File menu. 2 When the Print dialog appears, make sure the appropriate printer is indicated at the top of the dialog. To select a different printer, choose Setup. 3 Select what you want to print using the options in the dialog box. The options available vary depending on the document window, as you can see in the example dialogs below. 4 Choose OK to start the printing process. 5-42

110 common procedures changing layout options To review the layout options used to print a document: 1 Choose Layout from the Print dialog. 2 Use the options in the Page Layout dialog to specify page margins, size of graph, and order to print annotation, graph, and table. Margins. In the text box next to each margin (left, right, top, and bottom), type the width of the margin. The indicated width is added to the default print area set for the page by the printer. Refer to the document supplied with the printer if necessary. Note: The measurement system, mm or inches, is set using the International program in the Windows Control Panel. If you change the measurement system, it is used the next time you open UniPoint. Graph Height. Indicate the size of the graph as a percentage of the page. It is printed on a different page than the annotation or table if necessary. Position Order. The order in which the various parts shown in the document window are printed, by default: annotation, graph, table. 3 Choose OK to save parameters and return to the Print dialog. 5-43

111 unipoint user's guide Transferring information between files UniPoint allows you to transfer information between two control methods, two analysis methods, or two operations lists that are on-screen at the same time. You can also copy text from a UniPoint document window into a Windows word processing program, such as Write. The graph displayed in a document window can be copied and pasted into a Windows drawing program, such as Paintbrush. copying rows of text between UniPoint windows To copy the information in one or more rows for inclusion in another document window s table: 1 Select the rows by clicking then dragging the mouse pointer to highlight them in the table. Or, click on a row then choose Select All from the Edit menu. 2 Choose Copy from the Edit menu. This copies the information into an intermediary Windows program called the Clipboard. 3 In the designated document window, click on the row before which the copied row(s) are inserted. (If no rows exist, click on the first row.) 4 Choose Paste from the Edit menu. Note: UniPoint checks the information copied between windows and makes sure that the necessary parameters are set in the destination window. You see a message if UniPoint cannot place the information. For example, if you are copying events for a detector between two control methods, UniPoint verifies that the detector is named in the destination method. If the detector is not named, you must assign a description before UniPoint can paste the events. copying text into another Microsoft Windows program To copy text into a Microsoft Windows word processing program: 1 While viewing document window, select the table rows to copy, as described above. Or, copy one or more cells, as described below. A cell is the rectangle at the intersection of a column and row. a) To select one cell, move the mouse pointer ( ) so it is positioned over the cell and click the left mouse button. To indicate that the cell is selected within the highlighted row, the cell has a box around it, as shown in the following diagram. 5-44

112 common procedures To select a range of cells, click and drag the mouse pointer over the cells. The selected cells have a box around them, as shown in the following diagram. b) Click the right mouse button to display the menu shown at the left. c) Choose the Copy Cells command. The Copy Cells command copies the contents of the selected cell(s) into the Windows Clipboard. 3 Once the text has been copied to the Clipboard, choose Minimize from the Control menu (or click on the minimize box) to iconize UniPoint. 4 Start the Windows program. 5 Position the cursor within the window and choose Paste from the Edit menu. copying graph into another Microsoft Windows program To copy the graph into another Microsoft Windows program: 1 Manipulate the appearance of the graph as described earlier in this section. 2 Click on the graph so it is selected. 3 Choose Copy from the Edit menu. This copies the graph into the Clipboard. 4 Start the Windows drawing program. If you are pasting into Paintbrush, maximize the application window. Also enlarge the work area by removing the tools and palette from display. Choose the appropriate commands in the View menu to do this. 5 Choose Paste from the Edit menu. 6 Modify the image as necessary. 5-45

113 unipoint user's guide Getting software version number To view version information for UniPoint: 1 Choose About from the Help menu. 5-46

114 6 Control Methods In this section, you learn how to set and modify control method conditions. A control method contains system configuration information and describes a procedure to execute during a chromatography run. In the control method, you identify the Gilson devices in the HPLC system and indicate time-based commands to send to them during a run. These commands can include such things as setting mobile phase flow rate and concentration, detector parameters, and data collection start and stop. Control methods can also be created that get system components up and running. Other control methods may shut down HPLC devices at the end of the day or at the detection of a system error. To control HPLC devices, you need to do the following: assign a description, or use the default description assigned by Gilson, indicate any additional settings required for device control, and send timed events to, or wait for input from, the device. The procedures for doing the above are outlined in this section. Plus there is information about the Control Method window. 6-1

115 unipoint user's guide Control Method window Using the options in the Control Method window, you specify method conditions for controlling devices. You access this window by creating a control method file or by opening an existing file. The following illustration shows an example Control Method window, with events already set. Refer to Section 5, Common Procedures for more information on manipulating parts of the table or graph displayed in a document window. menu bar toolbar table pane graph pane annotation box legend box status bar menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Menu File Edit Device Event Table Graph Window Help Description Create, save, print, delete files or exit a window or UniPoint. Locate, make changes to, or transfer information. Name installed devices and other resources used by the method. Issue timed instructions to devices. Display or modify appearance of the table pane. Display or modify appearance of the graph pane. Re-arrange windows or icons, or activate a window; display file annotation or toolbar. Learn about UniPoint. 6-2

116 control methods table pane Lists the timed events currently set for the control method. If you see the message **invalid command** in the table, the device for which a command was previously set is not listed in the configuration for the control method. Before executing the method during a run, check the configuration for the control method using the System Configuration command in the method's File menu. Double-clicking on a row for a timed event automatically displays the dialog box used to set up the event. Using the right mouse button, you can double-click on a row that specifies the timed event for a protocol and display the window that shows the instructions set in the protocol. Double-clicking on the top (header) row displays a dialog for changing the format of the columns shown in the table. graph pane toolbar Shows a graphic representation of timed events and mobile phase concentration information set in the method. Provides quick access to some options available in the Control Method window. To activate a tool, just click on its icon. navigator tool open tool save tool print tool mobile phase tool data channels tool contact tool injector tool detector tool fraction collector tool generic device tool system tool Displays Navigator. Displays Open dialog. Stores method to disk. If it has not been saved, displays the Save As dialog. Displays Print dialog. Displays Mobile Phase Event dialog. Displays Data Channels Event dialog. Displays Contact Event dialog. Displays Injector Event dialog. Displays Detector Event dialog. Displays Fraction Collector Event dialog. Displays Generic Device Event dialog. Displays System Event dialog. To turn off/on the toolbar, choose Toolbar from the Window menu. 6-3

117 unipoint user's guide annotation box Displays configuration information for and parameters set in the control method. To turn on/off the annotation, choose its command from the Window menu. To add your own comments, use the Notes command in the Edit menu. Any comments specified in the text editor appear at the bottom of the annotation portion of analysis reports. legend box Identifies the symbols shown in the graph. To turn on/off the legend, choose its command from the Graph menu. By double-clicking on an item in the legend, you can access the Attributes dialog. Use that dialog box to remove events from display or to change the size of symbols and lines shown in the graph. Note: As a default description, UniPoint assigns Injector to an installed injector and its dilutor. That is why Injector is listed twice in the legend: once for the injector and once for the dilutor. status bar Displays help message if a menu command is highlighted. 6-4

118 control methods Checking configuration file To control Gilson devices connected along the GSIOC, UniPoint must know about those devices. It finds this information in the configuration file. If you are running multiple HPLC systems from UniPoint, check that the default file is appropriate before you create a control method. 1 If necessary, close any open document windows (the Navigator, if it appears, can remain on-screen.) 2 From the File menu, choose Default System Configuration. The Default System Configuration dialog appears. Its list box identifies the devices that the software thinks are currently connected along the Gilson Serial Input/Output Channel (GSIOC). The path to and name of the configuration file are listed at the bottom of the dialog box. 3 Use the options in the dialog box to modify or select a different configuration file. See System Configuration appendix for details. link between configuration and method When you save a control method, the path, name, and information in the configuration file is stored in the control method file. Each time you open the control method, UniPoint searches for the associated configuration file and checks the information in that file against the information in the control method. Refer to Configuration checks on page A-7 if you see a message box indicating that the configuration information does not match. 6-5

119 unipoint user's guide Assigning descriptions In the Control Method window, use the commands in the Device menu to review and assign descriptions to system components. For all resources except data channels and contacts, UniPoint assigns a default description when the device is identified in the method s configuration file. You can review and modify these Gilson-assigned descriptions to meet your needs. Whenever the device is referred to in UniPoint, it is identified by its description. For example, when specifying your own descriptions for pumps, the description can identify the fluid being pumped, such as, Acetonitrile. For data channels, you can identify the wavelength and sensitivity for each channel, for example, 254 nm at 0.1 sens. Note: A default description may not be assigned if you add a device to the configuration file after the control method has been created or opened. You therefore need to specify a description before issuing events to the device. To access the dialog box for assigning or changing a description: 1 Choose the device or resource from the Device menu. The Generic command enables you to set descriptions for additional devices not included under one of the other commands, such as 506C System Interface, 817 Valve Actuator, or 831 Temperature Regulator. Following are example dialog boxes used to set descriptions. The appearance of the dialog boxes and procedures for setting descriptions are the same. The top area of the dialog is used for description assignment and modification. As you can see in the examples, additional areas and options may be available depending on the device. See Additional device control parameters on page 6-9 for details. 6-6

120 control methods specifying description To assign a description if one has not been set: 1 Choose the device name in the Unit ID/Name list box. 2 Type a description in the Description box. Or, you can type the same text that appears next to the Name field if it meets your needs. You can use upper and lower case letters, numbers, and spaces in the description. 3 Click on the device name in the Unit ID/Name list box to enter the description. UniPoint assigns the description Injector to an injector and its dilutor. This combines their command sets and makes issuing timed events to them easier. You can assign different descriptions to the injector and dilutor and separate their command sets. The only devices that can be assigned the same description are an injector and its dilutor. modifying description To change a description: 1 Click on the device name in the Unit ID/Name list box. 2 Type the new description in the Description box. 3 Click on Change to update the list box. 6-7

121 unipoint user's guide removing description To delete a description for a device not used in the method, refer to the steps below. For example, if you have an HPLC system with three pumps but only two are required for the method, remove the description for the unused pump. Gilson recommends that you do not remove the pump from the configuration associated with the method. 1 Click on the device name in the Unit ID/Name list box. 2 If necessary, highlight the text in the Description box. 3 Press DELETE or BACKSPACE to remove the description. 4 Click on the device name in the Unit ID/Name list box to update the list box. You see a message box if you have removed a description for a device which has been issued timed events. Choose Yes to remove the events for the device. Choose No to keep the events. (You see **invalid device** and **invalid command** in the table. Before using the method in the run, you must enter a description for the device.) Choose Cancel to return to the dialog so you can re-enter the description. exiting dialog When you are finished specifying descriptions: 1 Click on OK to save the descriptions. Depending on the dialog box, you may see a message box if additional parameters are required before you can exit the box. These parameters are described in Additional device control parameters, below. 6-8

122 control methods Additional device control parameters In some of the dialog boxes used for setting device descriptions, you see elements for identifying additional parameters. These parameters identify such things as mobile phase conditions and the names of protocols and tray files. Mobile phase settings For each mobile phase, set compressibility and refill parameters. 1 Choose Mobile Phase from the Device menu. 2 Check the descriptions assigned to each pump. If you need information on changing a description, refer to Assigning descriptions on page 6-6. UniPoint assigns default descriptions to the pumps based on their unit IDs. For example, if the pump s ID is 1, its description is Pump 1. You may want to modify the description to reflect the liquid being pumped, for example, Water or Acetonitrile. 3 Choose the Settings button once the appropriate descriptions have been set. 4 When the Pump Settings dialog appears, select the name of the pump in the list box. 6-9

123 unipoint user's guide 5 Indicate or modify values in the text boxes, described below. When finished, choose OK to accept the values. Or, choose Cancel to revert to previous settings. Compressibility. How much a mobile phase can be compressed when placed under pressure. The software uses the compressibility setting to correct the flow characteristics of the system to reflect the mobile phase being pumped. Use the compressibility values (in Mbar -1 or 10-5 MPa) listed below. Temperature Liquid 20 C 25 C 30 C 40 C Water Benzene Chloroform Methylene Chloride Carbon Tetrachloride Ethanol Acetone Methanol n-heptane n-hexane Diethyl Ether Acetonitrile* Tetrahydrofuran* Water:Methanol, 10:90* Water:Methanol, 20:80* Water:Methanol, 40:60* Water:Methanol, 50:50* Water:Methanol, 60:40* Water:Methanol, 80:20* From the Handbook of Chemistry and Physics, CRC Press, 60 (1979). * Experimentally determined by Gilson. Water:Methanol mixtures are volume:volume mixtures. Refill. The duration (125 to 999 ms) of one piston refill stroke. For analytical flows (< 10 ml/min), a fast refill of 125 is usually appropriate. For preparative flows (>10 ml/min), use a value in the range of 250 to 400. Slower speeds may be necessary when pumping viscous mobile phases. Inlet Pressure P0. (For users of UniPoint Software with SF Pressure Control, ordered separately) After setting information for the supercritical fluid pump, choose the SF Setting button. In the Inlet Pressure P0 text box, type the pressure at the inlet of the supercritical fluid pump head. It is the vapor pressure of CO 2 at ambient temperature, typically 6 MPa (60 bar or 870 psi) at 22 C. A table of inlet pressures at other temperatures is available in the UniPoint on-line help, accessed via the Pump Settings dialog. 6-10

124 control methods Data channel parameters UniPoint can acquire and display several channels of data during a run. You need to identify the channels being acquired and set initial run-time scaling options for those channels. Then you can set timed events for starting and stopping data collection via those channels, as described on page To acquire data, you must make the appropriate connections in the HPLC system. See the UniPoint System Installation Guide for details. Or, if you have a 170 Detector, refer to its user's guide. If you have a Gilson model 116 through 119 Detector, up to three channels of data are sent along the GSIOC cable. Specify a description for each detector channel being collected. For a fourth channel of data to be transferred, make an analog connection between the 116 through 119 Detector and the other detector. Specify a description for the 116 through 119 analog input being used. For a Gilson 112, 115, 122, or 133 Detector or a non-gilson detector, make the analog connections between the detector and a Gilson 506C System Interface. Then set descriptions for each 506C analog channel being collected. For a Gilson 170 Detector, you can monitor up to 8 data channels via the RS-232 connection between the computer and the 170 Detector. Specify a description for each data channel to be monitored. setting channel information To indicate descriptions and scaling options for each data channel, refer to the procedures below. These options control the appearance of channel data collected and shown on-screen at run time. Note: If data is being collected via a 170 Detector, additional options are available in the Data Channel Options dialog. For information on those options, refer to the user's guide for the detector or UniPoint's on-line help. 1 Choose Data Channel from the Device menu. 2 Indicate a description for each channel to acquire. If necessary, refer to Assigning descriptions on page Choose the Options button. The Data Channel Options dialog appears. 6-11

125 unipoint user's guide 4 Select the name of the data channel in the list box. 5 Indicate values in the areas of the dialog box, described below. 6 Repeat steps 4 and 5 for each channel. Then choose OK to accept the settings. Or, choose Cancel to revert to previous settings. Following are descriptions of the areas in the Strip Chart Options dialog. The auto range and display time options pertain to all data channels being collected. The other options relate to the data channel whose description is currently highlighted in the list box. Auto Range. If this check box is selected, UniPoint automatically plots data to the minimum and maximum peak height determined across all collected data channels. Selection of this box overrides any other limits set for individual data channels in the dialog box. Channel Scale Parameters. Lists the data channels currently set for the control method. Select the channel to review or modify its options. Data Rate. The number of data points collected per second for all channels during the run. The maximum value is 80 points per second. However, the default of 20 is appropriate for most runs. Auto Zero. Select this option to set the first detected data point to zero, at the start of data collection. % Offset. Use this parameter to move the channel s trace away from the horizontal axis (and from other channel traces) on the screen and on the strip chart printout. Express the offset as a percent displacement from the horizontal axis (0%). The default value is 0% offset. Minimum and Maximum. The lower and upper limits for the signal axis displayed on-screen during the run. Be aware of the following: If the Auto Range check box is selected the software disregards these values and uses the minimum and maximum heights for all channels to determine the signal axis. If Auto Range is not selected and you indicate different minimum and maximum values for each channel being collected, the software uses the lowest and highest values entered to determine the signal axis. For example, if 0 and 10 mv were entered for the minimum and maximum values for the 254 nm channel and 0 and 100 mv were entered for the 280 nm channel, the signal axis would be 0 to 100 mv. Scale. How UniPoint scales the channel s data during the run: mv or user units. If you indicate a scale other than mv for a channel, make corresponding changes to the scaling for each of the other channels being collected. 6-12

126 control methods Display Time. The time period to display on the X axis of the strip chart display (shown in the graph pane of the Operations window). mvolt (displayed for User Units scale). The number of mvolts that correspond to the value indicated in the User Units text box. User Units (displayed for User Units scale). The number of user units that correspond to the value indicated in the mvolt text box. Label (displayed for User Units scale). A description of the units. customizing appearance of graph To review and modify the color assigned to data channel traces and turn on or off gridlines in the graph: 1 From the Data Channel Options dialog, choose Attributes. 2 When the Attributes dialog appears, select a channel from the list box and check the options for it. If necessary, make any necessary modifications. When finished, choose OK or Cancel. Data Channel. Lists the description for each data channel. Style. Lists available styles for the line: solid, dashed, or dotted. Color. Lists available color choices for the line. Thickness. Identifies the width of the line in pixels. Signal Grid. Indicates which gridlines appear for the Y axis. Major gridlines are aligned with numbered tick marks while minor gridlines are positioned between the numbered tick marks. Time Grid. Indicates which gridlines appear for the X axis. Major gridlines are aligned with numbered tick marks while minor gridlines are positioned between the numbered tick marks. 6-13

127 unipoint user's guide Injector options Additional control parameters for a Gilson injector and its dilutor may include specifying a protocol file and tray file. These conditions are set in the Injector dialog (Device menu/injector command). Additional control parameters for an injection pump include compressibility and refill rates. To see what additional command buttons may be available for a device, choose the description for the device in the Devices list box. protocol file An injector protocol consists of a set of untimed sequential instructions executed by an injector and its dilutor. When the control method is executed during a run, UniPoint starts the injector protocol at the indicated time point. Once the protocol starts, it executes independently from the control method unless synchronization events have been set between the two. See Protocols section for more information on creating a protocol and synchronizing it with a control method. To select the protocol file(s), use the Browse button. To execute a protocol when the control method is being run, specify a timed event for it. See page tray file A tray file identifies the groups of tubes in the rack(s) installed on an injector. When you issue a command using the Event menu that moves the injector s needle, you must indicate what position it moves to. That position is specified in the associated tray file. Choose the Tray Editor button to access the Tray Editor Software. That software enables you to set up the file or use its File menu to select the name of an existing tray file. See Tray Editor section for more information. 6-14

128 control methods injection pump options When you choose the name of an injection pump in the Devices list box, you see an Options button. Clicking on this button accesses a dialog for setting refill and compressibility. Refer to page 6-10 for details on these parameters. Detector options You can set detection parameters as timed events or in a generic protocol file. See Protocols section. Use the Browse button in the Detector dialog to select the file(s). To execute a protocol when the control method is being run, specify a timed event for it. See page Fraction collector options Additional control parameters for a fraction collector may include specifying a protocol file and tray file and setting channel and mv full scale information for peak collection. 6-15

129 unipoint user's guide protocol file You can set collection mode parameters as timed events or in a generic protocol file. See Protocols section for information on creating protocols. Also, be aware that Start and Stop Collection commands must be timed events in the control method. To select the protocol file(s), use the Browse button. To execute a protocol when the control method is being run, specify a timed event for it. See page tray file A tray file identifies the groups of tubes in the rack(s) installed on the fraction collector. When you issue a command that moves the collector s dispense head, you must indicate what position it moves to. That position is specified in the associated tray file. Choose the Tray Editor button to access the Tray Editor Software. That software enables you to set up the file or use its File menu to select the name of an existing tray file. Refer to the Tray Editor section for more information. monitor channel, mv full scale, and delay volume Choose the Options button to access the Fraction Collector Options dialog. In this dialog, indicate the data channel to monitor and the mv full scale of the analog signal sent to the fraction collector if you are collecting fractions by peak. The mv full scale value specified in this dialog box must be the same as that set in the Data Channel Options dialog; see page Using the Delay Volume text box, you can set the amount of eluent to be diverted to waste before collection into the current fraction site during the run. For a table listing delay times for specific lengths of tubing, refer to UniPoint s on-line help. Interface only and contact event option Within a control method, you indicate the timed events to issue via contacts. As a default, the Contacts dialog only shows contacts available on the 506C System Interface. To see all contacts available for Gilson devices, remove the X from the Interface Only check box. If necessary, you can then assign descriptions to any other contacts being used. 6-16

130 control methods After naming an input contact, select the Event check box if you want an event mark to appear in chromatograms. UniPoint automatically generates the event mark when the contact goes from an open to a closed state. On the chromatogram plot, UniPoint places a triangle and the contact description at the time of the event. High and low temperature limits If you have a Gilson 831 Temperature Controller installed, UniPoint can monitor for high and low temperatures. 1 Choose Generic from the Device menu. 2 Choose the 831 Temperature Controller in the list box and choose Options. 3 When the Generic Device Options dialog appears, select the temperature controller in the list box. Then indicate temperature limits in the text boxes. 4 When finished choose OK to accept the values. Or, choose Cancel to revert to previous settings. What UniPoint does when it detects a temperature error is set in the Error handling dialog. Refer to Error handling on page

131 unipoint user's guide Scheduling timed events Each command to issue at a specified time during a run is referred to as an event. You can specify events that set the gradient profile, start and stop data collection, run a protocol file that controls the injector, or send or wait for a contact signal. Using the dialogs within the Event menu, you tell UniPoint when to issue commands to Gilson devices via the Gilson Serial Input/Output Channel (GSIOC). The GSIOC is a communications channel that connects UniPoint to Gilson devices such as pumps, detector, injector and fraction collector. You can display an Event dialog box by doing one of the following: 1 Choose the device from the Event menu. Or, click on an event tool in the control method s toolbar (see page 6-3). Or, double-click on an event in the control method s table. Hint: While an Event dialog box is on-screen, you can quickly access the dialog for another device type by clicking its tool in the toolbar. The appearance and procedures for setting events within each dialog box are similar. Following are example Event dialogs. 6-18

132 control methods Within an Event dialog box, there is a Time box for indicating the time point at which to issue the event and a Comment box for entering an optional user description of the event. If you enter a comment, it appears in the status box at run time. Depending on the device, UniPoint displays a combination of various text boxes, option buttons, and list boxes. You use those boxes to specify the device, command, and any command parameters. The commands available for each device are described later in this section. When sending commands to the same device, allow enough time for the device to perform the operation indicated by the command before issuing it another command. As a suggestion, separate the commands by a minimum of 0.1 min. If you indicate more than one event, for different devices, at the same time point, they are sent in the order they appear in the table. The up and down arrows in the upper right corner enable you to step through events for the device type listed in the dialog s title bar. The text next to the arrows reads New Event if a new event is being inserted, or it reads Event x of x to identify the event whose information is shown. adding event To add a timed event: 1 If necessary, clear information from some of the text boxes (such as the Comment box) by choosing New. This also displays New Event next to the up and down arrows in the upper right corner of the dialog. 2 Indicate time and command parameters. Note: Instead of entering a value in the parameter box for most commands, you can enter an expression consisting of a variable and mathematical operations. For example, instead of specifying the wavelength value for the detector, you could indicate Wavelength. When you set the operations list for the run, UniPoint prompts you for the wavelength and you can indicate it at that time. 3 Choose Insert. The event appears in the control method s table and time line. Since each event has an associated time, the software places it in chronological order in the control method s table. 6-19

133 unipoint user's guide deleting event To remove an event: 1 Use the up or down arrow so the information for the event is displayed. 2 Choose Delete. The control method s table and time line are updated to reflect the removal of the event. modifying event To change parameters for an event: 1 Use the up or down arrow so the information for the event is displayed. 2 Make the necessary changes to the event. 3 Choose Change. exiting dialog When you are finished specifying events: 1 Choose Done. Constructing a mobile phase profile With a mobile phase profile, every event describes the flow rate and composition of the mobile phase components at a particular time. Follow these guidelines for constructing a profile: 1 Display the Mobile Phase Event dialog using the Event menu or toolbar. Note: If you are using UniPoint with SF Pressure Control (ordered separately), you will also see a text box for setting the P2 Pressure gradient for the 821 Pressure Regulator. For the P2 Pressure, indicate the pressure to achieve at the column outlet. Values between 0 and 40 MPa (400 bar or 8700 psi) are valid. 6-20

134 control methods 2 Begin the gradient profile at time 0.00 min to set initial conditions. Note: If pumps may already be running before the start of this method, set the first gradient and flow rate at a time later than 0.00 min. By doing this, the pumps can ramp smoothly from the current conditions to the new conditions. 3 Indicate the initial flow rate to achieve and percent composition to derive from each pump at 0.00 min. 4 Insert the event into the control method table and time-line graph. 5 Complete the mobile phase profile by adding additional time and composition values into the text boxes. Remember to choose Insert to define each event! 6 When you finish constructing the profile, exit the Mobile Phase Event dialog. previewing gradient UniPoint can display a simulation of the actual gradient that can be achieved during the run using the gradient profile that you set. Note: If your HPLC configuration does not include a Gilson 811 Mixer, UniPoint cannot display the gradient simulation. 1 From the Graph menu, choose the Gradient Simulation command. It may take a few seconds for UniPoint to display the gradient. 2 To remove the gradient, choose the Gradient Simulation command again. Device commands The following pages have more information on the available commands for controlling Gilson devices. For event dialogs that display listings of commands, the commands are presented in alphabetical order. 6-21

135 unipoint user's guide Pumps mobile phase For mobile phase pumps, you can indicate the flow rate and composition of the mobile phase components. See Constructing a mobile phase profile on page injection pump If you are controlling an injection pump for an autopreparative system, set events using the following commands. Note: For additional information, obtain a copy of the Autopreparative System Control Technical Note from your Gilson representative. Command Description Lock/Unlock Front Panel Record Display Set Dispense Volume x Rate Set Dispense Volume x Time Inhibits/provides use of front panel during a run. Copies display into report. Indicates sample injection amount. Use this or the Set Dispense Volume command. Indicates sample injection amount. peristaltic pump Following are the commands available for a Minipuls 3 Peristaltic Pump. You access these commands using the Generic command in the Event menu. Command Lock/Unlock Front Panel Prime Record Display Set Pump Speed Start Clockwise/ Counterclockwise Stop Description Inhibits/provides use of front panel during a run. Starts/stops priming of tubing. Copies display into report. Identifies the pump speed in hundredths of revolutions per minute (RPM). Starts the pump by indicating the desired direction of flow. Stops flow. Data channels For all data channels that have been identified, you can issue commands that start collection and later stop collection via the channels. During a run, the software automatically begins integrating data at the start of data collection if: an analysis method has been indicated and an inhibit integration event has not been set at time 0.00 in the analysis method. 6-22

136 control methods Contacts For an output contact, you can open, close, or pulse the output signal. For an input contact, you can Wait or Wait with Alarm (meaning UniPoint beeps until the input contact receives a signal from the peripheral device). To control devices via contacts, the device (injector, detector, chart recorder, fraction collector, and so on) must accept and sometimes send contact closure signals. And, the appropriate contact connections must be made between the devices. Note: Some peripheral devices cannot respond to a pulsed signal. In those cases, schedule an event to close the output contact followed by an event to open the same contact. If you fail to open the contact, it remains closed in subsequent methods. Detector 116 or 117 UV detector and 119 UV/VIS detector As a guideline for controlling detectors, you set events that identify the detection mode and set wavelength and sensitivity parameters for that mode. Note: You can also set detection parameters in a generic protocol file. See Protocols section. Following are the commands for Gilson detectors. The most commonly used commands are available. If you require additional functionality, see GSIOC command strings on page Command Autozero Channels Lock/Unlock Front Panel Record Display Record Parameter Set Dual Peak Width Set Dual Ratio Multiplier Set Dual Ratio Threshold Description Zeroes data channels. Inhibits/provides use of detector s front panel during a run. Copies display into report. For 116 Detector, copies first line of display only. For 117 or 119 Detector, copies the value of the indicated software parameter into the analysis report. Refer to the information on the P command in GSIOC appendix of the user's guide for the detector. Value between 4.0 and 99.0 seconds. If all peaks for wavelengths 1 and 2 are small, set the ratio multiplier to a number greater than 1. If all peaks are not of similar heights, set the multiplier to a number less than 1. Set the threshold (in AU) higher than the highest baseline noise level. Set Dual Sensitivity 1 Value depends on flow cell; see listing for Set Single Sensitivity 1. Set Dual Sensitivity 2 Value depends on flow cell; see listing for Set Single Sensitivity 1. Set Dual Wavelength 1 For 116 or 117 Detector, value between 190 and 380 nm. For 119 Detector, value between 190 and 720 nm. 6-23

137 unipoint user's guide Set Dual Wavelength 2 Set Lamp Saver Option On/Off Set Mode Set Scan End Wavelength Set Scan Out Time Set Scan Peak Width For 116 or 117 Detector, value between 190 and 380 nm. For 119 Detector, value between 190 and 720 nm. For 119 Detector, when Lamp Save Option is on, the detector automatically turns off a lamp if it is not being used by the detector. Identifies the operating mode for the detector. For the selected mode, sends the following information if the channel has been defined and collection initiated. Single Mode: Channel A - Wavelength/Sensitivity 1 Channel B - Wavelength/Sensitivity 2 Channel C - % Transmittance Dual Mode: Channel A - Wavelength 1/Sensitivity 1 Channel B - Wavelength 2/Sensitivity 2 Channel C - Ratio Scan Mode: Channel A - Wavelength/Sensitivity 1 Channel B - Wavelength/Sensitivity 2 Channel C - Scan For 116 or 117 Detector, value between 190 and 380 nm. For 119 Detector, value between 190 and 720 nm. Time it takes chart recorder to trace a wavelength scan on strip chart paper (doesn t affect time to scan a peak). Value between 0.1 and 20 minutes. Value between 4.0 and 99.0 seconds. Set Scan Sensitivity 1 Value depends on flow cell; see listing for Set Single Sensitivity 1. Set Scan Sensitivity 2 Value depends on flow cell; see listing for Set Single Sensitivity 1. Set Scan Sensitivity 3 Value depends on flow cell; see listing for Set Single Sensitivity 1. Set Scan Start Wavelength Set Scan Wavelength Set Single Peak Width Set Single Sensitivity 1 For 116 or 117 Detector, value between 190 and 380 nm. For a 119 Detector, value between 190 and 720 nm. Monitor wavelength. For a 116 or 117 Detector, value between 190 and 380 nm. For 119 Detector, value between 190 and 720 nm. Value between 4.0 and 99.0 seconds. Value depends on flow cell installed. 5 mm: range to 2.0 AU 2 mm: range to 5.0 AU 0.2 mm: range to 50.0 AU 0.05 mm: range to 200 AU Set Single Sensitivity 2 Value depends on flow cell; see listing for Set Single Sensitivity 1. Set Single Wavelength Start Scan Start File Turn Lamp On/Off or Turn Lamps On/Off or Toggle Lamp State For 116 or 117 Detector, value between 190 and 380 nm. For 119 Detector, value between 190 and 720 nm. Initiates scan between Scan Start Wavelength and Scan End Wavelength. Value between 1 and 9. File must be created using the detector s software. For 117 Detector, turns on/off UV lamp. For 119 Detector, turns on/off UV and if applicable VIS lamp. For 116 Detector, turns UV lamp on or off depending on current state. 6-24

138 control methods 118 UV/VIS detector Command Autozero Channels Lock/Unlock Front Panel Record Display Record Parameter Set Peak Width Set Sensitivity 1 Description Zeroes data channels. Inhibits/provides use of detector s front panel during a run. Copies display into report. Copies the value of the indicated software parameter into report. Refer to the information on the P command in GSIOC appendix of detector s User s Guide. Value between 4.0 and 99.0 seconds. Value depends on flow cell installed. 5 mm: range to 2.0 AU 2 mm: range to 5.0 AU 0.2 mm: range to 50.0 AU 0.05 mm: range to 200 AU Set Sensitivity 2 Value depends on flow cell; see listing for Set Sensitivity 1. Set Wavelength Start File Set Lamp Save Option On/Off Turn Lamps On/Off Value between 190 and 720 nm. Value between 1 and 9. File must be created using the detector s software. When Lamp Save Option is on, the detector automatically turns off a lamp if it is not being used by the detector. Turns on/off UV lamp. 122 fluorometer Note: For details on how to control the 122 via UniPoint, obtain a copy of the 122 Fluorometer Control Technical Note from your Gilson representative. Command Autozero Event Marker Run Program Scan Emission Spectrum Scan Excitation Spectrum Set Emission Bandwidth Set Emission Wavelength Set Excitation Wavelength Set CR Filter w/ Response Speed Description Zeroes analog outputs. Adds event marker to recorder output. Starts timed program created using the fluorometer s software. Scans emission spectrum. To set parameters for and output the scan, use the fluorometer s software. Scans excitation spectrum. To set parameters for and output the scan, use the fluorometer s software. Select between 10 nm (to improve wavelength selectivity), 18 nm (standard setting), or 40 nm (to enlarge the fluorescence signal). Indicate a value between 200 and 900 nm. Indicate a value between 200 and 900 nm. Selects the CR filter and identifies the response speed: fast, standard, or slow. 6-25

139 unipoint user's guide Set Digital Filter w/ Response Speed Selects the digital filter and identifies the response speed (in seconds): 3, 5, 10, 20, or 40. The digital filter method reduces baseline noise more than the CR filter method. A digital filter speed of 3 is approximately equivalent to a CR response speed of standard. A digital speed of 5 is approximately equivalent to a CR filter response speed of slow. A period of approximately 3 times the set value is required before the effect of the digital filter is seen. Set Lamp Off Timer Turns off lamp when specified time period (in hours) has elapsed: 99.9 hour maximum. Set PMT Gain Select 1, 10, 100, or Set Signal Attenuation Select 0 (which is the same as S in the fluorometer s software), 1, 2, 4, 8, 16, 32, 64, 128, or 256. Note: The attenuation setting affects the recorder output. It has no influence on integrator output. Recorder output is 0 when attenuation is set to 0. Integrator output can be considered as recorder output with the attenuation fixed at 1. Stop Program Stops program created using fluorometer s software. 170 diode array detector Note: For details on how to control the 170 via UniPoint, refer to the 170 user's guide. Command Balance Detector Description Reads the current signal value at each wavelength and tells the analog-to-digital converter that the value corresponds to 0 AU. This command affects the onscreen plotting of the diode array channels. Caution! Up to 1 minute may be required to execute a Balance Detector timed event. Therefore, you may need to insert the Balance Detector command as part of a start-up method. Or, ensure that you allow enough time for this event to occur before issuing later timed events. Set Analog Channel 1 Sets the following parameters: Monitor (nm) - The wavelength being monitored. Reference (nm) - The wavelength used to correct for background and for instabilities in the detector. Indicate a wavelength close to the monitor wavelength but that is in a region of non-absorbance. UniPoint automatically uses a 10 nm bandwidth for the monitor wavelength and a 40 nm bandwidth for the reference wavelength. Set Analog Channel 2 Set DAD Slit Width Sets the same parameters as described for the Set Channel 1 command. Follow these guidelines when selecting a slit width: Use 4 nm slit width for normal applications. Use narrow slit width (for example, 1 nm) if your analytes have narrow absorbance bands and high concentrations. Use a wide slit width (for example, 16 nm) to detect very low concentrations. Note: Do not change the slit width during the run. Insert the Set Slit Width command at the beginning of the control method or as part of a start-up method. Turn DAD Lamps On/Off Powers on the deuterium and tungsten lamps. 6-26

140 control methods Injector and its syringe pump or dilutor Injector Depending on how you are controlling your injector and its syringe pump or dilutor, you may need to set additional files before you can indicate timed events. The instructions used to control the injector system may be created using one of the following: Protocol file in UniPoint File stored in injector (non-xl version) Gilson Sampler Manager Software (model 719) In most cases, the events in the control method start a file that controls the injector and its syringe pump or dilutor and perform any synchronization between the injection of the sample and the control method. Following are the UniPoint commands available for injectors and dilutors. (These same commands are available for use in protocols. See Protocols section for details.) Since the injector and syringe pump or dilutor can be assigned the same description, their command lists are combined under the Event menu. This makes issuing commands to them easier. Command Home Needle Lock/Unlock Keypad Move Arm to Injection Port Move Arm to Rinse Station Move Arm to Sample Location Description Moves needle to left rear of injector. For non-xl version injector, inhibits/provides use of keypad during a run. Moves and lowers needle into injection valve's port. Moves and lowers needle into rinse station. Use the Dispense command to clean. Since some models of Gilson injectors have multiple ports in the rinse station and some models have multiple rinse stations, rinse station commands are model dependent. The commands available for your injector are shown in the Injector Event dialog. Moves the injector's needle into the specified sample tube. If you are selecting the sample to be injected, Gilson recommends that you enter a variable (for example, SampleLocation or Tube) in the Sample text box. Entering a variable name will cause a column to appear in an operations list that uses the control method. (See page 6-37 for more information on variables.) In the operations list, you can then indicate the zone and tube. If you are doing sample preparation steps, choose the sample location. To do this, double-click in the Sample text box. When the Browse Tray File window appears, click in the appropriate zone so its tubes become light blue. Then click on the tube location so its circle becomes red. Choose OK. When the Injector Event dialog re-appears, the selected zone and tube appear in the Sample text box. Note: If a tray file is not associated with the injector, a Browse Tray dialog appears. Use its options to select the file and then choose OK to display the Browse Tray File window. Select the Track Sample check box if you want to turn on UniPoint's sample tracking feature. Sample tracking provides for post-run viewing of the tube location associated with an injected sample. See Appendix B for details. For the Height parameter, 0 mm accesses the bottom of the tube. 6-27

141 unipoint user's guide Move Arm to Switching Port Move Arm to Top For 232 with two valves or 233 XL, moves and lowers needle into switching valve s port. Raises needle to top height. Move Arm to Transfer Port Moves and lowers needle into transfer port. Not available for 234. Move Arm to X/Y Position Moves the arm to a designated position set in mm. Height sets the position of the needle while moving. If you want to lower the needle before moving the arm, you can indicate a Height. However, use the Height parameter with caution. Lowering the needle may cause it to hit tubes during the move. For all Gilson injectors except the 215, the default Height is 0 mm which sets the needle s tip to its highest point. For the 215, UniPoint calculates a default Height that aligns the needle s tip with the bottom of the guide foot. To modify the default Height, manually lower the needle and then measure the distance between the bottom of the Y arm and the tip of the needle. Enter the distance, in mm, in the Height box. Move Arm to Z Position Record Display Respond to Input Set Collection Valve Position Set Injection Valve Position Set Low Pressure Valve On/Off Set Switching Valve Position Start File Start Timer Clock Synchronize Wait for Controller Wait for Display Message Wait for Injection Wait Time 6-28 Raises or lowers the needle without moving the arm. For the 215, UniPoint calculates a default Z Position that aligns the needle s tip with the bottom of the guide foot. To modify the default value, lower the needle and then measure the distance between the bottom of the Y arm and the tip of the needle. Enter the distance, in mm, in the Z Position box. Copies display into report. For answering prompts required by injection files stored in injector memory. For 233 XL or 215 being used as both the injector and fraction collector, sets a valve position to divert or collect before or after fraction collection. For the 215, it controls the low pressure valve. For the 233 XL, it controls the left-side valve. Note: During run-time fraction collection, UniPoint automatically changes the position of the valve to collect fractions or divert eluent to waste. Rotates injection valve to load or inject position. For XL version injector or 215, controls 3-way valve installed on the horizontal arm (or Y arm). For 232 with two valves or 233 XL, controls switching valve. For non-xl version injector, value between 1 and 9. File must be set up using injector s keypad. Initiates internal clock. For synchronization with protocols written using UniPoint. For synchronization between injector program and UniPoint. During the run, UniPoint enters and remains in a wait state until it receives a signal from the injector s program to release the wait. When it receives the signal, UniPoint continues the run. For XL version injector, this is the EGo command. For synchronization between injector program and UniPoint. During the run, UniPoint enters a wait state until the indicated message appears. When the message appears, UniPoint continues the run. For synchronization between injector program and UniPoint. During the run, UniPoint enters and remains in a wait state until it receives a signal from the injector s program to release the wait. When it receives the signal, UniPoint continues the run. For XL version injector, this is the EReady command. Instructs injector to wait for a preset length of time before continuing.

142 control methods 401/C dilutor Command Aspirate Dispense Home Syringe Prime Rotate Valve Description Draws specified amount of liquid into needle and transfer tubing. Dispenses specified amount of liquid. Moves syringe to top position. Starts/stops priming of syringe and transfer tubing. To aspirate or dispense liquid from the dilutor s solvent bottle, rotate the valve to reservoir. To aspirate from or dispense into a tube or transfer port, rotate the valve to needle. 402 syringe pump Note: The commands available to you depend on how your 402 Syringe Pump is configured in the UniPoint configuration file. Command Aspirate, Aspirate Left Syringe, or Aspirate Right Syringe Aspirate Right Syringe from Reservoir Dispense, Dispense Left Syringe, or Dispense Right Syringe Home Syringe, Home Left Syringe, Home Right Syringe, or Home Both Syringes Prime, Prime Left Syringe, Prime Right Syringe, or Prime Both Syringes Rotate Valve, Rotate Left Valve, or Rotate Right Valve Set Aspirate for Left Syringe, Set Aspirate for Right Syringe, or Set Aspirate for Both Syringes Set Dispense for Left Syringe, Set Dispense for Right Syringe, or Set Dispense for Both Syringes Set Sequential Dispense Start Both Syringes, Start Left Syringe, or Start Right Syringe Timely Start Left Syringe or Timely Start Right Syringe Description Sets flow rate and volume and then draws liquid into the syringe. (For Dual Syringe Configuration with Tee) Tells the pump to fill the left syringe from the reservoir, then fill the right syringe by dispensing the specified volume from the left syringe. Sets flow rate and volume and then dispenses liquid. Moves the syringe(s) to top position. Starts/stops priming of the syringe(s) and transfer tubing. To aspirate or dispense liquid from the solvent bottle, rotate the valve to Reservoir. To aspirate from or dispense into a tube or transfer port, rotate the valve to Needle. Sets flow rate and volume but does not draw liquid into the syringe(s). Use a Start command or Timely Start and Start command sequence to aspirate liquid. Sets flow rate and volume for the syringe(s) but does not dispense liquid. Use a Start command or Timely Start and Start command sequence to dispense liquid. Sets flow rate and volume for both syringes but does not dispense liquid. When the command is followed by a Start Both Syringes command, the pump dispenses liquid from the right syringe first, followed by the left syringe. The Set Sequential Dispense command could be used for sample dilution. Aspirate diluent into the left syringe (perhaps from reservoir). Then, aspirate sample into the right syringe. The sequential dispense will deliver sample from the right syringe first, followed by diluent from the left syringe. Starts aspirate or dispense as defined by the Set Aspirate or Set Dispense command. Delays motion of the specified syringe. UniPoint uses the parameters set in the last Set Aspirate or Set Dispense command. To actually aspirate or dispense liquid, follow the Timely Start command with a Start command. 6-29

143 unipoint user's guide Fraction collector Following are the commands for fraction collectors. The most commonly used commands are available. If you require additional functionality, see GSIOC command strings on page As a guideline, set events that identify how effluent is collected: by time, volume, peak, or combination of those. You must also indicate the location at which collection begins (see Set Fraction Site command). UniPoint assumes that the fraction collector has a 3-way valve installed. The dispense head does not travel to the drain trough during periods of non-collection but is always located over the current tube. Effluent diverts to drain through the 3-way valve s outlet tubing. Note: The Fraction Collector Control Technical Note includes additional information on collecting fractions. It is available, upon request, from your Gilson representative. Command Advance Collect Negative Peaks Collect Non-Peaks Collect Positive Peaks Home Collection Head Lock/Unlock Front Panel Record Display Set Collection and Travel Depths Description Moves collection head to next tube. Turns on collection of negative peaks. Indicates if all effluent is collected into tubes. Turns on collection of positive peaks. Moves dispense (collection) head to left rear of the tray. Inhibits/provides use of front panel during a run. Copies text on display into report generated for sample. (This command only appears for the 233 XL or 215 as fraction collector.) Use the Set Collection and Travel Depths command to customize needle movement during fraction collection. The collection depth is the needle height used when dispensing into fraction sites. The travel depth is the needle height used when moving between fraction sites. For the 233 XL, the default collection depth and travel depth is 2 mm below the guide foot. (0 mm is the bottom of the guide foot.) If you are using deep well microplates, the default collection and travel depths should be appropriate. However, if you are using standard microplates, you may want to adjust the collection depth and/or travel depth. To do this, manually move the 233 XL s needle so it is positioned over a well then lower the needle to the desired depth. Measure the distance between the bottom of the guide foot and the tip of the needle to identify the depth. Because the 215 s Z Drive can be installed at different heights, UniPoint calculates and places default values in the command s Collection Depth and Travel Depth parameter boxes. The default depths place the needle s tip slightly below the guide foot. If you are using deep well microplates, the default collection and travel depths should be appropriate. However, if you are using standard microplates, you may want to adjust the collection depth and/or travel depth. To do this, manually move the 215 needle so it is positioned over a well then lower the needle to the desired depth. Measure the distance between the bottom of the Y arm (or horizontal arm) and the tip of the needle to identify the depth. (0 mm is the bottom of the Y arm.) 6-30

144 control methods Note: If you omit the Set Collection and Travel Depths command from the control method or fraction collector protocol, UniPoint uses the default collection and travel depths during the run. For the 215, UniPoint calculates the default collection and travel depths, in mm, using the following formula. The calculated depth places the needle at 2 mm below the guide foot. Depth = Y arm height from 215 locator plate - Height at which Z Drive is clamped + 2 Y arm height from 215 locator plate is a constant value of 176 mm. Height at which Z Drive is clamped is a user-entered value stored in the system configuration file. Set Collection Valve Enables you to set the position of a 3-way valve before or after fraction collection. Note: During run-time fraction collection, UniPoint automatically changes the position of the valve to collect fractions or divert eluent to waste. Set Fraction Site Identifies where fraction collection will begin for an injected sample. In the Site text box for the Set Fraction Site command, Gilson recommends that you enter a variable (for example, FRACTION_SITE). Entering a variable name will cause a column to appear in an operations list that uses the control method. (See page 6-37 for more information on variables.) You can then indicate in the operations list the zone (for example, FRACTIONS) or the zone and tube (for example, FRACTIONS:1) into which collection begins. However, instead of a variable name, you may indicate the zone name and if necessary the tube number at which to start collection. (A column does not appear in the operations list if you indicate where to begin collection in the control method.) If you enter the zone name and tube number (for example, FRAC- TIONS:1), collection will begin in the specified tube for each sample injected during the run. Gilson refers to this as specific site collection. If you enter the zone name followed by a colon (for example, FRAC- TIONS:), collection will begin in the first tube of the zone for the first injected sample. For the second and each subsequent sample collected into the zone, collection will begin in the next available numbered tube. Gilson refers to this as continuous collection. (If you omit the colon after the zone name, UniPoint identifies the zone name as a variable and will display a column in the operations list.) To select the first fraction site, into which eluent will be collected for a sample, you can click in the Site text box and then click the Browse button. When the Browse Tray File window appears, click within the zone, on the tray image, so its tubes become blue. Click on the desired tube so it becomes red. Click on OK. When the Fraction Collector Event dialog re-appears, the zone:tube location is shown in the Site text box. If you want continuous collection, remove the tube number after the colon. Note: If a tray file is not associated with the fraction collector, a Browse Tray dialog appears. Use its options to select the file and then choose OK to display the Browse Tray File window. If no tray file exists, use the Tray Editor Software supplied with UniPoint to set it up. Note: If you set fraction collection commands in the control method but omit the Set Fraction Site command, UniPoint will still collect fractions during a run. UniPoint will look for a FRACTIONS zone in the fraction collector s tray file during the run and perform continuous collection for the injected samples. UniPoint will also collect fractions as described above if you do not enter anything into the Site text box. However Gilson recommends that you use the Set Fraction Site command and indicate a variable name for the Site parameter in your control method (or fraction collector protocol). 6-31

145 unipoint user's guide Set Fraction by Time Inside a Peak Subdivides peaks by time. Set Fraction by Volume Inside a Peak Subdivides peaks by volume. Set Non-Peak Time per Tube Set Non-Peak Volume per Tube Set Peak Level Set Peak Sensitivity Set Peak Width Set Time per Tube Set Volume per Tube Start Collection Stop Collection If collecting non-peaks, use this command or the Set Non-Peak Volume per Tube command. If collecting non-peaks, use this command or the Set Non-Peak Time per Tube command. To collect more effluent than is collected into tubes for peaks, indicate the tube volume (in ml) in the parameter box. The mv value above which all effluent is collected. This value is especially useful if the detector signal goes off-scale during collection. Height of smallest peak of interest, expressed as a percentage of a trace s fullscale height. Set a peak sensitivity value that is slightly greater than the highest baseline noise. The value can range from 0.5% to 10000%. Width of average peak at half height. The amount of time the dispense head dispenses into a tube before going to the next tube or drain. The maximum volume to collect into each tube. Initiates collection. Ends collection; dispense head does not move to home position. System interface System (computer) control For the 506C System Interface, you can select to zero the analog offset voltage for each available channel. To access this command, choose Generic from the Event menu and select the System Interface in the list box. The following computer control commands are available. Command Announcement Run Program Start Timer Clock Synchronize Wait Time Description Displays a message on-screen during the run. Enables UniPoint to execute a.gex file. See the Gilson Executable (.GEX) File appendix. Initiates internal clock; starts timer running for Wait Time command. Enables you to synchronize the computer with a protocol written using UniPoint. The computer enters a wait state until it receives a synchronize command from a protocol. Causes the computer to enter a wait state during the run; the wait is released when the specified time expires. Also starts timer unless previously started via the Start Timer Clock command. 6-32

146 control methods Valve actuator 817 Valve Actuator Following are the commands for an 817 Valve Actuator. To access these commands, choose Generic from the Event menu and select the valve actuator in the list box. The most commonly used commands are available. If you require additional functionality, see GSIOC command strings on page Command Lock/Unlock Keypad Record Display Set Valve Position Turn Solenoid 1 On/Off Turn Solenoid 2 On/Off Description Provides/inhibits use of keypad during a run. Copies text on display into report generated for sample. Rotates valve to selected position. Activates/deactivates solenoid valve connected to 817 rear panel. Activates/deactivates solenoid valve connected to 817 rear panel. 819 Valve Actuator When the 819 is configured as an injection valve, the following command is available in the Injector Event dialog. Be aware that the 819 is assigned the default description of Injector; therefore, its command list is combined with the command list for the sampling injector and syringe pump or dilutor. The following UniPoint Technical Notes, available from your Gilson representative, include additional information on 819 control: Using the 215/819 for Injection and Using the 215/819 for Injection and Collection. Command Set Injection Valve Position Description Rotates the valve to the selected position: load or inject. When the 819 is configured as a switching valve, the following command is available in the Generic Event dialog. Command Set Switching Valve Position Description Rotates the valve to the selected position: load or inject. VALVEMATE Following are the commands for a VALVEMATE. To access these commands, choose Generic from the Event menu and select the valve actuator in the list box. The most commonly used commands are available. If you require additional functionality, see GSIOC command strings on page Command Lock/Unlock Front Panel Move Valve to Home Position Record Display Description Provides/inhibits use of keypad during a run. Rotates the valve to its home position. Copies the upper or lower line of the display into the data notes section of the analysis report s annotation. And, displays the information in the status box during the run. 6-33

147 unipoint user's guide Record Hours of Power On Record Valve Position Record Valve Turn Count Set Valve Position Set Valve Position Variable Copies the number of hours that the VALVEMATE has been in use into the data notes section of the analysis report s annotation. And, displays the information in the status box during the run. Copies the current valve position into the data notes section of the analysis report s annotation. And, displays the information in the status box during the run. Copies the number of valve rotations into the data notes section of the analysis report s annotation. And, displays the information in the status box during the run. Rotates the valve into the selected position. For a 2-position valve, you can select between Home (position 0) and Switched (position 1). For a multi-position valve, you can select between 1 and the number of available positions for the installed valve. Enables you to set the valve position as a variable. When you create the operations list, acceptable values for the variable are 0 (for Home position) and 1 (for Switched position) if a 2-position valve is installed. For a multi-position valve, acceptable values are 1 through the number of available positions for the installed valve. Temperature regulator (or column oven) Following are the commands for an 831 Temperature Regulator. To access these commands, choose Generic from the Event menu and select the temperature regulator in the list box. The most commonly used commands are available. If you require additional functionality, see GSIOC command strings on page Note: UniPoint refers to the 831 as a temperature controller. If you want UniPoint to monitor the 831 for high or low temperatures, indicate those temperatures as described on page Command Lock/Unlock Front Panel Record Display Set Target Temperature Start/Stop Heating Description Inhibits/provides use of keypad during a run. Copies text on display into report generated for sample. Temperature (between 40 and 200 C) to obtain. When to begin or stop heating. Rack temperature regulator Following are the commands for an 832 Temperature Regulator for Racks A and B. To access these commands, choose Generic from the Event menu and select Rack A or Rack B in the list box. You see the following commands. Command Lock/Unlock Keypad Record Rack Status Description Inhibits/provides use of keypad during a run. Copies display into report. Set Temperature Specify temperature between 4 and 40 C. Start/Stop Temperature Begins/ends temperature control. 6-34

148 control methods Pressure regulator (controller) If you have UniPoint with the SF Pressure Control module (ordered separately), you can set the P2 Pressure gradient for the 821 Pressure Regulator in the Mobile Phase Event dialog. For help on using the Mobile Phase dialog, refer to page You indicate the system pressure to maintain in the P2 Pressure text box. GSIOC-to-RS-232 converter Following are the commands for a 606 GSIOC-to-RS-232 Converter. To access these commands, choose Generic from the Event menu and select the converter s description in the list box. Note: If you are using the 606 Converter to issue commands to a Gilson 133 Detector, obtain a copy of the 133 Refractive Index Detector Control Technical Note from your Gilson representative. Command Description Read FIFO Channel A Gets first in, first out data for Channel A. Read FIFO Channel B Gets first in, first out data for Channel B. Read Status Channel A Read Status Channel B Request Identification Reset Reads the status of the pins for Channel A s RS-232 port. Returns a 0 (false) or 1 (true) for the following abcde, and a value between 0 and 1536 for f,g where: a - CTS (Clear to Send) status b - DSR (Data Set Ready) status c - CD (Carrier Detect) status d - DTR (Data Terminal Ready) status e - RTS (Request to Send) status f - Number of characters in Tx (Transmit) buffer g - Number of characters in Rx (Receive) buffer Reads the status of the pins for Channel B s RS-232 port. See Read Channel A command for a description of what is returned. Identifies the model and version of the 606 Converter that is installed in the HPLC system. Restarts the 606 Converter. 6-35

149 unipoint user's guide Send String Channel A Sends ASCII character string to the device connected to Channel A. For the text string, refer to the documentation for the RS-232 device. That documentation should identify the commands that can be sent to the device via its RS-232 port. Place the text string within double quotes unless it contains a variable. For could be the RS-232 command that sets the wavelength to 280 nm for a non-gilson detector connected to the 606 Converter. If you wanted the wavelength to be a variable, you could WAVELENGTH for the text string. Note: If the command being sent requires a non-printing character, such as a key press, use the hexadecimal number for the non-printing character and place that number within brackets. For example, using [0D] in a command string indicates a carriage return. Send String Channel B Sends ASCII character string to device connected to Channel B. Refer to information for Send String to Channel A command. Set Status Channel A Specifies pin-out settings for Channel A. Set Status Channel B Specifies pin-out settings for Channel B. GSIOC command strings Each device has a set of GSIOC commands that it understands. Most of these commands are provided in the dialog boxes accessed from the Event menu. If there is another command that you would like a device to perform, refer to its User s Guide to see if the command is available. A complete list of GSIOC commands is listed in the device s User s Guide. The Generic Device Event dialog lets you specify commands for a Gilson device which does not have its own menu command or to specify GSIOC command strings for a device. To display the Generic Device Event dialog: 1 Choose Generic from the Event menu. Or, click on a generic event in the table. Or, click on its tool in the toolbar. 2 If you need help using the dialog, refer to Scheduling timed events on page 6-18 and indicating GSIOC command strings, below. 6-36

150 control methods indicating command strings GSIOC commands consist of strings of no more than 40 characters that specify instructions to a specified device. Each Gilson device has a set of commands that it understands. The complete list of GSIOC commands for any Gilson device is given in its User s Guide. To set an event for sending a GSIOC command string to a device: 1 If necessary, choose the Custom> button in the Generic Device Event dialog. If the Custom> button is already active its label becomes List>. Therefore, if you see a List> button, proceed to the next step because the options necessary for indicating a GSIOC command string are already shown. 2 Type the time at which the command is sent. 3 Optionally indicate a comment in the Comment box. 4 Select the name of the device in the Device list box. Or, type the unit ID of the device in the Unit ID box if the device is not listed. 5 Type the command string into the GSIOC string box. Place the entire command string within double quotes unless it contains a variable. For example, F1 is the buffered command to start file 1 on a 119 UV/VIS Detector. If you wanted the file number to be a variable, you could indicate F FILE_NUMBER for the command string. And, then answer the variable when setting up the operations list for the run. 6 Using the options buttons, choose the type of command. The documentation for the GSIOC command tells what type of command it is. Buffered commands send instructions to a device. These commands are executed one at a time. Immediate commands request status information from a device. These commands are executed immediately, temporarily interrupting other commands in progress. 7 Choose Insert. The event registers in the control method s table and time line. 8 When you finish adding events, choose Done. 6-37

151 unipoint user's guide Variables To make your control methods more flexible, you can indicate a variable name instead of entering a value in the command parameter box for a timed event. A variable name can be any combination of letters and numbers and can contain special characters (_, as long as it is not the first character or a mathematical operator (+, -, *, /, %). It cannot include spaces. Valid variable names would be Wavelength#, Sample_Vol1. UniPoint does not differentiate between upper and lower case letters in a variable name. For example, it views the names Wavelength, wavelength, and WAVELENGTH as identifying the same value. The command parameter specified for an event can also be the result of performing a mathematical function on two variables or a variable and number. For example, AirGap + SampVol, or SampVol + 15 are valid command parameters. You can add (+), subtract (-), multiply (*), divide (/), and return the remainder (%). When setting the Operations window for a run, UniPoint prompts you for a value to assign to any undefined variables used by a control method. To see what variable names have been set for a control method and to indicate lower and upper limits for and a default for each variable, do the following. 1 From the Event menu, choose Variables. 2 When the dialog box appears, choose the variable name in the list box. 3 Check the prompt that appears whenever the software references the variable. The prompt can be any combination of letters and numbers and can contain special characters and spaces. 4 To indicate minimum and maximum values for the variable, indicate the numbers in the Values Limits box. Separate the values with a colon, for example, 1:100. If the input is required, type an R before the minimum value, for example, R1:100. You can also indicate the variable name of SYRINGE for syringe volume or Z_ARM for Z_arm height as a limit. 6-38

152 control methods 5 To set a default value for the variable name, type a value in the Default Value box. 6 To remove an unused variable name, choose it in the list box and the choose Delete. 7 Choose OK when finished. if you add, change, or delete variable names If you add, change, or delete variable names and the control method is used in an operations list, you need to recreate the operations list or modify each step in the list that uses the control method, as outlined below. 1 Open the operations list. 2 Double-click on the step that executes the control method. 3 In the Step Entry dialog, click in the Control Method box and use the Browse to reselect the control method. The parameter text boxes at the bottom of the dialog update to reflect the changes in the control method. If a variable is no longer used by the method, its parameter box is grayed. 4 Enter the correct value in each parameter box and click on Change to update the operations table. 5 Use the scroll arrows to display information for the next step that uses the control method. 6 Repeat steps 3-5 for each step that uses the control method. 7 Choose Done when finished modifying steps. 8 To remove the table column for a variable that is no longer used: a) Display the Format Columns dialog by choosing its command from the Table menu or double-clicking on the header row in the table. b) In the Column Contents list box, choose the variable name and choose Delete. c) Choose OK to exit. 9 Save and close the operations list using the File menu. 6-39

153 unipoint user's guide Error handling UniPoint can automatically respond to the occurrence of defined errors during a run. To make this happen, set up control methods that tell UniPoint what to do if it encounters one of those errors. In most cases, these methods include system shutdown commands. To access the dialog for indicating error handling information: 1 Choose Error Handling from the Event menu. In the dialog box, error handling capabilities are available for the following. High and Low Pressure. Error occurs if UniPoint detects that the low or high system pressure limit set on the manometric module or in this dialog box has been surpassed. Wait Time-Out. Error occurs if the time period is exceeded. Wait timeout is the period during which UniPoint is in a programmed hold and is waiting for a GSIOC or contact signal. If the wait time-out is 0.00 min, the system waits indefinitely. The valid range is 0.00 to 999 min. GSIOC. Error occurs if a scheduled GSIOC command fails to execute or if UniPoint loses communication with a device located along the GSIOC. Contact. Error occurs if UniPoint receives a signal from the indicated contact. Temperature. Error occurs if UniPoint senses a low or high temperature limit set for an 831 or 832 Temperature Regulator. Leak. Error occurs if UniPoint detects error in the 831 Temperature Regulator. Tray Location. Error occurs if UniPoint cannot locate the next tube for sample injection or fraction collection during a run. 6-40

154 control methods activating error handling Entering a value in the text box next to an error type activates it. What happens during the run then depends on whether an error method is entered and whether the Continue on error check box is selected. If neither a method nor the Continue check box is selected, the run is stopped. If both a method and the Continue check box are selected, UniPoint executes the error handling method and then proceeds to the next step in the operations list. If a method is indicated but the Continue check box is not selected, UniPoint executes the error handling method and then stops the run. If a method is not indicated, but the Continue check box is selected, UniPoint finishes the current step and proceeds to the next step in the operations list. specifying error handling parameters To use the areas in the Error Handling dialog to activate the error handling capability: 1 In the text box next to each error, type the complete path and file name of the control method executed when that error is detected. To search the disk for the control method and have its location automatically entered, click in the text box and then on the Browse button. (Or, double-click in the text box.) You can specify the same method in each of the text boxes. 2 Add or remove the check from the Continue check box. Refer to activating error handling, above, to see how this check box affects what happens during the run. 3 If necessary, indicate the limit to use or the contact to monitor. To indicate the contact description, click in the text box and then choose the Browse button. A dialog listing currently named contacts appears. Use it to select the error contact. 4 When finished, choose OK to store the conditions. Or choose Cancel to return to original settings. before the run Create the necessary error-handling method(s) before the control method is indicated in the operations list for a run. Note: In an error-handling method, you may want to set the first gradient and flow rate at a time later than 0 min. By doing this, the pumps ramp smoothly from the current conditions to the conditions in the method. 6-41

155 7 Analysis Methods In the last section, you learned how to identify and set timed events for controlling devices during a run. This section describes how to set conditions for analyzing peaks in collected data. Each analysis method indicates how collected data is analyzed during a run or re-analysis processing. Analysis includes setting peak parameters, naming peaks, and identifying the reports to generate. 7-1

156 unipoint user's guide Analysis Method window Using the options in the Analysis Method window, you indicate how collected data is analyzed and reported. Whether the data is analyzed at run time or afterwards, UniPoint uses the information in the analysis method to report on peaks detected in samples. The following illustration shows the parts of an Analysis Method window, with information already set up. Refer to Section 5, Common Procedures for more information on manipulating parts of a table or graph displayed in a document window. menu bar toolbar table pane annotation box graph pane status bar legend box menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Menu File Edit Analysis Report Table Graph Window Help Description Create, save, print, delete files or exit a window or UniPoint. Locate, make changes to, or transfer information. Set peak integration, baseline, scaling, variable, and error handling options. Specify report type, format, and output. Display or modify appearance of the table pane. Display or modify appearance of graph pane. Re-arrange windows or icons, or activate a window; display file annotation or toolbar. Learn about UniPoint. 7-2

157 analysis methods table pane Lists the timed events currently set for the method. The events are based on the data collection start time indicated in the control method executed at the same time as the analysis method during a run. When you create an analysis method, the following integration events automatically appear at 0.00 minutes in the window. Default Baseline Disable Negative Peak Integration Peak Width: 0.20 min Peak Sensitivity: 2% You can modify the times at which these take place and the values for peak width and sensitivity, if necessary. To make the changes, see Integration events on page 7-5. Double-clicking on a row for a timed event automatically displays the Integration Events dialog used to set up the event. Double-clicking on the top (header) row in the table displays a dialog for changing the format of the columns shown in the table. graph pane toolbar Shows a graphic representation of timed events and peak retention time windows. Provides quick access to some options available in the Analysis Method window. To activate a tool, just click on its icon. navigator tool open tool save tool print tool integration tool channel scales tool peak table tool report type tool report output tool Displays Navigator. Displays Open dialog. Stores method to disk. If it has not been saved, displays the Save As dialog. Displays Print dialog. Displays Integration Events dialog. Displays Data Channel Scales - Analysis dialog. Displays Peak Table dialog. Displays Report Type dialog. Displays Report Output dialog. To turn off/on the toolbar, choose its command from the Window menu. 7-3

158 unipoint user's guide annotation box Lists analysis and reporting conditions set in the method. To turn on/ off the annotation, choose its command from the Window menu. To add your own comments to the annotation, use the Notes command in the Edit menu. legend box Identifies the symbols shown in the graph. To turn on/off the legend, choose its command from the Graph menu. Double-clicking on an item in the legend accesses the Attributes dialog. Use that dialog box to remove events from display or to change the size of symbols and lines in the graph. status bar Displays help message if a menu command is highlighted. Setting analysis conditions The Analysis menu in the method window provides for setting analysis conditions plus indicating variable and error handling information. Integration Events sets integration parameters for distinguishing peaks from baseline interference and sets alternate baseline techniques. Channel Scales specifies chromatogram plot conditions. Background Removal indicates if a blank chromatogram is subtracted from data analyzed by the method. Peak Table identifies information about the components in injected samples. Error Handling tells UniPoint what types of checks to do on the calculated peak amounts and how the software responds if it detects an error. Variables sets limits and default value for any variables identified in the analysis method. 7-4

159 analysis methods Integration events In the Integration Events dialog, you specify times at which to modify integration parameters. When you first open an Analysis Method window, the following integration events are immediately set at 0.00 minutes: Default Baseline Disable Negative Peak Integration Peak Width: 0.20 min Peak Sensitivity: 2% For most separations, these are the only events to set. However, you can delete or modify these events if necessary using the Integration Events dialog. Note: Removing the Disable Negative Peak Integration command does not turn on negative peak integration. Insert the Enable Negative Peak Integration command at the time point at which to start negative peak integration. To be integrated, a peak must elute during data collection. Furthermore, every contour rising from and falling to the baseline must meet the peak detection criteria. An unlimited number of peaks per run can be integrated. The software s ability to integrate peaks depends on the peak width and peak sensitivity settings. These settings give information to UniPoint about the size and shape of your peaks and the noise level riding the baseline. In most cases, the default values for peak width and sensitivity meet your needs. However, if one or more peaks of interest are not integrated, modify one or both of these parameters before subsequent runs or re-analysis processing. Following is a description of the available integration events and how to set timed events. 7-5

160 unipoint user's guide integration parameters Note: Changes to the integration parameters remain in effect until changed again. Peak Width. Peak width is the duration of a peak, in minutes, at its halfheight. The default value is 0.20 min and can be modified. The peak width value should be slightly longer than the width of the narrowest peak of interest. If UniPoint cannot locate peaks during an analysis, adjust the peak width value. Use the chromatogram plot or strip chart to measure the narrowest peak at its half-height. Then using the time scale, determine its duration. If all peaks are no more than twice the width of the narrowest peak, indicate a peak width that is slightly longer (for example, 0.05 min) than the narrowest peak. If the range of peak widths on your example trace does not allow the above approach, select a mid-range peak. Then determine whether any peaks are more than twice or less than half of its duration. If all peaks fall within that range, use the mid-range peak for setting peak width. Peak Sensitivity. Peak sensitivity is expressed as a percentage of a trace s full-scale height. This setting maximizes the amount of the peak that is integrated while minimizing the integration of baseline artifacts. The peak sensitivity value should be slightly greater than the highest baseline noise. The value can range from 0.5% to 10000%. UniPoint bases the full-scale range of peak sensitivity settings on a 10 mv default input signal. If your data device provides a full-scale input signal other than 10 mv, adjust the sensitivity setting accordingly. Full-scale input Peak sensitivity setting at full-scale height 10 mv 100% 100 mv 1000% 1000 mv 10000% If few or no peaks are integrated, set a lower sensitivity value. If too many peaks are integrated, indicate a higher sensitivity value before subsequent runs or re-analysis processing of samples. Enable/Inhibit Integration. If a region of a trace is uninteresting to you or if a region contains many unresolved peaks, you can inhibit integration during that period. Schedule one timed event to inhibit integration. Then schedule another event for the time at which to resume integration. This technique can eliminate a large number of uninteresting peaks from being listed in reports. 7-6

161 analysis methods baseline construction techniques UniPoint provides several baseline construction techniques. If you change the construction technique, the software uses the first affected peak to determine when to use the technique. The first affected peak is the peak whose end follows the scheduled event time. Therefore, when applying an alternate construction technique within a series of peaks, enter an event time that coincides with the peak retention time. Use these techniques sparingly and carefully so that data are analyzed in a meaningful way. Accurate peak integration depends on the shape of the baseline, which in turn depends on the peak shape and on the resolution from neighboring peaks. Note: Changes to the baseline construction technique remain in effect until the next baseline event is scheduled. The drawings on the following pages demonstrate changes to a default baseline that can result from changing the peak parameters. Review the changes to the peak start and end points determined by the software. The arrow in each drawings indicates the analysis event time. Default Baseline. To draw the baseline beneath qualified peaks, UniPoint connects the start and end of each integrated peak. If the software encounters merged peaks (no baseline resolution between peaks), it draws a taut string baseline from the first peak start to the next peak end that it finds. If any deep valleys interrupt the taut string baseline, the baseline is drawn from the peak start to the bottom of each valley and then to the peak end. The software then drops perpendiculars from all remaining valleys to the baseline to determine the baseline beneath each peak. The default baseline technique is used unless you specify another technique. If the default baseline does not report optimal peak heights (or areas), choose one of the other techniques, described on the following pages. 7-7

162 unipoint user's guide Horizontal Baseline. UniPoint draws a forced horizontal baseline. Then, to determine the baseline beneath each peak, it drops perpendiculars from each peak start and each peak end to the horizontal baseline. If the software encounters merged peaks (no baseline resolution between peaks) while using this technique, the horizontal baseline is maintained and perpendiculars are drawn from the valleys between peaks, rather than from peak starts and ends. If any peak end or valley falls below the horizontal, the baseline drops to that level and continues to be drawn at that level. Next Valley /All Valleys. UniPoint connects valleys between peaks to correct for temporary and gradual baseline drift respectively. The Next Valley option forces the baseline to the next valley only, whereas the All Valleys draws the baseline from valley to valley until the next baseline option is set. Next Valley All Valleys 7-8

163 analysis methods Tangent Skim. When you select this option, UniPoint draws a baseline under the solvent front peak in addition to component peaks. Default Baseline Tangent Skim adding event To add a timed event: 1 If necessary, clear information from some of the text boxes (such as the Comment box) by choosing New. This also displays New Event next to the up and down arrows in the upper right corner of the dialog. 2 Indicate time and command parameters. The limit of resolution is 0.01 min. Note: During a run, the event time is based on the time at which data collection begins in the control method. 3 Choose Insert. The change is applied to the first peak whose end follows the event time. When applying an alternate baseline in a series of close peaks, choose an event time that corresponds to the retention time of the first peak of interest. deleting event To remove an event: 1 Use the Event up or down arrow to display the information for the event. 2 Choose Delete. modifying event To change parameters for an event: 1 Use the Event up or down arrow to display the information for the event. 2 Make the necessary changes to the event. 3 Choose Change. 7-9

164 unipoint user's guide Channel scales In the Channel Scales dialog, you indicate which data channel is analyzed by the method. You also use this dialog box to indicate how traces from any other data channels are shown on the chromatogram plots in reports generated by the method. Peak amounts in reports are for data collected via the designated analysis channel. To specify data channel parameters: 1 From the Analysis menu, choose Data Channels. The Data Channel Scales dialog appears. 2 To display data channels in the list box, use the Browse or Edit button. Clicking on Browse displays the Browse Control Method dialog. Use the dialog to select a control method which already defines data channel information. After you select the control method, its data channel descriptions, and channel scaling parameters, will appear in the Data Channel Scales dialog. Clicking on Edit displays the Edit Channels dialog. Use the dialog to enter channel descriptions. To enter a channel description, type the description in the text box and click on Add. When you return to the Data Channel Scales dialog, you will see the data channel description(s) that you just set up. 3 Choose a channel in the Channel Scale Parameters list box. 4 Review the information in the areas of the dialog box, described below. Modify the information, if necessary. 5 Repeat steps 3 and 4 for each channel. Then choose OK to exit. Following are descriptions of the areas in the Data Channel Scales dialog. The Auto Range option pertains to all data channels being collected. The other options relate to the data channel whose description is currently highlighted in the Channel Scale Parameters list box. 7-10

165 analysis methods Auto Range. If this check box is selected, UniPoint automatically plots data to the minimum and maximum peak height determined across all collected data channels. Selection of this box overrides any other limits set for individual data channels in the dialog box. Channel Scale Parameters. The available data channels. If none are listed, click in the list box and then click on the Browse button. Use the dialog box that appears to choose the control method that describes the data channels being collected. Analysis Channel. After choosing the channel in the list box, place an X in this check box to indicate its data is analyzed by the method. Only one channel can be indicated as the analysis channel. Any reports generated by the method pertain to this channel. % Offset. Use this parameter to move the channel s trace away from the horizontal axis (and from other channel traces) on chromatogram plot. Express the offset as a percent displacement from the horizontal axis (0%). Minimum and Maximum. The lower and upper limits for the signal axis. Be aware of the following: If the Auto Range check box is selected the software disregards these values and uses the minimum and maximum heights for all channels to determine the signal axis. If Auto Range is not selected and you indicate different minimum and maximum values for each channel being collected, the software uses the lowest and highest values entered to determine the signal axis. For example, if 0 and 10 mv were entered for the minimum and maximum values for the 254 nm channel and 0 and 100 mv were entered for the 280 nm channel, the signal axis would be 0 to 100 mv. Scale. How UniPoint scales data when it is plotted: mv, user units, peak name, or tallest peak. If you indicate a scale other than mv for a channel, make corresponding changes to the scaling for each of the other channels being collected. mvolt (displayed for User Units scale). The number of mvolts that correspond to the value indicated in the User Units text box. User Units (displayed for User Units scale). The number of user units that correspond to the value indicated in the mvolt text box. Label (displayed for User Units scale). The label to identify the user units. 7-11

166 unipoint user's guide Peak Name (displayed for Named Peak scale). If you already created the peak table, described on page 7-13, select the name of the peak using the list box. If you have not created it, you can type in the name of the peak as it will be entered in the peak table. The specified peak extends to 100% on the chromatogram plot. All other peaks are plotted in relation to that peak. Smaller peaks are less than the full-scale height; larger peaks are displayed as off-scale. modifying data channel descriptions To add, change, or remove a data channel description: 1 From the Data Channel Scales dialog, choose the Edit button. The Edit Channels dialog appears. If any data channels were already named in the Data Channel Scales dialog, their names appear in the list box. 2 To add a name, type the description in the text box of the Edit Channels dialog and click on Add. Repeat for each channel. To change a channel name, click on its description in the list box and type the new description in the Name box. Then click on the previous description in the list box to update the list. To remove a channel name, click on it in the list box and then click on Delete. 3 Click on OK to exit the Edit Channels dialog. The names of the data channels appear in the list box in the Data Channel Scales dialog. 4 Select the analysis channel and set scaling parameters for that channel and any other data channels, as described previously. checking attributes for lines and symbols To review and modify the color assigned to data channel traces or to turn on or off gridlines in the chromatogram plot: 1 From the Data Channel Scales dialog, choose Attributes. 2 When the Data Channel Display Attributes dialog appears, select a channel from the list box and check the options for it. If necessary, make any necessary modifications. When finished, choose OK or Cancel. 7-12

167 analysis methods Data Channel. Lists the description for each data channel. Style. Lists available styles for the line: solid, dashed, or dotted. Color. Lists available color choices for the line. Thickness. Identifies the width of the line in pixels. Signal Grid. Indicates which gridlines appear for the Y axis. Major gridlines are aligned with numbered tick marks while minor gridlines are positioned between the numbered tick marks. Time Grid. Indicates which gridlines appear for the X axis. Major gridlines are aligned with numbered tick marks while minor gridlines are positioned between the numbered tick marks. Peak table The Peak Table dialog is where you tell UniPoint about the peaks in data analyzed by the method. If you name peaks in the peak table, your reports refer directly to those peaks when reporting areas, heights, amounts, and so on. In the Peak Table dialog, you also provide information about the amount of material in calibrators. Remember, you must run calibrators at one or more calibration level to obtain an internal standard, external standard, or percent normalization report. To display the Peak Table dialog: 1 Choose Peak Table from the Analysis menu. The top of the dialog shows the Time/Name list box that identifies any peak information that has been set. As a default, the Names option button is selected and you see the Peak Names area at the bottom of the dialog. However, the options in this area change depending on the option selected in the View area. 7-13

168 unipoint user's guide Naming peaks When you select the Names option button in the View area, options appear in the Peak Table dialog for indicating peak names and their retention times. You get retention time information by injecting and acquiring traces for test samples. Time. The retention time for the peak. The limit of resolution is hundredths of a minute (0.01 min). Peak retention time is based on start of data collection indicated in the control method executed at the same time as the analysis method. If the beginning of data collection and injection are not synchronous, retention times reported deviate from actual retention times by their difference. Name. Identify each peak for the component it represents. Reference Peak (optional). The software uses reference peaks to correct for retention time shifts that can occur during runs that analyze multiple samples. When the software notices a difference between a reference peak s expected and actual retention times, it adjusts the retention times of the preceding non-reference peaks in its internal peak table. You can designate an unlimited number of reference peaks in both calibration and unknown samples. An ideal reference peak is a large isolated peak with a consistent retention time. You can designate several reference peaks with retention times at the beginning, middle and end of the run. If you designate just one reference peak, it should be a late-eluting peak. Color. Available color fills for the peak. The default is black. If you select a color, choose a pattern other than <None>. Pattern. Available design fills for the peak. The default is <None>. Absolute Error and Relative Error. These are the retention time window parameters. To compensate for possible retention time drift, UniPoint defines a window around the retention time for each peak. If a peak elutes at any time during that window, the software identifies it. 7-14

169 analysis methods Absolute error is a constant amount of time on either side of a peak s nominal retention time. The software automatically searches for eluted peaks at every named retention time plus or minus this amount of time. Relative error is a percentage of peak retention time. Relative error, and, therefore, the width of retention time windows, increase with increasing retention time. Because late-eluting peaks are often broader than early peaks, the software uses this correction factor to improve the likelihood of locating late-eluting peaks. UniPoint uses the following formulas to calculate the beginning and end of any peak s time window. Each peak's time window appears in the graph pane, if displayed. Beginning: End: Ret. Time - [Abs. Error + (Ret. Time x Rel. Error)] Ret. Time + (Abs. Error + Ret. Time x Rel. Error) identifying peaks To name and set retention time for peaks: 1 Check that the Peak Names area is displayed at the bottom of the Peak Table dialog. If not, choose the Names option button. 2 Type the retention time for the first peak. 3 Type the name of the peak. 4 Optional - Choose the Reference Peak check box. 5 Optional - Indicate a color and pattern fill for the peak. 6 If necessary, modify the absolute error and relative error. 7 Choose Insert to add the peak information to the Time/Name list box. 8 Repeat the above for each peak. modifying peaks To modify information for a peak: 1 Choose the peak s name in the Time/Name list box. 2 Modify the appropriate information in the Peak Names area. 3 Once again, choose the peak s name in the Time/Name list box, and its information is updated. deleting peaks To delete the information for a peak: 1 Choose the peak name in the Time/Name list box. 2 Choose Delete. 7-15

170 unipoint user's guide Entering level information for peaks When you select the Levels option button in the View area, the options described below appear in the Peak Table dialog. These options are for entering the amount of material present at each calibration level. Note: To identify internal standard(s) and set amount(s), follow the instructions on page Level/Amount. This list box shows the levels set for the peak selected in the Time/Name list box. (If information is grayed, the peak has been identified as an internal standard.) Amount. The amount of material injected for the peak. Enter the amounts from smallest to largest. Unit Label. The unit label associated with the amounts, usually mass units. This label appears next to the amount axis on calibration plots. specifying levels To set amounts for calibration levels: 1 Choose the peak name in the Time/Name list box in the upper part of the dialog. 2 If necessary, choose the Levels option button. The Peak Levels area appears at the bottom of the dialog. 3 Type the amount injected at the first level. 4 Choose Insert. 5 Continue inserting amounts for each calibration level for the peak. 6 Repeat steps 3-5 for each additional peak. Remember if the peak is an internal standard, refer to Indicating internal standard peak(s) on page 7-18 to indicate amount(s). 7 To associate a units label with amounts, indicate the label before exiting the Peak Table dialog. 7-16

171 analysis methods changing level amount To change an amount indicated for a level: 1 Click on the amount in the Level/Amount list box. 2 Type the correct value in the Amount box. 3 Click in the Level/Amount list box to update its information. deleting level To delete a level: 1 Click on the level and amount in the Level/Amount list box. 2 Click on Delete. 7-17

172 unipoint user's guide Indicating internal standard peak(s) When you select the Int Std option button in the View area, you can indicate one or more peaks as an internal standard and indicate amount of material and the time frame during which to use the standard. Internal Standard. When checked, indicates that the peak highlighted in the Time/Name list box is an internal standard. Amount. The amount of internal standard material injected for the internal standard peak. Start Time. The time at which to begin using the internal standard. End Time. The time at which to stop using the internal standard. It must be later than the Start Time. Internal Standard Actual Amounts. Select this check box if you want to indicate the actual amount of the internal standard added to an unknown injected during a run. When you set up an operations list that uses this analysis method, UniPoint automatically provides a text box so you can indicate this amount. specifying internal 1 If necessary, choose the Int Std option button. standard peaks 2 Choose the name of an internal standard peak in the Time/Name list box. 3 Choose the Internal Standard check box. 4 Enter the amount for the internal standard. 5 Enter the time range in which UniPoint uses the internal standard to normalize sample amounts. 6 Click on the name of an internal standard peak in the Time/Name list box to update its information. 7 Repeat steps 2 through 6 for each internal standard peak. Make sure that the Start and End Times for each internal standard peak do not overlap. 7-18

173 analysis methods 8 If you want to indicate the internal standard actual amount when you set up an operations list that uses this method, place an X in the Internal Standard Actual Amounts check box. Grouping peaks When you select the Groups option button in the View area, options for grouping peaks appear in the Peak Table dialog. UniPoint reports total area or height amounts for all peaks selected in addition to the area or amount of each individual peak. setting up group names To indicate group names: 1 Choose the Groups option button. 2 In the Group box, type the name to assign to a collection of peaks. 3 Choose Insert to add the name to the Group Names list box. 4 Repeat steps 2 and 3 to set up a group name for each collection of peaks. 5 If you discover a mistake, select the name in the Group Names list box and revise the text in the Group box. When you click on another area of the dialog, the change is automatically made. If you want to delete a group name, select it in the Group Names list box and choose Delete. grouping peaks To place peaks into groups: 1 Choose the name of the peak in the Time/Name list box. 2 Choose the group name in the Group Names list box. 3 Choose the Group command button. 4 Repeat steps 1 through 3 to assign other peaks to groups. 5 To remove a peak from a group, choose its name in the Time/Name list box and choose Ungroup. 7-19

174 unipoint user's guide Additional peak options UniPoint provides additional options for keeping track of and reporting on peaks. 1 Choose the Options command button in the Peak Table dialog. 2 Check and if necessary modify the settings in the Peak Table Options dialog see descriptions below. Then choose OK to return to the Peak Table dialog. Track Retention Times. When this box is checked, UniPoint automatically corrects for retention time shifts that may occur during a run in which multiple samples are injected. When the software notices a difference between a peak s expected and actual retention times, it adjusts the retention times for all peaks so they are integrated. Void Volume Retention Time. The retention time of unretained effluent. This value is used to calculate capacity factor (k ). 7-20

175 analysis methods Background removal The Chromatogram Background Removal dialog lets you identify the blank chromatogram whose data is subtracted from sample data during a run or during re-analysis processing. 1 Choose Background Removal from the Analysis menu. 2 Set the parameters, described below, in the dialog and choose OK. Blank Location. None indicates that you do not want UniPoint to perform background subtraction. Choose Internal if the first injected sample in the run is the blank. Choose External if the information for the blank is located in a data file. Data File. The name of the file containing the blank chromatogram. Click in this field and then use the Browse button to locate the file, sample, and data channel to use for background subtraction. Sample. The description indicated in the operations list for the sample. Channel. The channel via which the data for the external sample was collected. 7-21

176 unipoint user's guide Producing reports To set reporting conditions and set report formats, use the Report menu. Report Type identifies how collected data are reported. Report Output indicates what reports are saved to disk or printed. Calibrator Format, Unknown Format, Calibration Summary Format, Unknown Summary Format identify the column information printed for each type of report; let you select from additional choices to customize reports. Report type The Report Type dialog identifies the kind of report to generate for each sample analyzed by the method. To set report type information: 1 Choose Type from the Report menu. 2 Set the parameters, described below, in the dialog and choose OK. report type The report type is the technique used to determine the amounts of components in a sample. Choose the report type that gives you the most relevant information about your samples. Area or Height Percent. This report type can be used during method development while you try to stabilize chromatographic conditions and determine component retention times. In unknown samples, the actual area or height of each peak is reported. The area or height is also given as a percentage of the total area or height of all integrated peaks in the sample. This report type assumes that the detection properties of all peaks are identical. Percent Normalization, External Standard, and Internal Standard. These are calibration report types. These report types require that you inject calibrators and provide information in the Peak Table dialog about each component of interest before injecting unknown samples. Calibrators account for differences in absorbance properties among peaks. They provide data that UniPoint uses to generate a calibration table that is then used to calculate and report amounts for unknowns. 7-22

177 analysis methods Additional options appear in the dialog box when you select one of the calibration report types. quantify by UniPoint quantifies peaks by measuring either the area or height of integrated peaks. Peak area and height measurements are a function of many chromatographic variables, including mobile phase composition and flow rate. Peak Area. Because peak area is relatively independent of mobile phase composition changes, it is the preferred measurement technique for gradient elution. Peak area determination is less subject to variations in instrument conditions or operator technique than is peak height. The major disadvantage is that peak area measurements are affected by the presence of neighboring peaks. Peak Height. Peak height is less subject to interference by adjacent, overlapping peaks and is, therefore, the method of choice when measuring overlapping peaks, particularly small peaks on the tailing edge of a larger peak. Peak heights are also relatively independent of flow rate changes. include By default, UniPoint reports information about every peak that meets its integration requirements. However, for some applications, you may want to get information only about the peaks of interest. Unnamed Peaks. If you want UniPoint to report information about every peak that meets its integration requirements, place an X in this check box. If you only want information on peaks named in the peak table, do not place an X in the check box. If the software integrates peaks that have not been named in the peak table, it assigns the peak a name based on the order in which it eluted. For example, the peak table named two peaks, Phenol and Benzaldehyde. However during the run, the software detects a peak between those two peaks. The unnamed peak would be labeled Peak 2. Min. Area or Height. You can specify a minimum valid peak area or height to exclude insignificant peaks from reports. Peaks with area or height smaller than the specified value are not reported and baselines are not drawn for those peaks. 7-23

178 unipoint user's guide calibration When using one of the calibration report types, UniPoint requires additional information about the calibration procedure. Arrangement. Indicate when calibrators are injected during the run (or ordered for re-analysis processing). Sequential: All calibrators are injected before unknowns. Bracketed: A group of calibrators is injected before and after each group of unknowns. Both groups of calibrators are used for analysis of the unknowns. Random: Calibrators are dispersed throughout the run. Preloaded: Calibrators were injected and analyzed during a previous run. The software uses the specified Preloaded Calibration File and Calibration Table Number within the file to analyze samples. Curve Fit. Indicate how to construct calibration curves. The software generates curves using data collected from calibrators and uses the curves to determine component amounts in unknowns. Point to Point: UniPoint connects the mean amount at each calibration level using a linear segment. For a one-level calibration plot, UniPoint generates a linear segment between the origin (0 area, 0 amount) and the mean peak area at the calibration level. Point to Point through Zero: UniPoint generates a linear segment between the origin (0 area, 0 amount) and the mean peak area at the first calibration level. It then connects the mean amount at each additional calibration level using a linear segment. Linear: UniPoint constructs a line that best represents the set of means. Quadratic: UniPoint generates a smooth curve that best represents the set of means. The curve may have one bend. Cubic: UniPoint generates a smooth curve that best represents the set of means. The curve may have two bends. 7-24

179 analysis methods After the run or before re-analysis, you should print or view the calibration curves to make sure that only one amount can be determined for a given area (or height). See Section 12, Calibrations. Preloaded Calibration File. If you indicated preloaded in the Arrangement list box, click in this box and then choose the Browse button. Use the dialog that appears to select the calibration file. Cal. Number. If multiple calibration tables are stored in the preloaded calibration file, indicate the table to use. options When you select the Options button, the Report Type Options dialog appears. Sample Amount. Select this check box if you want to indicate the total amount of material present in the injection volume of an unknown sample. When you set up an operations list that uses this analysis method, UniPoint automatically provides a text box for indicating this amount. If you indicate a sample amount, the percent concentration (as related to the total sample amount) of each reported peak is included in each report. The sample amount is unitless UniPoint lets you use any unit appropriate to the separation. Usually, sample amount is expressed in mass units. Nominal/Actual Amounts. If you want to indicate nominal and actual amounts for unknowns injected during a run, select this check box. When you set up an operations list that uses this analysis method, UniPoint automatically provides text boxes for indicating these amounts. Actual amount is the amount you actually measured to prepare a particular unknown sample. Nominal amount is the amount you wanted to measure. Using the actual and nominal amount, UniPoint automatically adjusts the amount of material in each calculated peak and reports instead the amount that would have been found if the sample had contained exactly the nominal amount of unknown: Unknown Peak Groups. Select this check box if you want to group unknown samples and obtain statistical data, such as standard deviation and standard error, for their peaks. When you set up an operations list that uses this analysis method, UniPoint automatically provides a text box for indicating the group number (1, 2, 3...) to assign to an unknown sample. 7-25

180 unipoint user's guide Report output The Report Output dialog identifies which reports should be saved to disk and which should be printed when the analysis method is executed. 1 Choose Output from the Report menu. 2 Choose whether to save, export, and print reports for samples analyzed by the method and what to print. Choose OK to exit. Each calibrator, unknown, and summary report has default information, identified by Gilson, that are inserted into its report. You can modify the report format as described on page Each time you analyze raw data, a report file and summary file can be saved to disk. In some cases, it is easier to save reports to disk during the run and then print them later. When you print reports at run time, each report may include the following: annotation that identifies the data file and how it was collected and analyzed. table of peak information and amounts if calibrators were injected or if a pre-stored calibration table was specified. graph of chromatogram plot for acquired traces or calibration plot for a calibration summary report. The check boxes in the Export area let you select which reports should be saved to a text file. The File Name box indicates the path and name to which the report information is saved. You can use the Browse button to locate an existing export file. If you want to overwrite the previous file with new information, do not modify the name in the File Name box. If you do not want the file overwritten, indicate a different file name. 7-26

181 analysis methods setting layout options For report printouts generated during a run or re-analysis processing, you can specify the width of page margins, size of chromatogram plot, and printing order for report components. 1 Choose Output from the Report menu. 2 In the Report Output dialog, choose the Layout button. 3 When the Report Layout dialog appears, review and if necessary modify the current settings. Margins. In the text box next to each margin (left, right, top, and bottom), specify the width of the margin. The software adds the indicated width to the default print area set for the page by the printer. The measurement system, mm or inches, is set using the International program in the Windows Control Panel. Graph Height. Indicate the size of the chromatogram plot as a percentage of the page. It is printed on a different page than the annotation or report if necessary. Position Order. The order in which the various parts of the report are printed, by default: annotation, graph, then table. 4 Choose OK to save any changes. 7-27

182 unipoint user's guide report annotation You can choose which parts of the analysis report s annotation will be viewed and printed. To make your selections: 1 Choose Output from the Report menu. 2 In the Report Output dialog, choose the Annotation button. 3 When the Annotation Contents dialog, use the check boxes to turn off or on parts of the annotation or enter a custom calculation. The next 2 pages identify the various parts of a report annotation so you can determine which parts to include in reports. See page 7-37 for information on entering a custom calculation into the annotation. 7-28

183 analysis methods Example annotation Data File: c:\gilson\tstmx\tstmxlvl.gdt Date acquired: Tue Apr :14:35 Control Method: C:\JEAN\FC_205\TSTMXLVL.GCT Analysis Method: C:\GILSON\TSTMX\TSTMX254.GAN Sample name: Step 1 Injection Number: 1 *Unknown* Analyzed on: Mon Jan :17:13 Events 1 0 Default Baseline initial default 2 0 Disable Negative Peak Integration initial default 3 0 Peak Width 0.2 initial default 4 0 Peak Sensivity2 initial default Fractions Collected: 5 (FRACTIONS:1-5) W0= AU at 254 nm at 1.05 min Number of Peaks Detected: 5 Channel Scales <Auto range> 254nm 0.00 to mvolts (0 %offset), Data rate: (points/second) <Analysis Channel> 10 mvolts == 0.01 AUFS 220nm 0.00 to mvolts (0 %offset), Data rate: (points/second) 10 mvolts == 0.01 AUFS Reporting External Standard Report (quantify by area) Min area reported: 0 Calibration type: Sequential Calibration curve fit: Linear Include summary statistics for unknown group samples Include individual values for unknown group samples Include summary statistics for standard samples Include individual values for standard samples Use actual sample amount from operations list Associated files Sample description Analysis description Analysis events Sample description Realtime event Sample description Channel scales Report description Calibrator Report Format Columns Inj. Number (Inj. Number) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Peak Name (Peak Name) Factor:1.00, Width:18, Justify:Center, Format:Decimal, Decimals:2 R. Time (R. Time) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Area (Area) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Level Amount (Level Amount) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Sample Descrip. (Sample Descrip.) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Unknown Report Format Columns Inj. Number (Inj. Number) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Peak Name (Peak Name) Factor:1.00, Width:18, Justify:Center, Format:Decimal, Decimals:2 R. Time (R. Time) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Area (Area) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Amount (Amount) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Amount % (Amount %) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Sample Descrip. (Sample Descrip.) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Report columns Calibrator Summary Report Format Columns Inj. Number (Inj. Number) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Peak Name (Peak Name) Factor:1.00, Width:18, Justify:Center, Format:Decimal, Decimals:2 R. Time (R. Time) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Area (Area) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Level Amount (Level Amount) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Sample Descrip. (Sample Descrip.) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Unknown Summary Report Format Columns Inj. Number (Inj. Number) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Peak Name (Peak Name) Factor:1.00, Width:18, Justify:Center, Format:Decimal, Decimals:2 7-29

184 unipoint user's guide R. Time (R. Time) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Area (Area) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Amount (Amount) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Amount % (Amount %) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Sample Descrip. (Sample Descrip.) Factor:1.00, Width:12, Justify:Center, Format:Decimal, Decimals:2 Report columns Report Output Save report to file Save calibration summary Save unknown summary Print: <nothing> Peak Analysis Error Conditions Report description Background Blank Removal <None> Track peak retention time Variables Name: S_NOM_AMT, Prompt: Nominal Amt, Limits: <None>, Default: <None> Name: S_ACT_AMT, Prompt: Actual Amt, Limits: <None>, Default: <None> Peak Table 1) 2.80 Peak 1 Lvl. Amount Lvl. Amount Lvl. Amount [ 1] 5 0 [ 2] 9 0 [ 3] 180 2) 3.70 Peak 2 Lvl. Amount Lvl. Amount Lvl. Amount [ 1] 50 [ 2] 100 [ 3] 220 3) 4.80 Peak 3 Lvl. Amount Lvl. Amount Lvl. Amount [ 1] 2 0 [ 2] 4 0 [ 3] 9 0 4) 5.60 Peak 4 Lvl. Amount Lvl. Amount Lvl. Amount [ 1] 4 0 [ 2] 8 0 [ 3] 150 Peak table Peak Detection Parameters Relative Error: 5 (%) Absolute Error: 0.1 (min) Control Method Notes Notes entered into the control method print here. Data Notes Notes entered into the operations list print here. Analysis Method Notes: Notes entered into the analysis method print here. Data notes Analysis notes 7-30

185 analysis methods Report format review and modification If you want to vary the default format for reports, use the Format commands under the Report menu. You can add or remove column information. checking columns To view what is printed for unknown, calibrator or summary reports: 1 From the Report menu, choose Column Formats. 2 When the Report Column Format dialog appears, click on the option button to see the columns available for calibrator, unknown, and summary reports. Column Contents. The contents of each column currently specified for the report type. The names of the columns are listed in the order, from left to right, that they appear on the report. Contents (or Expression if you clicked on the Custom check box). The contents of the selected column. The types of contents that can appear in a report type are set by Gilson. To view additional selections click on the down arrow. You see the Expression label if you indicated a custom calculation for the column. You can then type the formula that UniPoint uses to generate the amount that appears in the column, or click on the Builder button to display the Expression Builder dialog. See page 3-31 for procedures on specifying a custom calculation. Note: You must add the column for the custom expression to each report format if you want UniPoint to generate the information. Highlight the text in the Expression text box and press CTRL+C to copy the text to the Clipboard. Then choose the option button in the Report Type area. Click on the Custom button and click in the Expression text box and press CTRL+V. Then click on Insert. Use the up or down arrow button to move the new column to the appropriate location. 7-31

186 unipoint user's guide Custom. Choose the Custom check box if you want to include a column that is not part of UniPoint's default columns. When you click on the Custom check box, a Builder button appears and the Contents text box becomes the Expression text box. If you click on the Builder button, you see the Expression Builder dialog. See page 3-31 for procedures on specifying a custom calculation. Heading. The descriptive text that appears at the top of a column and identifies the type of information that appears within the column. Width. The width of the column in which the information is placed. For most columns, the default value is appropriate. However, you may need to widen the columns for sample description and peak name depending on your application. Factor. A number by which each value in a column is multiplied. Justify. How information in the column is aligned. Format. How numbers are displayed: decimal, with or without commas, or scientific notation. Decimals. The number of positions displayed after the decimal point. Individual Peaks (for summary report formats only). When selected, causes the information specified in the Column Contents list box to be reported for each integrated peak. Statistics (for summary report formats only). When selected, causes the mean, standard deviation, standard error, and number average to be reported for each numerical entry specified in the Column Contents list box. (This includes items such as injection number and level amount in addition to area and height.) Note: If you remove the X from both the Individual Peaks and Statistics check boxes, no summary report information is generated. 3 If you want to customize a report's contents, refer to the sidebars below. Otherwise, choose OK to exit the dialog. Note: If you are controlling a Gilson fraction collector via UniPoint, add the Fraction Site(s) column to report formats. The column lists tube locations for eluent collected for a peak. changing an attribute To assign a new attribute to a report column: 1 Select the column in the Column Contents list box. The current attributes for the column appear in the text and drop-down list boxes. 2 Use the text and drop-down list boxes to make the necessary changes. 7-32

187 analysis methods 3 Choose Change. 4 If you are done making modifications, choose OK to exit the dialog. When the report is generated, UniPoint formats text in the column using the new attributes. renaming a column To change a column heading in a report: 1 Select the column in the Column Contents list box. 2 Type a new heading in the Heading box. 3 Choose Change. 4 If you are done making modifications, choose OK to exit the dialog. When the report is generated, UniPoint assigns the new heading to the column. inserting a column In the table, columns are inserted to the left of the column selected in the Column Contents list box. You can place the highlight after the last column to insert a column there. To add a column: 1 Select the appropriate column in the Column Contents list box. Or, click beneath the last column to add a column there. 2 Use the text and drop-down list boxes to set the attributes for the new column. 3 Choose Insert. 4 If you are done adding columns, choose OK to exit the dialog. When the report is generated, UniPoint includes the column. removing a column To remove a column: 1 Select the column in the Column Contents list box. 2 Choose Delete. 3 If you are done making modifications, choose OK to exit the dialog. When the report is generated, UniPoint does not include the column. If you need that column later, use the inserting a column procedures. changing the position of a column To move a column: 1 Select the column in the Column Contents list box. 2 Click on the up and down buttons to change the position of the column in the report. 7-33

188 unipoint user's guide Report customization In UniPoint, you can specify custom calculations that enable you to generate report results to meet your needs. You can indicate custom calculations in report columns, report annotations, and peak labels. Note: For a description of the calculations that UniPoint currently uses to generate its chromatography calculations, refer to Appendix F. inserting custom calculation into report column The following example shows you how to include a column in individual and summary reports that uses the United States Pharmacopoeia calculation to generate the plate number for each peak. The United States Pharmacopoeia (USP) calculation is as follows: Note: UniPoint uses the British Pharmacopoeia calculation to generate plate number, which is as follows: To insert a report column that will generate the USP plate number for each reported peak: 1 Create or open the analysis method. 2 Choose Column Formats from the Report menu. 3 Select the option button of the report to which the column will be added. In this example, the Unknown report type is selected. 7-34

189 analysis methods 4 Click in the Custom check box. 5 Click on Builder to access the Expression Builder dialog which will assist you in specifying your custom calculations. The Expression Builder dialog lists UniPoint quantities, mathematical operators, peak names, and variable names that can be used in your calculation. To learn more about the dialog, click on its Help button. For the items in the Calculable Quantities list box, the description includes syntax and usage information. 6 In the Expression text box, type the custom calculation to generate the USP plate number for each reported peak: RETENTION_TIME/WIDTH(0)^2*16 The (0) argument in the equation identifies the fraction peak height, which in this example is 0 because the calculation requires the width at the baseline. Be aware that instead of typing the calculation, you can double-click on an identifier (such as WIDTH) and have it appear at the cursor location in the Expression text box. 7 Click on OK to return to the Report Column Format dialog. 8 In the Heading text box, indicate a description for the custom calculation. For this example, the heading could be USP Plate. 9 Click on Insert. 7-35

190 unipoint user's guide 10 Use the up and down buttons to position the column in the report. 11 To include the column in another report type, highlight the calculation in the Expression text box and press CTRL+C to copy the calculation. 12 In the Report Type area, choose the option button for another kind of report. 13 Click on Custom. 14 If necessary, click in the Expression text box and then press CTRL+V to paste the calculation. 15 Use the up and down button to position the column in the report. 16 Repeat steps if you want to include the calculation in summary reports. The following example shows the USP Plate column and the results generated for each peak. 7-36

191 analysis methods adding custom calculation into report annotation A calculation in the annotation pertains to the entire report while a calculation for a report column pertains to each peak listed in the report. To include a custom calculation in the annotation: 1 Create or open the analysis method. 2 Choose Report Output from the Report menu. 3 When the Report Output dialog appears, choose the Annotation button. The Annotation Contents dialog appears. 4 Type the text for the calculation in the Custom Information text box. The calculation must be within curly brackets, {}. See the examples below. Or, click on Builder to access the Expression Builder dialog and use its options to specify the calculation. Once again, the calculation entered in the Expression text box must be within curly brackets. Example 1: To display the total area for three integrated peaks, the custom calculation would be: {AREA(1)+AREA(2)+AREA(3)} Example 2: To insert text identifying the custom calculation, type the text but do not place it within brackets, for example: Total Peak Area = {AREA(1)+AREA(2)+AREA(3)} 7-37

192 unipoint user's guide adding custom calculation into peak label UniPoint allows you to modify what is displayed next to a peak in the chromatogram plot printed in reports. You can include the peak name plus additional information next to each integrated peak, for example, the peak area can be listed. To identify what you want to appear next to peaks: 1 Create or open the analysis method. 2 Choose Peak Table from the Analysis menu. 3 Use the Peak Table dialog to set peak information. 4 Choose the Options button to display the Peak Table Options dialog. 5 In the Peak Annotation box, type text or indicate a calculation that will be performed and its results listed next to the peak label. Refer to the following examples. Example 1: To display the peak name and its area, indicate the following in the text box: {PEAK_NAME}{AREA} Example 2: To insert text identifying the expression after the peak name, type the text but do not place it within brackets, for example: {PEAK_NAME} Area={AREA} The following diagrams show the peak name and area as it would appear depending on what you entered in the Peak Annotation box. The captions under the diagrams identify what was entered in the Peak Annotation box. Instead of area, you could display amount, asymmetry, plates, and so on, or your own custom calculation after the peak name. You can use the Expression Builder to create the calculation used to generate the result that will appear next to the peak. To access the Expression Builder, select the Builder button in the Peak Table Options dialog. Remember to place the calculation within brackets. 7-38

193 analysis methods Variables To make your analysis methods more flexible, you can indicate a variable name instead of entering a value in a command parameter box. A variable name can be any combination of letters and numbers and can contain special characters (_, as long as it is not the first character or a mathematical operator (+, -, *, /, %). It cannot include spaces. Valid variable names would be PeakWidth and Peak_Sensitivity. UniPoint does not differentiate between upper and lower case letters in a variable name. For example, it views the names PeakWidth, peakwidth, and PEAKWIDTH as identifying the same value. The command parameter specified for an event can also be the result of performing a mathematical function on two variables or a variable and number. For example, PeakWidth * 2 is a valid command parameter. You can add (+), subtract (-), multiply (*), divide (/), and return the remainder (%). When setting up the Operations window for a run, UniPoint prompts you for a value to assign to each variable. To see what variable names have been set for an analysis method and to indicate lower and upper limits for and a default for each variable, do the following: 1 From the Analysis menu, choose Variables. 2 When the dialog appears, choose the variable name in the list box. 3 Check the prompt that appears whenever the software references the variable. The prompt can be any combination of letters and numbers and can contain special characters and spaces. 4 To indicate minimum and maximum values for the variable, indicate the numbers in the Values Limits box. Separate the values with a colon, for example, 1:100. If the input is required, type an R before the minimum value, for example, R1:100. You can indicate variable names instead of limit values, for example, 1:PeakWidth. 7-39

194 unipoint user's guide 5 If you want to see a default value for the variable name, type that in the Default Value box. 6 To remove an unused variable name, choose it in the list box and choose Delete. 7 Choose OK when finished. if you add, change, or delete variable names If you add, change, or delete variable names and the analysis method is used in an operations list, you need to recreate the operations list or modify each step in the list that uses the analysis method, as outlined below. 1 Open the operations list. 2 Double-click on the step that executes the analysis method. 3 In the Step Entry dialog, click in the Analysis Method box and use the Browse to reselect the analysis method. The parameter text boxes at the bottom of the dialog update to reflect the changes in the analysis method. If a variable is no longer used by the method, its parameter box is grayed. 4 Enter the correct value in each parameter box and click on Change to update the operations table. 5 Use the scroll arrows to display information for the next step that uses the analysis method. 6 Repeat steps 3-5 for each step that uses the analysis method. 7 Choose Done when finished modifying steps. 8 To remove the table column for a variable that is no longer used: a) Display the Format Columns dialog by choosing its command from the Table menu or double-clicking on the header row in the table. b) In the Column Contents list box, choose the variable name and choose Delete. c) Choose OK to exit. 9 Save and close the operations list using the File menu. 7-40

195 analysis methods Error handling UniPoint can perform validation and system performance checks on peaks in samples. The Error Handling dialog identifies the error conditions that UniPoint should be aware of when analyzing data using the method. To display the dialog box: 1 From the Analysis menu, choose Error Handling. activating error handling Selecting a peak name and indicating error conditions for it activates error handling. What happens during a run or re-analysis processing then depends on whether an error control method is entered and whether the Continue on error check box is selected. If neither a control method nor the Continue check box is selected, the run or re-analysis processing is stopped when the error condition is met. If both a control method and the Continue check box are selected, UniPoint does one of the following: * during a run, it executes the error handling method and then proceeds to the next step in the operations list. * during re-analysis processing, it proceeds to the next step in the re-analysis list. If a control method is indicated but the Continue check box is not selected, UniPoint does one of the following: * during a run, it executes the error handling method and then stops the run. * during re-analysis processing, it stops processing. If a method is not indicated, but the Continue check box is selected, UniPoint finishes the current step and proceeds to the next step in the operations list or re-analysis list. 7-41

196 unipoint user's guide indicating error conditions To specify error conditions: 1 In the Error Handling dialog, indicate parameters as described below. Peak Names. Choose the names of the peak to monitor. The list box shows the peaks set in the peak table. Error Method. Specify the path to and name of the control method to execute when the selected error condition is met. If necessary, click in this text box and then choose the Browse button to select the file. Continue on error. Choose this check box if the run or re-analysis processing should continue even if an error has been encountered. Refer to activating error handling, above, to see how this check box affects what happens during the run or re-analysis. Error Conditions. In the list box, choose the peak calculation or value to monitor. Using the option buttons, choose one of the mathematical operators: greater than, less than, or outside range. In the text box(es), type the monitor value(s). 2 Repeat for the next peak. 3 When finished, choose OK to exit the dialog box. removing error conditions To cancel an error condition set for a peak. 1 Choose the peak name in the list box. 2 Choose Clear. 3 Repeat steps 1 and 2 to remove any other error conditions. 4 Choose OK to exit the dialog box. 7-42

197 8 Operations Using the options in the Operations window, you specify the list of steps to execute during a run. A step in the list can identify a sample to inject and the control and analysis conditions to process that sample. Or a step may identify a control method to use for system startup or shutdown. This section describes the components of the Operations window and how to generate the operations list. The Operations window is also where you start and monitor the progress of a run. You learn more about that in Section 9. Plus, it is where you perform manual control for developing methods, described in Section

198 unipoint user's guide Operations window You access the Operations window by creating an operations file or by opening an existing file. Following is an Operations window. When you first display the window, the graph pane may not be shown. Choose Show Graph from the Graph menu. Refer to Section 5, Common Procedures for more information on manipulating parts of the table or graph displayed in a document window. menu bar toolbar table pane status box graph pane annotation box status bar legend box menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Menu File Edit List Entry Manual Run Table Graph Window Help Description Create, save, print, delete files or exit a window or UniPoint. Locate, make changes to, or transfer information. Set step-by-step instructions to perform during a run. Control HPLC system manually. Start, stop, pause run and indicate file naming parameters. Display or modify appearance of the table pane. Display or modify appearance of the graph pane. Re-arrange windows or icons, or activate a window; display annotation or toolbar. Learn about UniPoint. table pane Lists the steps to execute during a run. Double-clicking on a row automatically displays the dialog box used to set up parameters for the step. Using the right mouse button, you can double-click on the control or analysis method in a step and display the window that shows the instructions set in the method. 8-2

199 operations graph pane Shows the run-time chromatographic plot of acquired data, gradient profile of the control method, plus run-time events. (See page 5-27 for a description of the lines and symbols used for run-time events.) The trace begins at the data collection begin time (usually synchronized with injection). For the gradient profile, the mobile phase composition (Y axis labeled % Mobile Phase) is plotted with respect to time (X axis). toolbar Provides tools for manipulating the graph and for starting, pausing, and stopping the run and/or stopping pump flow. To activate a tool, just click on its icon. navigator tool open tool save tool print tool arrow tool magnifying glass tool cross-hairs tool full-scale tool original view tool stop tool pause tool go tool stop pumps tool Displays Navigator. Displays Open dialog. Stores operations list to disk. If it has not been saved, displays the Save As dialog. Displays Print dialog. Turns off magnifying glass and cross-hairs tools. For enlarging portion of graph. For identifying the mv (or user units) and time values for a point on the trace. For returning graph axes to minimum and maximum values for displayed trace(s). For returning graph axes to minimum and maximum values set in control method s Strip Chart Options dialog. See page For terminating run. For pausing run. For starting run. For stopping pumps. To turn off/on the toolbar, choose its command from the Window menu. 8-3

200 unipoint user's guide legend box Identifies the symbols and lines shown in the graph. To turn on/off the legend, choose its command from the Graph menu. For each trace being collected, the legend identifies the following: By double-clicking on an item in the legend, you can access the Attributes dialog. Use that dialog box to remove events from display or to change the size of symbols and lines shown in the graph. Note: The symbols and lines used for run-time events (such as detector autozero) are not identified in the legend. See page 5-27 for a description of those symbols and lines. status box Displays status information for the components of the HPLC system. You see pressure information and contact status in this box. Plus during the run, you see user comments indicated in the method or routines file being executed. For system control to occur, the message System Status must be displayed in this box. If the message Disconnected is shown, you can set up and modify the list; however, it cannot be run. To run the list, manually start the Method Executor, described on page 9-2. If you see the message No Control Method - Edit Only Mode, you must set up and save a control method before controlling system components via an Operations window. annotation box Displays user comments for the operations list. To turn on/off the annotation, choose its command from the Window menu. To add comments, use the Notes command in the Edit menu. status bar Shows operational status or help message if a menu command is highlighted. 8-4

201 operations Setting up an operations list You can set up and make changes to the operations list using the Run Entry dialog or using the commands in the Edit menu. If you are building a new operations list, you can use the Express Entry dialog or the Step Entry dialog to get started. If you are making changes to an existing operations file, use the commands in the Edit menu or Step Entry dialog. Multi-system users. Make sure only one configuration file is referenced by the steps in the list. before creating list Before you set up an operations list, create the control and, optionally, analysis methods to use during the run. If injected samples are analyzed and reported on by more than one analysis method, you need to add columns to the table. To add a column for an additional analysis method: 1 Double-click on the top line in the table to reveal the Format Columns dialog. (Or choose Format Columns from the Table menu.) 2 Select a column in the Column Contents list box. (In the operations table, the new column inserts to the left of this column.) 3 Choose Analysis Method 2 in the Contents drop-down list box. 4 Make any additional changes in the other text boxes. 5 Choose Insert. 6 Repeat steps 2-5 to insert another analysis method column if necessary. 7 When finished, choose Done to exit the dialog. The table displays the new column(s). If the operations list had information in it you see <Replace> in each row of the added column. You can use the Replace command in the Edit menu to quickly substitute <Replace> with the path to and name of the analysis method to use during the run. 8-5

202 unipoint user's guide Using express entry dialog for list setup To quickly generate the operations list, you can use the Express Entry dialog. This method of list setup is preferable if you are injecting all unknowns or calibrators, or you are injecting a group of calibrators followed by a group of unknowns. If you are randomly injecting calibrators and unknowns, refer to Using Step Entry dialog for list setup and modification on page Choose Express from the List Entry menu. 2 Use the options in the Express Entry dialog to set information for the operations list. 3 Choose Generate to create the list. If steps are currently set in the operations list, they are replaced by the newly generated steps. 4 Refer to Modifying the operations list using edit menu on page 8-13 for details on quickly assigning descriptions to samples. Following is a description of the options in the Express Entry dialog. Calib Levels. For calibration analyses, type the number of calibration levels being injected for calibrator samples. Repeats/Level. If you are making multiple injections of each calibrator at a level, type the number of repeats here. UniPoint averages each level s peak data and uses the average peak size to build the calibration curve for each standard peak. A value of 1 represents a no-repeat condition or in other words one injection per calibration level. Number of Unknowns. Tell UniPoint how many unknown samples are injected after the calibrators. 8-6

203 operations Repeats/Unknown. If you are making multiple injections of each unknown, type the number of repeats here. A value of 1 represents a norepeat condition or in other words one injection per unknown. Number of Cycles. If running a multi-cycle calibration and unknown analysis, indicate the number of cycles here. For example, a calibration analysis consists of 4 calibration levels with 3 repeats per level and 5 unknown samples with 2 repeats per unknown. How many steps are generated for 1 calibration cycle? For 2 cycles? 1 cycle: Steps = (4 levels x 3 repeats per level) + (5 unknowns x 2 repeats per unknown) = 22 2 cycles: Steps = (22 injections per cycle x 2 cycles) = 44 Control Method. Type, or use Browse to specify, the path to and file name of the control method used to control devices. Analysis Method. Type, or use Browse to specify, the path to and file name of the analysis method used to analyze collected data. Additional text boxes. Other text boxes may automatically appear depending on the parameters set in your control and analysis methods. For example, if an injector protocol requests inputs, such as sample volume and sample location, text boxes to answer those inputs appear. If one of the inputs requests the fraction site at which to begin collection, refer to Setting up operations list for fraction collection on page If you indicated in the analysis method that actual and nominal amounts should be entered for samples or that unknowns should be grouped for additional reporting, text boxes appear for those also. See page 7-25 for a description of actual and nominal amounts. If you added columns using the Format Columns dialog, you see text boxes to indicate information for those columns. For example, you may have added a column for a second analysis method. 8-7

204 unipoint user's guide Using step entry dialog for list setup and modification To indicate steps in the operations list, you can use the Step Entry dialog. This setup method is useful if you are randomly injecting calibrators. The Step Entry dialog can also be used to modify or delete steps in an existing operations list. 1 Choose Step from the List Entry menu. The Step Entry dialog appears. (If the table already has one or more steps, you can doubleclick on a row to quickly access the dialog.) 2 Use the options in the dialog box to create or modify the operations list. The text boxes that appear in the dialog box vary depending on conditions set in the control or analysis method specified for a step. The Step up and down arrows in the upper right corner enable you to scroll through existing operations steps. Description. Text describing the sample. If you enter a number in the description, you can have UniPoint automatically increment the number when multiple entries are generated. To see the Inc. box for incrementing the sample description, include a number in the Description box (for example, Sample 1) before you choose the Multiple button. Control Method. The path to and file name of the control method used to control devices. Double-click in the text box and then use the Browse dialog that appears to select the file. Analysis Method. The path to and file name of the analysis method used to analyze data collected for the sample. Double-click in the text box and then use the Browse dialog that appears to select the file. 8-8

205 operations Additional text boxes. Other text boxes may automatically appear depending on the parameters set in your control and analysis methods. For example, if an injector protocol requests inputs, such as sample volume and sample location, text boxes to answer those inputs appear. If one of the inputs requests the fraction site at which to begin collection, refer to Setting up operations list for fraction collection on page If you indicated in the analysis method that actual and nominal amounts should be entered for samples or that unknowns should be grouped for additional reporting, text boxes appear for those also. See page 7-25 for a description of actual and nominal amounts. If you added columns using the Format Columns dialog, you see text boxes to indicate information for those columns. For example, you may have added a column for a second analysis method. adding step To add the first step to a new list or add a step to the bottom of the list: 1 Make sure New Step appears in the upper right corner of the dialog box. If not, choose New. 2 Specify information in the text boxes. For file names, you must indicate the complete path to the file. Therefore, click in the text box and then use the Browse button to select the file. 3 Choose Insert when finished indicating the step s information. 4 Repeat to add more steps. inserting step between steps To insert a step before the first step or between two existing steps: 1 Use the Step up or down arrow to select the step before which the new step inserts. For example, to place a step between steps 3 and 4, display information for step 4 in the text boxes. 2 Use the text boxes to specify information for the step. 3 Choose Insert. generating multiple steps The easiest way to set up the operations list is to generate a list of all of the steps and then modify the steps as necessary. 1 Choose the Multiple button. You see additional options. Refer to the dialog box on next page. 8-9

206 unipoint user's guide Inc. An Increment box appears next to text boxes that prompt you for a value, such as Calib. Level. If you do not want to increment the value for each step, indicate 0 (zero) or leave the box blank. Num. Steps to Generate. How many steps to add to the bottom of the list. Num. Repeats per Step. How many multiples of the step to generate. This is especially useful for specifying multiple injections of the same calibrator or unknown. 2 Indicate information in the text boxes. 3 Choose Generate. If you already have one or more steps indicated, the steps are added to the bottom of the existing list. 4 Make the necessary changes to each step. See modifying step, below. Refer to Modifying the operations list using edit menu on page 8-13 for details on quickly assigning descriptions to samples. deleting step To remove a step: 1 Use the Step up or down arrow so the information for the step is displayed. 2 Choose Delete. modifying step To change options for a step: 1 Use the Step up or down arrow so the information for the step is displayed. 2 Use the text boxes to make the necessary changes to the step. 3 Choose Change. 8-10

207 operations Using the tray file to generate the operations list When creating an operations list, you can browse to the tray file, select multiple tube sites, and have UniPoint generate steps in the operations list. 1 Create or open an Operations window. 2 Choose Step from the List Entry menu. 3 In the Step Entry dialog, indicate control method and analysis method information. 4 Enter values for all variables except the sample location variable (usually labeled TUBE or SAMPLE_LOCATION). 5 Click in the text box for the sample location variable and click on Browse. Or, you can double-click in the text box. 6 When the Browse Tray window appears, choose multiple tube sites as described below. To select random tubes within one zone: a) If necessary, click within the zone to make it active. Its tubes turn from gray to blue to indicate the zone is active. b) Press the Ctrl key and select each tube. Selected tubes are red. To select a range of tubes within one zone: a) If necessary, click within the zone to make it active. Its tubes turn from gray to blue to indicate the zone is active. b) Click on the beginning tube and then press the Shift key and click on the last tube. Selected tubes are red. Note: UniPoint will allow you to click and drag over more than one zone. However, be aware that when the software places the tube sites in the operations list, it sorts the tube sites by number only. The software does not sort by tube site within zone. Therefore, you may need to move steps so they are ordered the way that you want them. 7 Choose the Add Step(s) button. A message box appears specifying the number of steps that were added. 8 Click on OK when the message appears that tells how many steps were added. Then click on Done to return to the Step Entry dialog. 9 Click on Done to exit the Step Entry dialog. 10 Review the operations list and if necessary enter or change the sample description for each sample. To enter sample descriptions, use the Step Entry dialog or the Paste Special command within the Edit menu. 8-11

208 unipoint user's guide Setting up operations list for fraction collection If you are doing fraction collection, UniPoint can keep track of the tubes used for each injection (referred to as continuous collection). Or, you can indicate the tube at which to start collection for each injection (referred to as specific site collection.) Note: The Fraction Collector Control Technical Note, available from your Gilson representative, describes how to set up UniPoint to control a Gilson fraction collector. With continuous collection, you indicate the zone in which collection occurs. For the first injection, collection starts in the first tube of the zone. For the second and each subsequent sample collected into the zone, collection will begin in the next available numbered tube. (See Examples 2 and 3, below.) With specific site collection, you indicate both the zone and tube at which collection starts for each sample. (See Example 1, below.) You may use both continuous collection and specific site collection within the same operations list. Following are example operations lists. In the operations lists, the TUBE column identifies the location of the sample to be injected. The FRACTION_SITE column identifies where collection begins for the injected sample. TUBE and FRACTION_SITE were variables in the control method. Example 1 Specific site collection: collection of each sample s eluent into same set of tubes (one zone) If the fractions for each injection are collected into the same set of tubes, ensure that the zone:tube for the FRACTION_SITE variable is the same for each sample, for example, FRACTIONS:1. Description Control Method Analysis Method TUBE FRACTION_SITE Injection 1 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:1 FRACTIONS:1 Injection 2 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:2 FRACTIONS:1 Injection 3 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:3 FRACTIONS:1 Injection 4 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:4 FRACTIONS:1 Injection 5 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:5 FRACTIONS:1 8-12

209 operations Example 2 Continuous collection: collection of each sample s eluent into its own set of tubes (one zone) If the fractions for each injection are collected into a different set of tubes, indicate the zone name, followed by a colon, into which collection begins in each step. Refer to the FRACTION_SITE column. Description Control Method Analysis Method TUBE FRACTION_SITE Injection 1 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:1 FRACTIONS: Injection 2 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:2 FRACTIONS: Injection 3 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:3 FRACTIONS: Injection 4 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:4 FRACTIONS: Injection 5 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN TUBE:5 FRACTIONS: Example 3 Continuous collection: collection of each sample s eluent into its own set of tubes (multiple zones) If the fractions for each injection are collected into a different set of tubes, indicate the zone name, followed by a colon, into which collection begins in each step. The following example uses two zones. Fractions from standard injections are collected into one zone (STD_FRACTIONS), and fractions from unknown injections are collected into another zone (UNK_FRACTIONS). Description Control Method Analysis Method TUBE FRACTION_SITE Injection 1 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN STD_TUBE:1 STD_FRACTIONS: Injection 2 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN STD_TUBE:2 STD_FRACTIONS: Injection 3 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN STD_TUBE:3 STD_FRACTIONS: Injection 4 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN UNK_TUBE:1 UNK_FRACTIONS: Injection 5 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN UNK_TUBE:2 UNK_FRACTIONS: Injection 6 C:\233XL\CONTROL.GCT C:\233XL\ANALYSIS.GAN UNK_TUBE:3 UNK_FRACTIONS: Generating operations list when re-injecting collected fractions When setting up an operations list to re-inject collected fractions, you can browse to an open Results window and select fraction site information to add to the operations list. This feature works with HPLC systems that use a 233 XL or 215/819 for sample injection and fraction collection. Following is a description of how to browse to the Results window to generate steps in the Operations window. Note: Before creating or opening the operations list, make any necessary modifications to the control and analysis methods. For example, in the control method, remove fraction collector events unless you are collecting eluent for the re-injected fractions. Choose Save As from the File menu and save the control method to a new name. 8-13

210 unipoint user's guide 1 Display the Results window by opening the data (.GDT) file or report (.GR?) file. 2 Create or open an Operations window. 3 Choose Step from the List Entry menu. 4 In the Step Entry dialog, indicate control method and analysis method information. 5 Enter values for all variables except the sample location variable (usually labeled TUBE or SAMPLE_LOCATION). 6 Click in the text box for the sample location variable and click on Browse. Or, you can double-click in the text box. 7 When the Browse Tray window appears, click on the Link to Results button. The Results window then appears, along with the Samples dialog, sample tray, and fraction collector tray. 8 Use the Samples dialog to select the appropriate sample. 9 Select fraction sites by doing one of the following: In the fraction collector tray, hold down the Ctrl key and click on each fraction site. Select the cross-hairs tool. Position the mouse pointer in the chromatogram plot. Then hold down the Ctrl key and click between the fraction collector begin and end marks to select fraction sites. Or, click and drag to draw a rectangle around the desired fractions. 10 Choose the Add Steps button. 11 Repeat steps 8-10 if you want to select fractions collected for other samples. 12 When finished, choose Done in the Fraction Collector Sites window. The Operations window appears and its table lists steps for the selected fraction sites. 13 Review the list and if necessary enter or change the sample description for each fraction. 8-14

211 operations Modifying the operations list using edit menu Once an operations list is set up, you can use the commands in the Edit menu to make changes. If you need help on deleting, copying, and pasting rows or on finding and replacing text, see page importing information into a column An additional command, Paste Special, in the Edit menu enables you to bring in a column of information from Notepad and insert it into the table. This command is useful for quickly entering sample IDs into the Description column. 1 Set up the operations list as described earlier in this section. 2 Choose Paste Special from the Edit menu. 3 When the Paste Special dialog appears, choose the Editor command button to open Windows Notepad text editor. 4 Use the text editor to identify the sample descriptions. Put each sample description on a separate line. Or if you have a file already set up, use the Open command in the File menu to select the file. 5 Select all of the text in the editor and choose the Copy command. 6 Choose Exit from the File menu. To save the list for future use, choose Yes when the message box appears. To discard the list, choose No. 7 When the Paste Special dialog re-appears, choose Description in the list box and then choose Paste to perform the insertion. 8 Choose Cancel to exit the dialog box. 8-15

212 unipoint user's guide Reviewing where run-generated files are stored During the run, UniPoint can generate data, report (which include chromatogram plots of acquired traces), calibration, log, and re-analysis list files. UniPoint automatically creates a data/report directory for these files and assigns default names to the run-generated files. 1 Choose Storage from the Run menu to display the Run File Storage dialog. Note: If you have not saved the operations list and assigned a name, you see a message box. Use the Save As or Save command in the File menu to store the operations list to disk. 2 In the Run File Storage dialog, check the names assigned to files. 3 To modify default names, choose <Templates> and make the changes. Then select <Files> to review how the changes affect the file names. If you indicate a directory name that does not exist, UniPoint creates it for you during the run. As a default, UniPoint uses the name of the operations list as the directory name. 4 Choose OK to accept the settings and exit the dialog. Following is a description of the areas of the Run File Storage dialog and how UniPoint assigns names. Output Directory. Specifies the data/report directory into which rungenerated files are stored. As a default directory name, UniPoint: uses the name assigned to the operations list adds a 3-digit extension (001 is assigned the 1st time the operations list is run, 002 is assigned the 2nd time, and so on) 8-16

213 operations Data File. Specifies the path and file name where information for collected data is stored. To assign the file name, UniPoint uses the name of the operations list but adds the.gdt extension. Information for all samples injected during the run is stored here. Log File. Specifies the path and file name where run-time events are stored. To assign the name, UniPoint uses the same naming scheme as for the data file but adds the.glg extension. Save Log. Indicates if a listing of run-time events is generated. Method File. If multiple analysis methods are used to analyze injected samples, select the appropriate method file name from the list box. You can then view and if necessary change the path and file name to use for storing report and calibration information. Report File. Specifies the path and file name where report information for all samples are stored. As a default file name, UniPoint: uses file name assigned to the analysis method assigns the extension.gr1 If data is later re-analyzed using the same analysis method, the software increments the number in the extension. (Up to 9 report files can be created using the same analysis method and stored in the same directory.) Calibration File. Specifies the path and file name for calibration plots generated for standard peaks. To assign the name, UniPoint uses the same naming scheme as for a report file but adds the.gb1 extension. Disk Space. Shows how much room is available on the disk and how much is required for the run. If there is not enough space, do either or both of the following: remove unneeded files from the disk before starting the run save the output files to another fixed or network disk drive. Templates. Allows you to change the default file or directory naming scheme used by UniPoint. When you choose the <Templates> button, the software places question marks in the output directory name and file names. The placement of the question marks identifies which part of the name automatically increments when the software assigns names. You can remove or change the placement of the question mark(s) in a name to meet your needs. However, do not modify the first two characters in the file name extension for any file type. Choose <Files> to see how a change to a template affects the name assigned to the file or directory. 8-17

214 9 Programmed Run The Operations window is where you start and monitor the progress of a run. The parts of the window were described in Section 8. While the run is in progress, you can: modify steps that have not yet been executed. This includes modifying a control method or analysis method file so updated conditions are used, or adding a step to inject a priority sample. use the toolbar to manipulate the appearance of the run-time graph. perform other types of processing in the UniPoint, for example, view the results from a previous run, create the operations list for the next run, or execute a re-analysis. access and use another Windows program, such as Microsoft Word. 9-1

215 unipoint user's guide Method Executor The Method Executor is an intermediary between UniPoint and Gilson GSIOC devices. It performs the steps in a run and enables the computer to perform other tasks while UniPoint is running. When you open or create an Operations window, UniPoint searches for and tries to start a Method Executor for programmed and manual control of the HPLC system. You see the message System Status in the status box of the Operations window if a Method Executor is available. UniPoint only allows one Method Executor to communicate with an HPLC system configuration at a time. If you open multiple Operations windows, another Method Executor is not started if the Operations window references a configuration that is currently monitored by a Method Executor. If you are viewing a new Operations window, UniPoint checks the configuration file associated with the last saved control method (the "default" control method) and determines if a Method Executor has been started for it. If you opened an existing operations list, the software checks the configuration associated with the control method named in the first step of the list to see if a Method Executor has been started for it. Once a Method Executor has been started for an HPLC system, it continues monitoring system components until you exit UniPoint. manually starting the method executor If an Operations window is "disconnected" from system components, you can manually start a Method Executor for it by doing the following: 1 If another Operations window is linked to the Method Executor, close that window. Or, disconnect it by choosing Method Executor from the File menu and choosing the Shutdown Method Executor button. 2 In the Operations window to connect to the Method Executor, choose Method Executor from the File menu and choose the Start Method Executor button. 9-2

216 programmed run Starting a run To get ready for and begin a run: 1 Make sure all system modules are turned on and are ready to run (pumps are primed, column is equilibrated, samples are in place, printer has paper and is turned on, and so on). 2 Check that the correct operations list is selected and all files used by the list exist. 3 Make sure a Method Executor is available. You see the message System Status in the status box. 4 Choose Start from the Run menu or the go tool ( ) from the toolbar. 9-3

217 unipoint user's guide During the run Manipulating run-time graph As data is acquired, the run-time plot is scaled according to the channel scaling parameters set in the control method for the current step. However, you can change the scaling by zooming. zooming You can use the magnifying glass tool to enlarge a region of interest. To zoom: 1 Choose the magnifying glass tool ( ). 2 Place the mouse pointer to one side of the region of interest in the graph. 3 Click and hold the left mouse button. 4 Drag the mouse to draw a zoom box around the desired peaks. For example: To enlarge the size of peaks and still have the chromatogram plot update as new data is collected, extend the box slightly past the end of the traces before releasing the mouse button. To scale peaks to a desired height, draw the box using the height of the peak of interest or the Y axis as a reference, and then release the mouse button. (See diagram on next page.) To display a time period of interest, draw the box using the X axis as a reference, and then release the mouse button. (See diagram on next page.) 9-4

218 programmed run 5 Be aware of the following: If the zoom box extended past the end of the displayed trace(s), the plot continues to update as additional data is acquired. However, the time period duration shown in the X axis remains the same. To display the entire chromatogram plot, choose the full-scale tool. If the zoom box did not extend past the end of the displayed trace(s), the plot does not update as additional data is acquired. The chromatogram plot remains static. It begins to update again if you choose the arrow tool ( ) or the full-scale tool ( ) or when data for the next injected sample begins to appear. moving plot along X axis To move the plot along the X axis: 1 Use the magnifying glass tool ( ) as described above to enlarge part of the plot. 2 Use the scroll bar that appears along the bottom of the plot. To examine peaks with longer retention times, move the scroll box to the left. To examine peaks with shorter retention times, move the box to the right. finding peak information To determine retention time and voltage information: 1 Click on the cross-hairs tool ( ). 2 Move the mouse pointer to the peak of interest. In the information line at the bottom of the window, you see: retention time mv height (or specified user-defined units) If you are looking at a test chromatogram, this is a convenient way to determine the retention time of a peak. 3 Click on the arrow tool ( ) to turn off the cross-hairs tool ( ). 9-5

219 unipoint user's guide returning X and Y axes Choose the original view tool ( ) from the toolbar or its command from to control method settings the Graph menu to return the X and Y axes to the minimum and maximum settings in the control method. UniPoint uses the values in the method's Strip Chart Options dialog (see page 6-11). removing/replacing traces and symbols from display To remove one or more traces or symbols: 1 Double-click on an item in the legend. Or, choose Attributes from the Graph menu. 2 When the Attributes dialog appears, choose the description for the item to remove from the Legend list box. 3 Remove the X from the Display/Symbol check box. 4 Repeat steps 2 and 3 for any additional items to remove. 5 To disable display of peak names, remove the X from the Display Peak Names check box. 6 To disable display of run-time events (such as detector autozero), remove the X from the Display Event check box. (For a description of symbols and lines for run-time events, see page 5-27.) 7 Choose OK. To re-display an item, follow the instructions above but insert the X in the Display check box. displaying/removing gridlines You can turn off and on the display of gridlines in the graph: 1 In the Graph menu of the document window, choose the Signal Axis (for the Y axis) and Time Axis (for the X axis). 2 When the dialog box appears, remove or insert the check mark in the Major or Minor check boxes and choose OK. 3 Repeat for the other axis. 9-6

220 programmed run Interrupting a run You can choose to pause, stop flow, or terminate a run in progress. pause run To pause a run, choose Pause from the Run menu or its tool ( ) from the toolbar. This option causes the run time and gradient progression to enter a timed hold. The duration of the pause is indicated in the status area. Mobile phase continues to flow at the current composition and flow rate. If you pause a run, data collection is not interrupted. It proceeds until the data collection time for the current control method elapses. To continue the gradient progression, choose Pause again from the Run menu or its tool from the toolbar. The run time and the gradient profile continue as programmed. stop flow To stop flow, choose Stop All Pumps from the Manual menu or its tool ( ) from the toolbar. This option causes the run time and gradient progression to enter a timed hold. The duration of the hold is indicated in the status area. However, unlike the Pause option, mobile phase does not flow. Like the pause option, data collection is not interrupted. It proceeds until the data collection time elapses. To restart the flow and remain in the timed hold, choose Mobile Phase from the Manual menu. Then click on Start Flow in the dialog box. To restart the flow and continue the run as programmed in the control method, choose Pause from the Run menu or its tool ( ) from the toolbar. 9-7

221 unipoint user's guide stop run To stop the run before it completes, choose Stop from the Run menu or its tool ( ) from the toolbar. This option stops the run. When asked, verify that you want to quit the run by clicking on Yes. If you choose No, the run continues. If you choose Yes, a message box appears that asks you whether or not to save collected data. Choosing Yes saves the data collected for the operations step on which the run was stopped to the data file, along with the data collected for any previous steps. If you choose No, UniPoint does not save the data collected for the step to the data file. Note: If you stop the run on the first data collection step and indicate that you do not want to save data, UniPoint asks if you want to delete the data/report output directory that was also created for the run and any files, such as the log file, stored in it. If you stop a run and no data was collected in any of the executed steps, UniPoint automatically deletes the data/report output directory and any files in it, such as the log file. The mobile phase composition and flow conditions in effect at the time of termination remain in effect. Flow does not stop automatically after a run! To stop flow, choose Stop All Pumps from the Run menu or its tool ( ) from the toolbar. Resuming a run after termination You can restart a run that was stopped while in progress. You can resume the run at the same step at which it was terminated. Or, you can resume the run at an earlier step if you want to repeat a series of steps. 1 Choose Resume from the Run menu. 2 Type the number of the step at which to resume. You can indicate the same step number at which the run was terminated. Or, specify the number of a step prior to that one so you can repeat steps within the run. 9-8

222 programmed run The information for each sample is added to the end of the data and report files created for the terminated run. If you repeat a series of steps, there is duplicate information for the repeated injections. To generate meaningful reports, you can do a batch re-analysis that omits any bad injections. 3 You will see a Fraction Collection area at the bottom of the Resume Run dialog if the data (.GDT) file specified in the File text box contains any fraction collection events. If the steps in your operations list identify the zone and tube number (specific site collection), UniPoint will collect into the specified tube when you resume the run and will not use the information in the Fraction Collection list box. Therefore, you can disregard the information in the Fraction Collection list box. If the steps in your operations list identify just the zone name (continuous collection), UniPoint uses the information in the Fraction Collection list box to identify the tube at which to start collection for a zone. You can review and if necessary modify the information in the Fraction Collection list box, described below. In the Fraction Collection list box, UniPoint lists each zone used in the data file specified in the File text box and lists the next tube to be accessed in the zone. Be aware that every zone required for the resumed run may not be listed if the previous run was terminated before all of the zones were used at least once. During the resumed run, collection begins in tube 1 for any unlisted zone. Also be aware that a listed zone may not be required by any of the steps in the resumed run. When the run is resumed, fraction collection will begin in the specified tube for the first step that uses a zone. For each subsequent step that identifies the zone, collection will begin in the next available numbered tube in the zone. If you want to change the start tube for a zone in the Fraction Collector list box, do one of the following. Click on the zone:tube item in the list box so it appears in the Fraction Collection text box. Replace the tube number with the new tube number; do not change the zone name. Click on Change to modify the zone:tube item in the Fraction Collection list box. 9-9

223 unipoint user's guide Click on the zone:tube item in the list box so it appears in the Fraction Collection text box. Use the Browse button to access the Tray Editor Browse dialog. To choose the fraction site, click within the zone, on the tray image, so its tubes become cyan (light blue). Click on the desired tube so it becomes red. Click on OK to return to the Resume Run dialog. When the dialog re-appears, the selected fraction site appears in the Fraction Collection text box. Click on Change to modify the zone:tube item in the Fraction Collection list box. After the run Status of UniPoint A run is complete once UniPoint has finished all of the steps in the operations list (or after you have selected Stop). The mobile phase composition and flow conditions in effect when the run is finished remain in effect. Flow does not stop automatically after a run! To stop flow, choose Stop All Pumps from the Run menu or its tool ( ) from the toolbar. If the method was set to save data and analysis reports to the fixed disk, those data and report files can be viewed and/or printed using the Results window; see Section 11. Stored data can also be re-analyzed using new analysis conditions; see Section 13. If the run was a calibration run, the internal calibration curves used to analyze unknowns were saved to a calibration file. This file is named for the analysis method and has the.gb? extension. See Section 12 for information on viewing calibration plots. 9-10

224 programmed run Shutting down the HPLC system Shutdown methods are control method files that include conditions that you want in effect after each run. A shutdown method might reduce the flow rate to zero and extinguish the lamp on a detector. You create shutdown methods as you would any other control method. To automatically shut down your HPLC system at the end of a run, specify the name of the shutdown control method as the last step in the operations list. If your operations list does not automatically execute a shutdown method, Gilson recommends the following shutdown procedure: 1 Choose Mobile Phase from the Manual menu. 2 In the Time box, specify the ramp time over which to achieve the desired shutdown conditions. 3 In the Flow and % boxes, specify shutdown conditions appropriate to your column. 4 Choose Begin. 5 After achieving the desired composition and flow rate, turn off the pumps and mixer. Note: When pumping salt solutions, leave the pump running at a very slow flow rate (0.05 ml/min) to avoid precipitating salt crystal deposits that might scratch the pump head s piston. If the system will remain unused for a long time, rinse the salt solutions from the pump heads with HPLC-grade water before shutdown. 6 Choose Done to exit the Manual Mobile Phase Control dialog. Viewing log file To display the log file produced during a run: 1 Choose Open from the File menu. 2 Locate and choose the name of the log file. It is stored in the data/ report output directory created for the run and has the extension.glg. 3 Choose OK. The file appears in a text editor box. You can add your own comments, change the font, and print the log file. See page 5-34 for more information on using a text editor. 9-11

225 10 Manual HPLC System Control In addition to timed runs, UniPoint also provides manual control over HPLC system components. Manual control is useful for getting the system up and running, for method development, and for checking HPLC system operation before a run. Note: Before running your system in manual mode, be sure that the devices in the HPLC system are turned on and the appropriate connections are made as described in the UniPoint System Installation Guide. Manual control is available from the Manual menu of an Operations window. The first command in the menu stops all pumps. The middle group of commands displays dialog boxes for issuing instructions to system components. The last command is for checking or selecting the manual control method. When a manual mode command is issued, the system responds immediately. There are no timed event schedules in manual mode, but you can instruct the pumps to achieve a specific flow over time. Note: A protocol file cannot be run at the same time as other types of manual control. When you open or create an Operations window, UniPoint searches for and tries to start a Method Executor for programmed and manual control of the HPLC system. You see the System Status message in the status box of the Operations window if a Method Executor is available. 10-1

226 unipoint user's guide Manual control method For manual control to occur, UniPoint needs the system configuration and device description information set in a control method. By default, the software uses the control method in the first step of the operations list. If no steps have been set, it recalls the name of the last control method saved to disk. Use the following procedures to see which method is used for manual control and change that method if necessary. 1 Choose Control Method from the Manual menu. 2 Review the name of the control method listed in the File Name box. This is the method used for manual operation. 3 Do one of the following: Click on Cancel if the control method listed is appropriate. Or, use the options in the dialog box to select a different control method and click on OK. modifying the control method During manual control, you may realize that a device or one of its resources, such as an output contact or data channel, has not been assigned a description. You can modify the manual control method and reflect these changes in the Operations window. 1 Open the document window for the control method. 2 Make the necessary changes using the Device menu. 3 Save and close the control method. 4 Reselect the method using the Control Method command in the Manual menu of the Operations window. UniPoint reads the information in the updated control method so it can be used for manual control. 10-2

227 manual HPLC system control Control of mobile phase pumps and manometric module In the Mobile Phase Control dialog, you can zero the manometric module and instruct Gilson pumps to achieve a desired mobile phase composition and flow rate over a specified time. To display this dialog box: 1 Choose Mobile Phase from the Manual menu. Note: If you are using UniPoint with SF Pressure Control (ordered separately), you will also see a text box for setting the Pressure for the 821 Pressure Regulator. Pressure is the pressure to achieve at the column outlet. Values between 0 and 40 MPa (400 bar or 8700 psi) are valid. zeroing the manometric module Caution: Before zeroing the manometric module, there must be no pressure on the HPLC system. Check that the pumps are not pumping and the prime/purge valve is open before following these procedures. To zero the manometric module: 1 Click on the Zero Mano. Module button. 2 When the caution box appears, once again ensure there is no pressure on the HPLC system. 3 Click on OK to zero the manometric module and return to the Mobile Phase Control box. Or, click on Cancel to return to the Mobile Phase Control box without zeroing the manometric module. 10-3

228 unipoint user's guide controlling the pumps To control the pumps: 1 In the Time box, type the amount of time to ramp from the current flow and composition conditions to the desired conditions. The limit of resolution is 0.01 min. Press BACKSPACE to correct typing errors. For immediate results, type 0 min. Use this with caution column shock can result from drastic shifts in composition or flow. 2 In the Flow box, type the desired flow rate. You can set a flow resolution of times the maximum flow of the pump head. At or below 10% of the maximum flow, settable resolution is times the maximum flow. 3 In the mobile phase composition box for each pump except the first one, type the desired percent composition from that pump. 4 After specifying all parameters, click on one of the command buttons. The labels of the some of the buttons change once the button is selected. For example, when you click on Begin, it changes to End. Begin/End. Choose Begin to ramp the pumps from the existing flow and composition conditions to the new conditions you just set. Choose End to stop ramping to new conditions. Mobile phase continues to flow at the flow rate and composition in effect when you chose End. To ramp again to new conditions, choose Begin. Hold/Resume. Choose Hold to temporarily stop the ramping of the pumps to new conditions. Mobile phase continues to flow at the flow rate and composition in effect when you chose Hold. To continue ramping after a hold state has been entered, choose Resume. Stop Flow. Choose Stop Flow to stop the pumps. Mobile phase does not continue to flow. To restart flow, click on Begin. Unlock/Lock. Choose Unlock to return control of the pumps to their front panels. The front panel settings on the pumps immediately become active. To restore control to UniPoint, choose Lock. 5 Choose Done to remove the Mobile Phase Control dialog from the screen. When you close this dialog box, the conditions indicated remain in effect. 10-4

229 manual HPLC system control Strip chart control The Manual Strip Chart dialog allows you to set scaling options for and to start and stop data collection. You can view a strip chart for one, two, or several data channels in the graph pane of the Operations window. 1 Choose Strip Chart from the Manual menu. 2 Review and if necessary modify the options in the dialog box. The default conditions are those set in the manual control method. The data rate, strip chart speed, and auto-range options pertain to data collection for all channels. The other options in the dialog relate to the data channel whose description is currently highlighted in the list box. Channel. Select the channel to set or modify its display, offset, or minimum and maximum signal axis options. Display. Remove the X from the check box if you do not want to see the channel once data collection is initiated using the Start command button. % Offset. Use this parameter to move the channel s trace away from the horizontal (time) axis and from other channel traces. Express the offset as a percent displacement from the horizontal axis (0%). The default value is 0% offset. Min (mvolt) and Max (mvolt). Set the lower and upper limits for the signal axis. Be aware of the following: If the Auto Range check box is selected the software disregards these values and uses the minimum and maximum heights for all channels to determine the signal axis. If Auto Range is not selected and you indicate different minimum and maximum values for each channel being collected, the software uses the lowest and highest values entered to determine the signal axis. For example, if 0 and 10 mv were entered for the minimum and maximum values for the 254 nm channel and 0 and 100 mv were entered for the 280 nm channel, the signal axis would be 0 to 100 mv. 10-5

230 unipoint user's guide Auto Range. If this check box is selected, UniPoint automatically scales incoming data to its minimum and maximum on the on-screen strip chart. Data Rate. The number of data points collected per second for all channels. The maximum value is 80 points per second. Display Time. Identifies the time period to display on the X axis of the strip chart. 3 To begin viewing data from the channels, choose Start. The traces for the channels appear in the graph pane of the Operations window. The legend identifies the channels. 4 To get a better view of the data in the graph pane, choose Done to remove the Manual Strip Chart dialog from the screen. Data collection continues until you choose Stop in the Manual Strip Chart dialog or close the Operations window. When data collection is halted, a dialog box appears that enables you to print or save the collected data. If you select to save the strip chart, it is stored to the output directory identified in the Run File Storage dialog. UniPoint assigns the file name STRIP??M.GDT where?? is a number (01-99) that is incremented each time a manual strip chart is saved to the directory. UniPoint also assigns the description Manual to the sample. If you select to print the strip chart, you see an Operations Strip Chart Print dialog. This dialog identifies the connected printer and enables you to indicate strip chart speed (mm/min) and set printer options. 10-6

231 manual HPLC system control Contact control The I/O Contact Control dialog offers control over output contacts. You can open, close, or pulse output contacts. Before opening the I/O Contact Control dialog, you can check the state of an output contact by referring to the status box. 1 Choose Contacts from the Manual menu. 2 To change the state of or pulse a contact, click on its name in the list box. Then choose Open, Close, or Pulse. 3 Choose Done to remove the I/O Contact Control dialog from the screen. When you close this box, each output contact remains in its current state. 10-7

232 unipoint user's guide Control of injector, detector, collector, computer, and other devices During manual control, you can send instructions to the injector, detector, fraction collector, and any other Gilson device that has been set in the configuration file of the manual control method. To issue a command to one of these devices: 1 Choose the device name from the Manual menu. The Generic command enables you to issue commands for devices not included under one of the other commands, such as for the 817 Valve Actuator. 2 Use the areas in the dialog to select the command to issue and choose the Send command. 3 When finished issuing commands, choose Done. 10-8

233 manual HPLC system control GSIOC command strings Each device has a set of commands that it understands. Most, but not all, of these commands have been provided in the dialog boxes accessed from the Manual menu. If there is another feature that you would like to perform with a device, refer to its User s Guide. A complete list of available GSIOC commands is listed there. To issue a command string to a device: 1 Choose Generic from the Manual menu. 2 If necessary, choose the Custom> button when the Generic Device Control dialog appears. If the Custom> button is already active, its label becomes List>. Therefore, if you see a List> button, proceed to the next step because the options necessary for sending a GSIOC command string are already shown. 3 Type the command string in the Command box. 4 Using the options buttons, choose the type of command. The documentation for the GSIOC command tells what type of command it is. Buffered commands send instructions to a device. These commands are executed one at a time. Immediate commands request status information from a device. These commands are executed immediately, temporarily interrupting other commands in progress. 5 Monitor the unit s response to your command in the Response area. 6 Choose Done to remove the dialog box from the screen. 10-9

234 11 Results The Results window enables you to view chromatogram plots and analysis information for collected data. You can do this by opening a data file or report file. Note: If your system includes a 170 Diode Array Detector, refer to the detector user's guide for information on viewing and manipulating acquired diode array data. difference between data and report files From a run, UniPoint produces a file containing the channel data collected and stored for all injected samples. This data file contains time and signal information for each sample. No analysis amounts are stored in a data file. Therefore when you open a data file, you do not see analysis information unless you associate an analysis method and optionally a calibration file with the data file. (Procedures for associating method and calibration files with a data file are in this section.) To view results of data analysis, you select the report file generated during a run or re-analysis processing. Along with the calculations, the report file identifies the data file, analysis method, and, if applicable, calibration file used to generate amounts. 11-1

235 unipoint user's guide Results window The following illustrations show the components of the Results window that can be displayed for a data file and a report file. Refer to Section 5, Common Procedures for more information on manipulating parts of the table or graph displayed in a document window. menu bar toolbar annotation box summary graph graph pane Results window for data file table pane Results window for report file legend box status bar 11-2

236 results menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Some menu items are not available if a report file is open. Menu File Edit View Linking Analysis Comparisons Table Graph Window Help Description Create, save, print, delete files or exit a window or UniPoint. Transfer information. Select sample data or report and associated tray file(s) to view. Associate method and calibration files with displayed data. Modify integration, peak, and data channel parameters. Generate new trace by subtracting or dividing two traces or removing baseline shift from a trace. Display or modify appearance of the table pane. Display or modify appearance of the graph pane. Re-arrange windows or icons, or activate a window; display file annotation or toolbar. Learn about UniPoint. table pane Shows information for integrated peaks. If no information is shown in the table for a report, no peaks were integrated. The columns in the table depend on the report type and format specified in the analysis method used to produce the report file or linked to the data file. Note: As you can see in the example data file window above, you do not see the table pane if you have not linked to an analysis method. For external standard, internal standard or percent normalization reports, note the following when looking at amounts: If a linear regression or point to point curve fit was used to determine amounts for an unknown sample, the software lists amounts for areas (or heights) that are up to 10% above the highest level amounts on the calibration curve. Beyond that, it lists ** high ** for the amount. If a quadratic or cubic curve fit was used, the software lists ** high ** or ** low ** for areas (or heights) that are above or below the highest and lowest level amounts on the calibration curve. If the calibration curve was invalid or the amount was calculated as a negative value, the software lists ** error ** for the amount associated with a peak. If an integrated peak was not named in the analysis method's peak table, the software lists 0.00 for its amount(s). If you are viewing a data file and a calibration table has not been linked, you see ** N/A ** for amounts, indicating they are not available. 11-3

237 unipoint user's guide graph pane legend box Shows the chromatogram plot for collected traces plus run-time events and peak names, if data file is linked to an analysis method or report file is displayed. (For a description of run-time symbols and lines, see page 5-27.) Identifies the traces shown in the graph. If there is a > in front of a description, that trace s peak information is reported in the table pane. By double-clicking on the legend, you can access the Attributes dialog to change how traces appear and remove traces, run-time events, and peak names from display. See page summary graph toolbar Shows the complete chromatogram plot for the selected traces. Allows for manipulating the appearance of the chromatogram plot plus provides quick access to file maintenance and selection options. To activate the tool, just click on its icon. navigator tool open tool save tool print tool arrow tool magnifying glass tool cross-hairs tool move trace tool adjust baseline tool full-scale tool Displays Navigator. Displays Open dialog. Displays Save Report dialog. (Not available if report file is open or if data file is not linked to an analysis method.) Displays Print dialog. Turns off magnifying glass, move trace, and cross-hairs tools. For enlarging area of graph. For identifying mv (or user units) and time values for point on trace. For moving trace(s) to different location in graph. For modifying the baseline beneath a peak. (Not available if report file is open or if data file is not linked to an analysis method.) For returning graph axes to minimum and maximum values for displayed trace(s). 11-4

238 results original view tool integration tool peak table tool analysis channel tool spectral library tool Returns graph to the settings shown when the window was first opened, or if a data file is linked to an analysis method to the settings in the method. Displays the Integration Events dialog. Displays the Peak Table dialog. Displays the Analysis Channel dialog. Displays the Spectral Library Entry dialog. To turn off/on the toolbar, choose its command from the Window menu. annotation box status bar Lists information for the displayed data or report. To turn off/on the annotation, choose its command from the Window menu. Displays help message if a menu command is highlighted. Displays mv (or user units) and time values for point on trace if cross-hairs tool is being used. 11-5

239 unipoint user's guide Opening report and data files To view results from a chromatographic run, you can choose a report file to view both trace and analysis information or a data file to view traces. Both file types display the Results window. Selecting report file information to view To select a report file to view: 1 From the File menu, choose Open. Or, use the Navigator to click on the Results option and then click on Open. 2 When the Open dialog appears, type or select a report file. If the file is not listed in the File Name box, do one or more of the following: In the Drives box, select a different drive. In the Directories box, select a different directory. When you select a report file, additional options appear in the Open dialog. 3 When the desired report file is shown in the File Name box, do one of the following: Click on OK to view report information for the first sample and then view report and summary report information for all other samples. Or, highlight the description of a sample or summary report in the Report list box and then click on OK. This enables you to initially display report information for a sample other than the first injection. The Results window appears, displaying the report information and chromatogram plot for the selected sample. The traces are scaled to the settings specified in the analysis method. 11-6

240 results The title bar identifies the name of the report file and the injection number of the sample whose information is shown. If multiple traces were collected for the sample, the table pane contains information for the analysis channel selected in the analysis method. Note: If the data file associated with a report file was deleted, only the report information appears; no chromatogram plot is shown. 4 To view the report for another sample, choose Samples from the View menu. In the list box, double-click on the description. It may take a few seconds for the report's information to display in the Results window. You can keep the Report dialog on-screen and browse through each report by selecting its name and then choosing View. To move the Reports dialog, click on its title bar and drag it to a new location. To size the Report dialog, position the mouse pointer at the side or corner of the box. Click and drag the box to the desired size. Choose Done to remove the Reports box. 5 If injector and/or fraction collector tray information is available for the report file, use the commands in the View menu to display the tray files. The Display All command automatically sizes and shows the Results window, Samples dialog, injector tray (if available), and fraction collector tray (if available). Refer to Appendix B for more information on setting pre-run parameters so you can view injector and fraction collector tray information after the run. 11-7

241 unipoint user's guide Selecting data file information to view You can select samples from one or more data files and view their data in the Results window. Note: If an analysis method is open, UniPoint uses the conditions in that method to try to integrate peaks. If you do not want analysis to occur, close the method before opening the data file. If you have multiple analysis methods open, you see the Analysis Method Link dialog and then you can select the method to use. 1 From the File menu, choose Open. 2 When the Open dialog appears, type or select a data file. If the file is not listed in the File Name box, do one or more of the following: In the Drives box, select a different drive. In the Directories box, select a different directory. 3 Refer to the side bars below depending on what you want to view. step through traces collected for each injected sample To view the data collected for the first injected sample and then view data collected for any additional injections: 1 When the desired data file is shown in the File Name box, do one of the following: Click on OK to see the trace(s) for the first sample. Or, highlight the description of a different sample in the list box and then click on OK. This enables you to initially display the trace(s) for a sample other than the first injection. 11-8

242 results The Results window appears, displaying the trace(s) for the first sample or the selected sample. The traces are displayed at full scale. The title bar identifies the name of the data file and the injection number of the sample whose information is shown. 2 To view the trace(s) for another sample, choose Samples from the View menu. In the list box, double-click on the sample description. It may take a few seconds before the sample information is displayed. You can keep the list box on-screen and browse through each sample by selecting its name and then choosing View. To move the Samples dialog, click on its title bar and drag it to a new location. Using the Samples dialog, you can also select a sample and add its trace(s) to the chromatogram plot in the graph pane. To do this, press the CTRL key then double-click on the sample's description. The chromatogram plot updates to show the additional trace(s) and realtime events for the selected sample. The title bar does not update. To size the Samples dialog, position the mouse pointer at the side or corner of the box. Click and drag the box to the desired size. Choose Done to remove the Samples dialog. 3 If injector and/or fraction collector tray information is available for the data file, use the commands in the View menu to display the tray files. The Display All command automatically sizes and shows the Results window, Samples dialog, injector tray (if available), and fraction collector tray (if available). Refer to Appendix B for more information on setting pre-run parameters so you can view injector and fraction collector tray information after the run. 4 To analyze displayed data and see report information, link to an analysis method. See page for details. view each trace in its own window To view each trace collected for a sample in its own document window: 1 When the desired data file is shown in the File Name box of the Open dialog, choose the sample(s) in the Sample list box. To select multiple samples, hold down the CTRL key while clicking on each sample name. 2 Choose the data channel(s) for the trace(s) to display. 11-9

243 unipoint user's guide 3 Choose the Add button to insert the sample and channel combinations into the bottom-most list box. 4 Continue adding information to the bottom-most list box. You can select samples from another file by choosing a different data file in the File Name box. 5 Choose OK when you are done selecting samples. A Results window appears for each of the selected traces. The traces are displayed at full scale. 6 Use the commands in the Window menu to re-arrange the document windows. Also use the Window menu to make a window active

244 results compare traces for different samples in the same window You can view traces collected for different samples in the same docu ment window by doing the following: 1 When the desired data file is shown in the File Name box of the Open dialog, choose the sample(s) in the Sample list box. To select multiple samples, hold down the CTRL key while clicking on each sample name. 2 Choose the data channel(s) of the trace(s) to display. 3 Choose the Add button to insert the sample and channel combination(s) into the bottom list box. Up to 8 trace(s) can be compared at one time. 4 Continue adding information to the bottom list box. To view sample information from a different data file, type or select the name of the data file so it appears in the File Name box. Then use the Sample and Channel list boxes. 5 Choose the Compare check box. 6 Choose OK. The Results window appears and displays full-scale trace(s) for the selected samples. And, the title bar displays COMPARE

245 unipoint user's guide Locating and opening a data file using sample description UniPoint can search for a string of text that appears in the sample description located in one or more data files. If the software finds the text string, it lists the complete path for the file. You can then choose to open the file and view the data collected for the sample. To search for a sample description: 1 From the File menu, choose Sample Search. The Sample Search dialog appears. 2 In the Sample Search Pattern box, type the string of text to find. 3 Use the Start Directory and Drives boxes to specify the path of the data files to search. The software searches all data files located in the path, including all subdirectories, listed above the Start Directory list box. 4 To further pinpoint the search: Select the Match Whole Word Only check box to find occurrences of the text that are complete words and not part of larger words. Select the Match Case check box to find occurrences of the search text that appear in the same upper and lower case combination indicated in the Sample Search Pattern box. 5 Choose Search. 6 To print a listing of the files whose sample descriptions include the search text, choose Print when the search completes

246 results 7 To view information associated with a sample description, choose it in the Matching Samples/Files list box. The Selected Sample Information area shows annotation information for the selected sample. To view the chromatogram plot for the sample, choose Open to display the Results window. 8 To remove the Sample Search dialog from display, choose Done

247 unipoint user's guide Overlaying (or merging) additional chromatogram data If a data file is open, you can select sample information from the same data file or another data file, and view it along with the trace(s) already displayed. (You cannot overlay or merge data into a report file.) overlaying data from the same file Using the Samples dialog, you can also select a sample and add its trace(s) to the chromatogram plot in the graph pane. 1 Choose Samples from the Edit menu. 2 When the Samples dialog appears, press the CTRL key then doubleclick on the sample's description in the list box. The chromatogram plot updates to show the additional trace(s) and real-time events for the selected sample. The title bar does not update. 3 To close the dialog box, double-click on its control-menu box. overlaying data from a different file To display sample data from a different file: 1 If multiple Results windows are displayed, choose the appropriate window by clicking on its title bar. 2 Choose Merge from the File menu. 3 Use the dialog to select a data file, the sample, and its trace(s). Then choose Add to insert its information into the list box. 4 Choose OK. The information is shown in the selected Results window (see next page)

248 results Before Overlaying Trace(s) After Overlaying Trace(s) removing overlaid trace(s) To remove one or more overlaid traces from the graph: 1 Choose Remove Trace from the Graph menu. 2 When the Trace Removal dialog appears, choose the description for the trace in the list box and then choose Remove. 3 Choose Done to exit the dialog

249 unipoint user's guide Analyzing data or analyzing it in a different way The Results window enables you to link displayed data with an analysis method or a calibration plot so the data can be analyzed using new conditions. It also lets you modify analysis parameters so you can see how the changes affect the integration of peaks. You can then store the outcome of the analysis to a report file. Linking to an analysis method If you opened a data file and want to integrate peaks, you need to link to an analysis method file. 1 Choose Analysis Method from the Linking menu. 2 Select the name of the method in the list box. If the method s name is not displayed: a) Choose Browse. b) When the Browse dialog appears, locate the method and choose OK. c) When the Analysis Method Link dialog appears again, select the method in the list box and choose OK. The software opens the document window for the method and puts its icon at the bottom of the window. Plus, the software uses the conditions in the method to scale the chromatogram plot and to generate new reporting information. If a calibration report type is indicated in the method but a pre-loaded calibration file is not, you need to link to a calibration table to see amounts for unknown samples, described on next page. In the legend, the trace with a > sign in front of its description is the one reported in the table pane. If an additional trace can be analyzed using the conditions in the linked method, choose the Analysis Channel command in the Analysis menu. The table pane reports areas (or heights) for the channel. If report information cannot be generated, the table pane is blank

250 results 3 If necessary, use the commands in the Analysis menu to modify analysis parameters. You can: change the peak table. See page change integration events. See page 7-5. change channel scaling parameters. See page If you made any changes to method conditions, you are prompted to save them when you close the Results window. 4 To save report information for the displayed information, chose Save Report from the File menu. (See Generating new report on page for information on using the dialog box.) Before Method Link After Method Link 11-17

251 unipoint user's guide Linking to a calibration file To report amounts for unknown samples, you can link to a calibration file previously generated during a run or re-analysis processing. Before linking to a calibration file, you must link to an analysis method, as described above. 1 Choose Calibration from the Linking menu. 2 Select the name of the calibration in the list box. If the calibration file s name is not displayed: a) Choose Browse. b) When the Browse dialog appears, locate the file name and choose OK. c) When the Calibration Link dialog appears again, select the method in the list box and choose OK. The software opens the document window for the calibration file and puts its icon at the bottom of the window. Plus, the software uses the calibration curve to generate amounts for unknown samples and displays that information in the table pane when an unknown sample is shown in the graph pane. 3 To save report information for the displayed information, chose Save Report from the File menu

252 results Before Calibration Link After Calibration Link 11-19

253 unipoint user's guide Linking to a control method To view the method conditions used to collect sample data, you can link to the control method. 1 Choose Control Method from the Linking menu. 2 Select the name of the method in the list box. If the method s name is not displayed: a) Choose Browse. b) When the Browse dialog appears, locate the method and choose OK. c) When the Control Method Link dialog appears again, select the method in the list box and choose OK. The software opens the document window for the method and puts its icon at the bottom of the window. And, the graph pane in the Results window is updated to display the events set in the method. You also see an additional Y axis that helps identify mobile phase percentage. Since the legend automatically sizes itself to display its information, the legend may block the chromatogram plot so it cannot be viewed. Choose Legend in the Graph menu to remove it, or drag the legend to a new location. 3 Notice that the beginning of the trace(s) is adjusted to reflect the start of data collection set in the control method. 4 To identify symbols and lines shown in the graph, choose Attributes from the Graph menu. Or double-click on an item in the legend. Note: The symbols and lines used for run-time events (such as detector autozero) are not identified in the legend. See page 5-27 for a description of those symbols and lines

254 results Before Method Link After Method Link 11-21

255 unipoint user's guide Manipulating graph and table Viewing and scaling techniques in graph pane The following procedures are applicable whether you are viewing a report file or data file. finding peak information To determine retention time and voltage information: 1 Click on the cross-hairs tool ( ). 2 Move the mouse pointer to the peak of interest. In the information line at the bottom of the window, you see: retention time mv height (or specified user-defined units) peak name, if the data has been linked to an analysis method or if you are viewing a report file If you are looking at a test chromatogram, this is a convenient way to determine the retention time of a peak. 3 Click on the arrow tool ( ) to turn off the cross-hairs tool ( ). zooming To enlarge an area of the graph: 1 Click on the magnifying glass tool ( ). 2 Place the mouse pointer to one side of the region of interest in either the graph or summary graph, if displayed. 3 Click and hold the left mouse button. 4 Drag the mouse to draw a box around the desired peaks. Release the mouse button. The region of interest now fills the graph, while the summary graph, if displayed, shows the plot of the entire data set. A box within the summary graph identifies the region that was enlarged

256 results 5 To display the entire chromatogram plot, click on the full-scale tool ( ). moving plot along X axis To move the plot along the X axis: 1 Zoom on part of the plot. 2 Use the scroll bar that appears along the bottom of the plot. To examine peaks with longer retention times, move the scroll box to the right. To examine peaks with shorter retention times, move the box to the left. moving traces within plot You can move one or more of the trace(s). This option can be used to offset a chromatogram that is overlapping another. It can also be helpful when subtracting or dividing chromatograms since you can adjust for varying retention times by aligning peaks in chromatograms. 1 Click on the move trace tool ( ). 2 In the legend, click on the description of each trace to move. The associated trace line in the chromatogram plot becomes highlighted. 3 Move the mouse pointer into the graph pane

257 unipoint user's guide 4 Click and hold down the mouse button to move the trace to the desired location. By holding down the CTRL key while moving the trace, the trace only moves vertically. By holding down the SHIFT key, the trace can only be moved horizontally. 5 To remove the highlight from each selected trace, click on its description in the legend. 6 Click on the arrow tool ( ) to turn off the move trace tool ( ). scaling peaks to a desired height To scale peaks to the same height, do either of the following: Zoom using the Y axis as a reference for the beginning and ending of the expansion box. When you release the mouse button, all peaks are scaled to that height. Display the Signal Axis dialog by choosing its command from the Graph menu or by clicking the right mouse button while the mouse pointer is on the Y axis in the graph. When the dialog appears, indicate the scaling values in the Minimum and Maximum boxes. Then choose OK. displaying time period of interest To display the traces within a certain time period, do either of the following: Zoom using the X axis as a reference for the beginning and ending of the expansion box. When you release the mouse button, only information within the selected time frame is shown in the graph. Display the Time Axis dialog by choosing its command from the Graph menu or by clicking the right mouse button while the mouse pointer is on the X axis in the graph. When the dialog appears, indicate the time period using the Minimum and Maximum boxes. Then choose OK

258 results displaying trace(s) at full scale settings To return to a full-scale display along the X and Y axis: 1 Choose the full-scale tool ( ) from the toolbar. If you moved any traces, they are returned to their original locations. offsetting traces To have UniPoint offset traces in the plot: 1 Optionally, remove the display of any real-time events because they are not offset along with the traces. Use the Attributes command in the Graph menu. 2 Click on the move trace tool ( ). 3 Move the mouse pointer into the graph pane. The mouse pointer resembles the move trace tool. 4 Click the right mouse button to reveal the pop-up menu, shown at the left. Then using the left mouse button, click on Stagger Traces. The mouse pointer changes to the following shape: 5 Using the left mouse button, click then drag the mouse pointer to draw the offset line (refer to the left-hand diagram below). This line identifies the angle at which each of the traces will be offset from the other. The starting point of the line is where the start of the first trace is positioned. The ending point of the line is where the start of the last trace is positioned. Each additional trace is evenly spaced between the first and last trace. When you release the mouse button, UniPoint staggers the traces (refer to the right-hand diagram below). 6 To unstagger the traces, re-display the pop-up menu and click on Remove Shifts. removing/replacing traces and symbols from display To remove one or more traces or symbols: 1 Double-click on an item in the legend. Or, choose Attributes from the Graph menu

259 unipoint user's guide 2 When the Attributes dialog appears, choose the description for the item to remove from the Legend list box. 3 Remove the X from the Display/Symbol check box. 4 Repeat steps 2 and 3 for any additional items to remove. 5 To disable display of peak names, remove the X from the Display Peak Names check box. 6 To disable display of run-time events (such as detector autozero), remove the X from the Display Event check box. (For a description of run-time event symbols/lines, see page 5-27.) 7 Choose OK. To re-display an item, follow the instructions above but insert the X in the Display check box. displaying/removing gridlines You can turn off and on the display of gridlines in the graph: 1 In the Graph menu of the document window, choose the Signal Axis (for the Y axis) or Time Axis (for the X axis) command. 2 When the dialog box appears, remove or insert an X in the Major or Minor check boxes and choose OK. 3 Repeat for the other axis. returning to original view To return the graph display to its original appearance: 1 Choose Original View from the Graph menu or its tool ( ) from the toolbar. If you are viewing a report file or a data file that is not linked to an analysis method, the graph returns to its appearance when the file was first opened. If you turned off any traces or modified the colors of traces, they are returned to their previous settings. If you are viewing a data file that is linked to an analysis method, the software uses the settings in the method to display the information in the graph

260 results Peak integration techniques When you have a data file open and have linked to an analysis method, you can modify peak information for the analysis channel. To store the results for future review, see Generating a report file on page The following procedures are not available if you are viewing a report file. checking/changing analysis channel To quickly determine which trace is reported in the table pane, look at the legend. The trace with a > sign before its description is the analysis channel. If an additional trace can be analyzed using the conditions in the linked analysis method, choose the Analysis Channel command in the Analysis menu. The table pane reports peak information for the data channel. If report information cannot be generated, the table pane is blank. adjusting baseline To adjust the baseline beneath a peak: 1 Zoom on the peak, if necessary. 2 Click on the adjust baseline tool ( ). Begin points (up triangle) and end points (down triangle) appear on the baseline under each integrated peak. 3 Click and drag the begin or end point for the peak s baseline. Peak information in the table pane automatically updates. 4 Click on the arrow tool ( ) to remove peak begin and end points from display in the chromatogram plot

261 unipoint user's guide integrating peak To integrate a peak: 1 Zoom on the peak, if necessary. 2 Click on the adjust baseline tool ( ). Begin points (up triangle) and end points (down triangle) appear on the baselines under each integrated peak. 3 Move the mouse pointer to the peak to integrate. Using the right mouse button, click on the peak. When the menu appears, choose Insert Baseline. 4 Move the mouse pointer to the begin point for the peak. 5 Click and drag the mouse pointer below the peak to draw its baseline. Release the mouse button to set the end point for the peak. To name the peak in the peak table, see changing peak table, on the next page. 6 Click on the arrow tool ( ) to remove peak begin and end points from display in the chromatogram plot. removing peak integration To inhibit integration of a peak: 1 Zoom on the peak, if necessary. 2 Click on the adjust baseline tool ( ). Begin points (up triangle) and end points (down triangle) appear on the baselines under each integrated peak. 3 Move the mouse pointer to the peak. Click on the right mouse button. When the menu appears, choose Delete Baseline. 4 Click on the begin or end point of the baseline to remove. 5 Click on the arrow tool ( ) to remove peak begin and end points from display in the chromatogram plot

262 results reverting to method baselines If you made modifications to baselines and want to return to conditions as set in the linked analysis method. 1 Click on the adjust baseline tool ( ). 2 Move the mouse pointer onto the chromatogram plot. 3 Click on the right mouse button. When the menu appears, choose Reset Baselines. 4 Click on the arrow tool ( ) to remove peak begin and end points from display in the chromatogram plot. changing peak table To modify the peak table: 1 Choose Peak Table from the Analysis menu. 2 Use the dialog as described on page Shortcut. To quickly specify the name and retention time for a peak, do the following: 1 Choose the cross-hairs tool ( ). 2 Move it to the peak. 3 Click the right mouse button. 4 When the dialog appears, check and if necessary modify the retention time and type the name of the peak. (The software puts a default peak name in the text box, for example, Peak 2.) 5 Choose OK. 6 Repeat steps 2-5 for each peak

263 unipoint user's guide changing integration events To modify integration events: 1 Choose Integration Events from the Analysis menu. 2 Use the dialog as described on page 7-5. changing channel scaling To modify channel scales set in the linked analysis method: 1 Choose Data Channels from the Analysis menu. 2 Use the dialog as described on page

264 results Generating new report file When you link a data file to an analysis method, and if appropriate, a calibration file, you can store the results for the currently displayed trace(s) to a report file. 1 Choose Report Save from the File menu. 2 Indicate information in the dialog box. And, choose OK. 3 To include information for another sample in the report file: a) Display the next sample using the Samples command in the Edit menu. b) Display the Report Save As dialog. The name of the report file automatically displays. c) Make sure the Append check box is selected. d) Choose OK. 4 Repeat step 3 for each additional sample whose information is included in the file

265 unipoint user's guide Comparison commands Using the commands in the Comparisons menu, you can perform mathematical calculations on one or two traces to produce another trace. (This menu is not available if report file information is displayed in the Results window.) subtracting two traces You can generate a chromatogram that represents the subtraction of the heights of two chromatograms. This option can be used for subtracting a background chromatogram from a chromatogram. 1 Choose Subtract from the Comparisons menu. 2 In the Channel 1 list box, choose the name of the trace from which data is subtracted. 3 In the Channel 2 list box, choose the name of the trace whose data is subtracted. 4 Choose OK. The resulting comparison trace appears in the chromatogram plot. The legend identifies the trace. 5 To save the comparison, create a report file for the information displayed in the window. See page dividing two traces You can generate a trace that represents the ratio of the heights of two chromatograms. The ratio scale is logarithmic. UniPoint uses each peak s baseline as the threshold value dividing the traces. (That is why you must link to an analysis method before this command becomes available.) UniPoint begins the calculation for the ratio 10% above the baseline. You can use the ratio feature to check peak purity. Since many variables affect the ratio, it is recommended that you refer to sources such as the following articles if you are going to use the Ratio command

266 results R. N. Yost, J. Stoveken, and W. Maclean. Positive peak identification in liquid chromatography using absorbance ratioing with a variablewavelength spectrophotometric detector. Journal of Chromatographic Science, 134: (1977). Paul A. Webb, Dean Ball, and Tony Thornton. Real-time absorbance ratio and absorbance difference measurements using dual-wavelength HPLC detectors. Journal of Chromatographic Science, 21: (1983). Haiyung Cheng and R. Rao Gadde. Absorbance ratio plots in high performance liquid chromatography. Journal of Chromatographic Science, 23: (1985). To display a ratio chromatogram: 1 Choose Ratio from the Comparisons menu. 2 In the Channel 1 list box, choose the name of the trace whose data is in the numerator. 3 In the Channel 2 list box, choose the name of the trace whose data is in the denominator. 4 Choose OK. The resulting comparison trace appears in the chromatogram plot. The legend identifies the trace. 5 To save the comparison, create a report file for the information displayed in the window. See page

267 unipoint user's guide removing shift from a trace Using the Shift Removal option, you can generate a new trace that represents a reduction of a trace s non-peak heights to zero. Any nonintegrated peaks are also reduced to zero. Since the resulting trace has no baseline artifacts, peaks are more defined. The Shift Removal option performs the same function as subtracting a baseline trace from another trace. 1 Choose Shift Removal from the Comparisons menu. 2 Choose the name of the trace in the Channel list box. 3 Choose OK. The resulting comparison trace appears in the chromatogram plot. normalizing traces Using the Normalize All command in the Comparisons menu, you can display traces on the same scale. When the traces are normalized, there is a maximum amplitude of 1000 units between the lowest and highest signal points. To turn off normalization, choose Original View from the Graph menu or its tool ( ) from the toolbar

268 12 Calibrations Calibration plots are generated during an HPLC run or a batch reanalysis of calibrator samples. To view calibration plots, you use the Calibration window. Using the options in the window, you can modify the appearance of a plot and save it to a new file name. Once saved, its name can be entered into an analysis method and used as a pre-stored calibration for future runs and re-analyses. Or, you can link to the calibration file while viewing a data file in the Results window and display peak amounts. 12-1

269 unipoint user's guide Calibration window The following illustration shows the Calibration window. Refer to Section 5, Common Procedures for more information on manipulating parts of the table or graph displayed in a document window. menu bar toolbar table pane annotation box graph pane legend box status bar menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Menu File Edit View Curve Fit Table Graph Window Help Description Create, save, print, delete files or exit a window or UniPoint. Transfer information. Select standard peak or calibration table to view. Select type of curve to display. Display or modify appearance of the table pane. Display or modify appearance of the graph pane. Re-arrange windows or icons, or activate a window; display file annotation or toolbar. Learn about UniPoint. table pane The table is the summary report generated for the displayed calibration curve. This report includes the statistical data used to produce the calibration curve for the peak. It also indicates if calibration data has been removed or is absent for a calibration level. If you ran a single level calibration, a response factor is the only thing listed in the table. The response factor is the amount at the calibration level divided by the area. The resulting ratio is multiplied by the area of an unknown to determine the amount listed in the unknown's report. 12-2

270 calibrations graph pane The graph pane shows the calibration plot: X axis Y axis solid vertical lines squares red lines Amount Area (or height) Level amounts entered into the peak table for the peak Peak area (or height) for a repeat Thicker red line is the mean, thinner red lines identify standard deviations from the mean toolbar Allows for manipulating the appearance of the calibration curve plus provides quick access to file maintenance and selection options. To activate a tool, just click on its icon. navigator tool open tool save tool print tool arrow tool magnifying glass tool cross-hairs tool full-scale tool Displays Navigator. Displays Open dialog. Displays Save As dialog. Displays Print dialog. Turns off magnifying glass and cross-hairs tools. For enlarging area of graph. For identifying amount and area value for point on plot. For returning graph axes to minimum and maximum values for displayed calibration curve. To turn off/on the toolbar, choose its command from the Window menu. legend box annotation box status bar Identifies the symbols shown in the calibration table. To turn off/on the legend, choose its command from the Graph menu. Lists information for each of the samples used to build the calibration plot. It also lists statistical information for the displayed calibration curve. To turn off/on the annotation, choose its command from the Window menu. Displays help message if a menu command is highlighted. 12-3

271 unipoint user's guide Building calibrations To build calibrations using data collected from calibrators, use the Re- Analysis window. See Batch Re-Analysis Processing for more information. Here is a review of the steps to perform: 1 Open the re-analysis file that contains information for the calibrators. Or, generate a new re-analysis list using the options in the Re- Analysis window. 2 Make any changes to the re-analysis steps. Each step lists information about the sample and the analysis method(s) to use during re-analysis processing. If you only want to re-analyze calibrators, delete any steps for unknown samples. 3 Execute the re-analysis. 12-4

272 calibrations Viewing calibrations Since calibration plots are used to calculate amounts for unknown samples, you should examine the calibration curve generated for each standard peak. Viewing calibration plots is especially important if quadratic or cubic was the curve fitting method used to generate them. opening file To open a calibration file: 1 From the File menu, choose Open. Or, use the Navigator to click on the Calibrations option and then click on Open. 2 When the Open dialog appears, type or select a calibration file. If the file is not listed in the File Name box, do one or more of the following: In the Drives box, select a different drive. In the Directories box, select a different directory. 3 If multiple calibrations were generated from a run and you want to view a table other than the first one generated, choose the specific table in the bottom list box. 4 Choose OK. The Calibration window shows the summary report and calibration plot generated for the first standard peak of the selected calibration. If you see an error message, refer to page

273 unipoint user's guide determining area (or height) at given point on curve Use the cross-hairs tool to determine the area (or height) at a given point on the curve: 1 Click on the cross-hairs tool ( ). Then move the mouse pointer to the calibration plot. You see curve intersect lines. These lines show where an area (or height horizontal arrow) and its associated amount (vertical arrow) intersect the curve. Amount and area (or height) values are shown in the message line at the bottom of the application window. By moving these lines, you can identify the amount calculated for an area (or height). 2 Move the mouse pointer along the Y axis. When you do, the curve intersect lines also move. 3 When the mouse pointer is in the desired location, refer to the area (or height) and its associated amount displayed in the message line. Because of screen resolution, you may not be able to position the mouse pointer exactly at the desired area (or height). 4 Click on the arrow tool ( ) to turn off the cross-hairs tool ( ). changing curve fitting method You can view a different curve fitting method for the calibration curve. 1 Choose the method from the CurveFit menu. 12-6

274 calibrations zooming To enlarge an area of the calibration plot: 1 Click on the magnifying glass tool ( ). 2 Place the mouse pointer to one side of the region of interest in the graph. 3 Click and drag to draw a box around the region. Release the mouse button. The region of interest now fills the graph. 4 To display the entire calibration plot, click on the full-scale tool ( ). getting file information for a repeat To learn about the injected sample associated with a repeat: 1 Zoom on the repeat's square, if necessary. 2 Click on the square using the right mouse button. 3 When the menu appears, click on Info. A dialog giving information on the repeat appears. 4 Choose OK to remove the dialog. 12-7

275 unipoint user's guide disabling/restoring repeats You remove or re-apply the value of one or more repeats from or to the calibration calculation. 1 Zoom on the repeat's square, if necessary. 2 Click on the square using the right mouse button. 3 When the menu appears, choose Disable Point. The calibration curve is automatically redrawn using the new mean and deviations, and the report information is updated. 4 To remove a calibration level, disable each repeat at that level. To enable one or more repeats: 1 Click on the repeat's square using the right mouse button. 2 When the menu appears, choose Restore Point. 3 To re-apply a calibration level, restore each repeat. displaying/removing gridlines You can turn off and on the display of gridlines in the graph: 1 In the Graph menu of the document window, choose the Area/ Height Axis (for the Y axis) or Amount Axis (for the X axis) command. 2 When the dialog box appears, remove or insert an X in the Major or Minor check boxes and choose OK. 3 Repeat for the other axis. selecting another peak To view and, if necessary, modify the calibration curve for another peak: 1 Choose Peaks from the View menu. 2 From the list box, select the peak name and choose View. (Or, doubleclick on the name.) It may take a few seconds for the calibration plot for the peak to display. You can keep the list box on-screen and view the calibration plot for each additional peak in the calibration. To move the Peaks dialog, click on the title bar and drag the dialog to the desired location. 12-8

276 calibrations To size the Peaks dialog, position the mouse pointer at the side or corner of the box. Click and drag the box to the desired size. 3 Choose Done to remove the dialog. selecting another calibration If multiple calibration tables were generated for injected samples: 1 Choose Calibrations from the View menu. 2 From the list box, select the calibration description and choose View. (Or, double-click on the description.) It may take a few seconds for the calibration plot for the currently selected peak to display. You can keep the list box on-screen and view the calibration plot for the selected peak in each calibration. To move the Calibrations dialog, click on the title bar and drag the dialog to the desired location. To size the Calibrations dialog, position the mouse pointer at the side or corner of the box. Click and drag the box to the desired size. 3 Choose Done to remove the dialog. Merging calibrations You can merge the calibration table information stored in another file with that for the currently open calibration file. 1 Choose Merge from the File menu. 2 Use the dialog to select a calibration file and choose OK. The table and calibration plot for the currently selected peak updates to incorporate the information from the merged file. 12-9

277 unipoint user's guide Use the features of the Calibration window as described earlier in this section to view calibration curves and reports for other standard peaks. 3 To save the new calibration table for future use, choose Save As from the File menu and indicate a new name for the file. Error messages If no calibration plot is drawn in the graph pane, an error message appears in the upper left corner. That message is Peak not identified or method is incompatible with data. If you get this message: 1 Use the Results window to view the sample data used to produce the calibration curves. After linking to the analysis method, change the analysis parameters until baselines are drawn beneath peaks. 2 Then use the Re-Analysis window to analyze the data again and rebuild calibration curves for standard peak. If all or a portion of a calibration curve is flat or contains a negative slope, an error message appears to the left of the peak name, for example, INVALID calibration of Phenol. If you get this message: 1 Remove one or more calibration levels from the calibration curve by removing each repeat at those levels. Or preferably, re-inject the calibrator samples and have the software generate new calibration curves for standard peaks

278 13 Batch Re-Analysis Processing The Re-Analysis window enables you to perform a post-run batch analysis on previously acquired data. The data to analyze can be from one or several data files. When you execute a run, UniPoint automatically generates a re-analysis file (.GAR) and places it in the data/report directory. The file contains a list of the injected samples and the analysis method(s) used during the run. You can use and if necessary modify the information in this file before executing a batch re-analysis. To determine if a batch re-analysis is required, refer to the reports generated at run time or view the data in the Results window and link to the appropriate analysis method. You may decide to do a re-analysis for any of the following reasons: peaks were not integrated correctly peaks were not named injections were not labeled correctly wrong actual and nominal amounts were entered for samples you want a different report type generated for samples you want to remove background information you want to analyze unknown samples using calibration curves in a calibration file (the name of the calibration file is set in the Report Type dialog) The following parameters set in an analysis method affect how data are analyzed: analysis events (peak width and sensitivity, baseline construction techniques) analysis channel quantification technique (area or height) peak table information reporting parameters (you may want to turn off report print until after the re-analysis) chromatogram scaling parameters background subtraction Refer to Section 7 for more information on how to set analysis parameters. Make the necessary changes to the analysis method so data is analyzed correctly. Then perform the batch re-analysis to generate new reports. To get ready for and start a batch re-analysis, you: 1 Open an existing re-analysis (.GAR) file. Or, create a re-analysis list using the List Entry menu in the Re-Analysis window. 2 Make any modifications to the re-analysis file using the Edit menu or Step Entry dialog. 3 Check that peaks are integrated by previewing the re-analysis. 4 Optionally, indicate that you want to manually adjust baselines during processing. 5 Check where re-analysis output is stored. 6 Start processing. 13-1

279 unipoint user's guide Re-Analysis window Using the options in the Re-Analysis window, you specify information that UniPoint uses to re-analyze data. You access this window by creating or opening a re-analysis file. The following illustration shows the Re-Analysis window. When you first access the window, the graph pane may not be displayed. Choose Show Graph from the Graph menu. Refer to Section 5, Common Procedures for more information on manipulating parts of the table or graph displayed in a document window. menu bar toolbar table pane annotation box summary window graph pane status box legend box status bar menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Menu File Edit List Entry Analyze Table Graph Window Help Description Create, save, print, delete files or exit a window or UniPoint. Locate, make changes to, or transfer information. Set step-by-step instructions to execute during a re-analysis. Start, stop, pause a re-analysis and indicate file naming parameters. Display or modify appearance of the table pane. Display or modify appearance of the graph pane. Re-arrange windows or icons, or activate a window; display file annotation or toolbar. Learn about UniPoint. table pane Lists the steps for re-analysis processing. Double-clicking on a row for a step displays the dialog box used to set up the step. 13-2

280 batch re-analysis Using the right mouse button, you can double-click on a step and display the window that shows the instructions set in the analysis method. (If multiple methods are listed, displays instructions for first method.) Double-clicking on the top (header) row displays a dialog for manipulating the columns shown in the table. graph pane During re-analysis processing or if previewing the re-analysis, shows a chromatogram plot of the data being re-analyzed. If previewing the re-analysis and there are multiple analysis methods named in the re-analysis list, you see an Analysis Method box with option buttons in it. Choose the option button for another method to see how peaks are integrated. legend box toolbar During re-analysis, identifies the traces shown in the graph. Provides quick access to options for starting and stopping processing, manipulating the chromatogram plot, and file maintenance and selection. To activate a tool, just click on its icon. navigator tool open tool save tool Displays Navigator. Displays Open dialog. Displays Save As dialog. print tool arrow tool magnifying glass tool adjust baseline tool full-scale tool stop tool pause tool go tool Displays Print dialog. Turns off magnifying glass tool. For enlarging portion of graph. For manual adjustment of peak baselines during re-analysis processing. For returning graph axes to minimum and maximum values for displayed data. For terminating re-analysis processing. For pausing re-analysis processing. For starting re-analysis processing. To turn off/on the toolbar, choose its command from the Window menu. status box annotation box Displays status information during re-analysis processing. Displays any user-entered comments about the re-analysis list. To turn off/on the annotation, choose its command from the Window menu. To add comments, use the Notes command in the Edit menu. 13-3

281 unipoint user's guide status bar Shows operational status or help message if a menu command is highlighted. Setting up a re-analysis list In most cases, the quickest way to set up a re-analysis list is to open and modify a previously created re-analysis list. At run time, UniPoint automatically generates a re-analysis file (.GAR extension) for the samples being processed and stores it in the data/report directory. To perform a re-analysis on those samples, open the file and make any necessary changes. You can make changes using the commands in the Edit menu (see page 13-10) or by double-clicking on a row and accessing the Step Entry dialog (see page 13-7). You can also build a new re-analysis list using the Express Entry dialog (see page 13-5) or using the Step Entry dialog. before creating or modifying the list Before you set up a re-analysis list, you should have created or modified each analysis method used during processing. To add a column for an additional analysis method: 1 Double-click on the top line in the table to reveal the Format Columns dialog. (Or, choose Format Columns from the Table menu.) 2 Select a column in the Column Contents list box. (The new column inserts to the left of the selected column.) 3 Choose Analysis Method 2 in the Contents drop-down list box. 4 Make any additional changes in the other text boxes. 5 Choose Insert. 6 Choose Done to exit the dialog. 13-4

282 batch re-analysis The table displays the new column. If the re-analysis table already had information in it, you see the text <Replace> in the new column. Use the Replace command in the Edit menu to quickly substitute <Replace> with the path to and name of the analysis method to use during re-analysis processing. 7 Repeat steps 2-6 to insert another analysis method column if necessary. Using express entry dialog for list setup For rapid list setup, you can use the Express Entry dialog. 1 Choose Express Setup from the List Entry menu. 2 Use the options in the dialog to set information for the re-analysis list. 3 Choose Generate to create the list. If steps are currently set in the reanalysis list, they are replaced by the newly generated steps. Following is the default Express Entry dialog and a description of its options. Additional fields may appear in the dialog if you added columns to the table or if additional columns were indicated in the reanalysis file generated by the run. Calib Levels. For calibration analyses, type the number of calibration levels being analyzed for calibrator samples. 13-5

283 unipoint user's guide Repeats/Level. If multiple injections were performed for each calibrator at a level, type the number of repeats here. UniPoint averages each level s peak data and uses the average peak size to build the calibration curve for each standard peak. A value of 1 represents a no-repeat condition or in other words one injection per calibration level. Number of Unknowns. Tell UniPoint how many unknown samples are analyzed after the calibrators. Repeats/Unknown. If multiple injections were performed for each unknown, type the number of repeats here. A value of 1 represents a no-repeat condition or in other words one injection per unknown. Number of Cycles. To repeat the entire calibration and unknown analysis more than once, indicate that here. Example. A calibration analysis consists of 4 calibration levels with 3 repeats/level and 5 unknown samples with 2 repeats per unknown. How many steps are generated for 1 calibration cycle? For 2 cycles? 1 cycle: Steps = (4 levels x 3 repeats per level) + (5 unknowns x 2 repeats per unknown) = 22 2 cycles: Steps = (22 injections per cycle x 2 cycles) = 44 Inj. Number. Type the number of the injection to use to generate the list, usually 1. Data File. Type, or browse to select, the path to and file name of the data file containing the sample information to analyze. Analysis Method. Type, or browse to select, the path to and file name of the analysis method used to analyze collected data. 13-6

284 batch re-analysis Additional text boxes. Other text boxes may automatically appear depending on the parameters set in the control and analysis methods, used during the run. For example, if an injector protocol requests inputs, such as sample volume, a text box to indicate that appears in the Express Entry dialog. UniPoint does not use the information in these text boxes during re-analysis. You can display the text boxes, if they provide helpful identification. Or, you can remove them from display using the Format Columns dialog accessed via the Table menu. If you indicated in the analysis method that actual and nominal amounts should be entered for samples, text boxes appear for those also. See page 7-25 for a description of actual and nominal amounts. If you added columns using the Format Columns dialog, you see text boxes to indicate information for those columns. For example, you may have added a column for an additional analysis method. Using step entry dialog for list setup and modification To indicate steps in the re-analysis list, you can use the Step Entry dialog. Use this setup method if sample data was collected via multiple runs. The Step Entry dialog can also be used to modify or delete steps in an existing re-analysis list. To display the dialog: 1 Choose Step from the List Entry menu. The Step Entry dialog appears. (If the table already has one or more steps, you can double-click on a row to quickly access the dialog.) 2 Use the options in the dialog to create or modify the re-analysis list. Refer to the instructions starting on page Following is a default Step Entry dialog and a description of its options. Additional fields may appear in the dialog if you added columns to the table or if additional columns were indicated in the re-analysis list file generated by the run. The Step up and down arrows in the upper right corner enable you to step through the re-analysis list. 13-7

285 unipoint user's guide Injection Number. The position at which the sample was injected during the run. This number helps to identify the sample if no description is indicated. Data File. The path to and name of the file containing the channel data for the sample. Double-click in the text box and then use the Browse dialog that appears to select the file. Analysis Method. The path to and file name of the analysis method used to analyze data collected for the sample. Double-click in the text box and then use the Browse dialog that appears to select the file. Description. Text describing the sample. If you enter a number in the description, you can have UniPoint automatically increment the number when multiple entries are generated. To see the Inc. box for incrementing the description, include a number in the Description box (for example, Sample 1) before you choose the Multiple button. See example dialog box on the next page. Calib. Level. If the sample is a calibrator, its calibration level. If the sample is an unknown, leave the text box blank or enter 0 (zero). Additional text boxes. Other text boxes may automatically appear depending on the parameters set in your analysis method(s). For example, if the analysis method requests that actual and nominal amounts should be entered for samples, text boxes appear for those. See page 7-25 for a description of actual and nominal amounts. If you added columns using the Format Columns dialog, you see text boxes to indicate information for those columns, such as for additional analysis methods. adding step To add the first step to a new list or add a step to the bottom of the list: 1 Make sure New Step appears in the upper right corner of the dialog box. If not, choose New. 2 Indicate information in the text boxes. For file names, you must indicate the complete path to the file. Therefore, click in the text box and then use the Browse button to select the file. 3 Choose Insert. 4 Repeat to add more steps. inserting step between steps To insert a step before the first step or between two existing steps: 1 Use the up or down arrow to select the step before which the new step inserts. For example, to insert a step between Steps 3 and 4, display information for step 4 in the text boxes. 2 Type information for the step. 3 Choose Insert. 13-8

286 batch re-analysis generating multiple step The easiest way to set up the re-analysis list is to generate a list of all steps and then modify the steps as necessary. 1 Choose the Multiple button. You see additional options. Inc. An Increment box appears next to text boxes that prompt you for a value, such as Calib. Level or Injection Number. If you do not want to increment the value for each step, indicate 0 (zero). Number of Steps to Generate. How many steps to add to the bottom of the list. Number of Repeats per Step. How many multiples of the step should be generated. This is especially useful for multiple injections at the same calibration level. 2 Indicate information in the default text boxes. 3 Choose Generate. If you already have one or more steps indicated, the steps are added to the bottom of the list. 4 Make the necessary changes to each step. See modifying step, below, and Modifying the re-analysis list using edit menu, on next page. deleting step To remove a step: 1 Use the Step up or down arrow so the information for the step is displayed. 2 Choose Delete. modifying step To change options for a step: 1 Use the Step up or down arrow so the information for the step is displayed. 2 Use the text boxes to make the necessary changes to the step. 3 Choose Change. 13-9

287 unipoint user's guide Modifying the re-analysis list using edit menu Once a re-analysis list has been setup, you can use the commands in the Edit menu to make changes. If you need help on deleting, copying, and pasting rows or on finding and replacing text, see page importing information into a column An additional command, Paste Special, in the Edit menu enables you to bring in a column of information from Notepad and insert it into the table. This command is useful for quickly entering sample descriptions into a column. 1 Set up the re-analysis list as described earlier in this section. 2 Choose Paste Special from the Edit menu. 3 When the Paste Special dialog appears, choose the Editor command button to open Windows Notepad text editor. 4 Use the text editor to identify the sample descriptions. Put each sample description on a separate line. Or if you have a file already set up use the Open command in the File menu to select the file. 5 Select all of the text in the editor and choose the Copy command. 6 Choose Exit from the File menu. To save the list for future use, choose Yes when the message box appears. To discard the list, choose No. 7 When the Paste Special dialog re-appears, choose Description in the list box and then choose Paste to perform the insertion. 8 Choose Cancel to exit the dialog box

288 batch re-analysis Indicating baseline adjustment During a re-analysis processing, you can view and possibly modify the baseline generated for samples. To set parameters for baseline adjustment: 1 Choose Baseline Adjust from the Analyze menu. 2 Indicate options, described below, in the dialog box. Then choose OK to exit. Analysis Method. Lists the name of each method used during reanalysis processing. Choose the method name and then select from among the other options in the dialog. Manual Baseline Adjust. Indicates if you want to modify the systemgenerated baseline for peaks during re-analysis. Use Manual Baseline. Indicates if you want to view a manual baseline used to generate peak information for the data. This baseline was generated either during a previous batch re-analysis or during an individual re-analysis using the Results window. Report File. Type, or use the Browse button to select, the name of the report file that contains the previously-generated manual baseline(s). Reviewing where files are stored During the re-analysis, UniPoint generates new report files (which include chromatogram plots of acquired traces) and, if appropriate, calibration files. 1 Choose Storage from the Analyze menu. If the re-analysis list is not named, you see a message box. Use the Save As or Save command in the File menu to store the list to disk

289 unipoint user's guide 2 When the Re-Analysis File Storage dialog appears, check the name of the data/report output directory in which the files are saved. Also check the names assigned to files. 3 To modify default names, choose <Templates> and make the changes. Then select <Files> to review how the changes affect the file names. If you indicate a directory name that does not exist, UniPoint creates it for you during the re-analysis. As a default, UniPoint uses the name of the re-analysis list as the directory name. 4 Choose OK to accept the settings and exit the dialog. Following is a description of the areas of the Re-Analysis File Storage dialog and how UniPoint assigns names. Output Directory. Specifies the directory into which files are stored. Method File. If multiple analysis methods are used to analyze data, select the appropriate method file name from the list box. View and if necessary change the path and file name used for storing report and calibration information. Report File. Specifies the path and file name where information for reports are stored. As a default file name, UniPoint: uses file name assigned to the analysis method, assigns the extension.gr? where the question mark is replaced with a number between 1 and 9 each time the analysis method is used to analyze the data. Calibration File. Specifies the path and file name for the calibration plots generated for standard peaks. To assign the name, UniPoint using the same naming scheme as for a report file but adds the.gb? extension. Disk Space. Shows how much room is available on the disk and how much is required for the re-analysis. If there is not enough space: remove unneeded files from the disk before starting the re-analysis save the files to another fixed or network disk drive

290 batch re-analysis Templates. Allows you to change the default file or directory naming scheme used by UniPoint. When you choose the <Templates> button, the software places question marks in the directory or file name. The placement of the question marks identifies which part of the name automatically increments when the software assigns names. You can remove or change the placement of the question marks in a name to meet your needs. However, do not modify the first two characters in the file name extension for the report or calibration file. Choose <Files> to see how a change to a file name s template affects the name to assign to the file. Previewing the re-analysis Before executing the re-analysis, you can view the chromatogram plot for some of the data and make sure the integration parameters are appropriate. 1 Open or set up the re-analysis list. 2 Choose the Analysis menu and make sure Preview Re-Analysis has a check mark next to it. 3 Make sure the graph pane is displayed. 4 Click on a step in the table. The graph updates and displays the chromatogram plot for the trace(s) collected for the sample. 5 Use the magnifying glass tool ( ) and full-scale tool ( ) as necessary to get a better look at the chromatogram plot. Zooming. Choose the magnifying glass tool ( ). Place the mouse pointer to one side of the region of interest in the chromatogram plot. Click and hold the left mouse button. Drag the mouse to draw a box around the desired peaks. Release the mouse button. The region of interest now fills the graph. A box within the summary graph identifies the region that was enlarged. Returning plot to full-scale. To display the entire chromatogram plot, choose the full-scale tool ( ). 6 If there are multiple analysis methods named in the re-analysis list, you see an Analysis Method box with option buttons in it. Choose the option button for another method to see how peaks are integrated. 7 Repeat steps 4-6 to view additional chromatogram plots to make sure peaks are integrated properly. 8 If necessary, open, make any changes to, and save the updated analysis method(s). You can then click on the same step or different step to see how the change affects peak integration

291 unipoint user's guide Executing the re-analysis 1 Choose Start from the Analyze menu or its tool ( )from the toolbar. The software begins processing the first sample using the conditions in the analysis method. The status box and status line in the document window identifies step and method information. If baseline adjustment was selected for the method, processing automatically pauses so you can make any necessary modifications to the baseline. See manually adjusting baselines below. Click on Continue in the message box when you are finished making changes. Before continuing to the next step, the software processes the sample using the conditions set in any additional analysis methods. The software displays the message Re-analysis finished when processing completes. manually adjusting baselines When re-analysis processing pauses for baseline adjustment, the software displays the begin point (up triangle) and end point (down triangle) for each integrated peak. You can adjust a peak s baseline, integrate a peak, or disable integration of a peak. When finished making adjustments, click on the Continue button in the Manual Baseline Adjust message box. Adjusting a peak s baseline. To adjust the baseline beneath a peak, zoom on the peak. Point and drag the peak s begin or end point. Integrating a peak. To integrate a peak not currently integrated, zoom on the peak. Using the right mouse button, click on the peak. When the menu appears, choose Insert Baseline. Move the mouse pointer to the starting point for the peak. Click and drag the mouse pointer below a peak to draw its baseline. Release the mouse button to set the end point for the peak. Removing peak integration. To inhibit integration of a peak, zoom on the peak. Using the right mouse button, click on the peak. When the menu appears, choose Delete Baseline. Then click on the begin or end point of the baseline to remove

292 batch re-analysis manually pausing re-analysis You can pause the re-analysis by choose Pause in the Analyze menu or the pause tool ( ). Pause mode is for viewing only; you cannot adjust the baseline beneath peaks and have those changes stored to the generated report file. stopping re-analysis To stop the re-analysis before it completes, choose Stop in the Analyze menu or the stop tool ( ). UniPoint completes the current step before stopping the re-analysis. Resuming a re-analysis after termination You can restart a re-analysis that was terminated while in progress. You can resume the re-analysis at the same step at which it was stopped. Or, resume the re-analysis at an earlier step to repeat a series of steps. 1 Choose Resume from the Analyze menu. 2 Type the number of the step at which the re-analysis resumes. You can indicate the same step number at which the re-analysis was terminated. Or, specify the number of a prior step prior to repeat steps within the list. The information for each sample is appended to the report files created for the terminated re-analysis. If you repeat a series of steps, there is duplicate information for the repeated steps

293 14 Protocols A protocol consists of a set of instructions executed one after another by a device. The protocol is started by indicating a timed event for it in a control method. Each step in the protocol is performed independently of what is going on in the control method, unless synchronization events have been established between them. A protocol can also be started in the Operations window using the Manual menu. This enables you to test the protocol before it becomes part of a control method. See Section 10 for information on manual control. There are two kinds of protocols that can be created: injector protocols to control an injector and its syringe pump or dilutor and generic protocols to control any Gilson device connected along the GSIOC. Gilson has supplied a set of injector protocols for you to use or modify to meet your needs. See page for a description of the supplied protocols. 14-1

294 unipoint user's guide Protocol windows There are two Protocol windows available in UniPoint: Injector Protocol window supplies options for controlling an injector and its syringe pump or dilutor Generic Protocol window supplies options for controlling any Gilson device menu bar toolbar table pane annotation box status bar Injector Protocol window Generic Protocol window menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Menu File Edit Device Event Table Window Help Description Create, save, print, delete files or exit a window or UniPoint. Make changes to or transfer information. Name installed devices and other resources used by the protocol. Issue instructions to devices. Display or modify appearance of the table pane. Re-arrange windows or icons, or activate a window; display file annotation or toolbar. Learn about UniPoint. table pane Lists the steps currently set for the protocol. If you see the message **invalid command** in the table, the device for which a command was previously set is not listed in the configuration for the protocol. Before executing the protocol during a run, check the configuration using the System Configuration command in the protocol's File menu. This message also occurs if a routines file is not available or listed in the Injector dialog or Generic Device dialog accessed via the Device menu. 14-2

295 protocols Double-clicking on a row for an event automatically displays the dialog box used to set up the event. Using the right mouse button, you can double-click on a row that specifies an event for a routine and open the file containing the instructions set in the routine. Double-clicking on the top (header) row displays a dialog for changing the format of the columns shown in the table. toolbar Provides quick access to some options available in the Injector or Generic Protocol window. To activate a tool, just click on its icon. (Not all of the tools below are available in the Injector Protocol window.) navigator tool open tool save tool print tool data channels tool contact tool injector tool detector tool fraction collector tool generic tool system tool Displays Navigator. Displays Open dialog. Stores protocol to disk. If not yet named, displays Protocol dialog and then Save As dialog. Displays Print dialog. Displays Data Channels Event dialog. Displays Contact Event dialog. Displays Injector Event dialog. Displays Detector Event dialog. Displays Fraction Collector Event dialog. Displays Generic Device Event dialog. Displays System Event dialog. To turn off/on the toolbar, choose its command from the Window menu. annotation box Displays configuration information for and parameters set in the protocol file. To turn off/on the annotation, choose its command from the Window menu. To add your own comments, use the Notes command in the Edit menu. status bar Displays help message if a menu command is highlighted. 14-3

296 unipoint user's guide Overview of creating a protocol Following is an overview of how to set up a protocol. 1 If running multiple configurations, ensure that the appropriate configuration file has been selected. To do this: a) Exit all open document windows. (The Navigator if it appears can be left open.) b) Choose Default System Configuration from the File menu. c) When the dialog appears, check the name of the file indicated at the bottom of the dialog to see if the correct one is chosen. If not, use the Browse button to select the appropriate file. 2 Create the document window for the protocol. You can do either of the following: Using the Navigator, choose Advanced to access the window for selecting the Injector or Generic Protocol option. Then choose New. Or choose New from the File menu. When the dialog appears, choose More Documents to access the list box containing the selections for protocols. Choose the type of protocol: Injector or Generic. 3 When the Protocol window appears, use the Device menu to check or assign descriptions to, and specify options for, system components. (See page 14-5.) If you created routines files, use the Injector and Generic commands to make the information in those files available for use in the protocol file. (See page 14-5.) 4 Use the Event menu to indicate steps for the protocol. (See page 14-6.) 5 Once the steps for the protocol are set, choose Save from the File menu. In the Protocol dialog, type a description for the protocol in the Description box and choose OK. (See page ) 6 When the Save As dialog appears, indicate the name and path to which the protocol file is saved and choose OK. 14-4

297 protocols Assigning descriptions Before you can issue commands to Gilson components, you must assign a user-defined description to them. To assign descriptions, use the command(s) under the Device menu. Device menu for injector protocol Following is an example dialog for assigning description(s). The procedures for checking and modifying descriptions are the same as those for a control method. See Section 6 for more information. Device menu for generic protocol Note: Gilson uses the special character * when assigning descriptions in the example protocol files supplied with the software. This causes the software to forgo the device configuration checks it performs when a protocol file is associated with a control method. Use this symbol with care in your files. For example, if the configuration for the protocol file has a 231 XL injector and the configuration for the control method has a 234 injector, the software does not warn you of any mismatches if the description for the 231 XL injector is Injector*. It warns you if the description is Injector. loading routines files Within a protocol file, you can access the routines set up in a routines file and use them to set up the protocol. (Routines are groups of named commands that make building protocols easier. See Section 15.) 1 Choose the Injector or Generic command. The dialog box that appears includes a Routines Files Description area. 2 Choose the Browse button to select the routines file and place its label in the Routines Files Description area. 3 Repeat step 2 for each routines file to use. When you choose Injector or Generic from the Event menu. You can insert a command that executes the instructions in a routine. 14-5

298 unipoint user's guide Issuing commands Each command issued when the protocol is executed is referred to as a step. You can specify steps using the dialog boxes in the Event menu. Plus you can quickly duplicate, remove, modify steps in the table using the commands in the Edit menu. To display an Event dialog box: Event menu for injector protocol 1 Choose the device from the Event menu. Or, click on a tool in the toolbar. Or, double-click on an event in the table. Hint: While an Event dialog box is on-screen, you can quickly access the dialog for another device type by selecting its tool from the toolbar. Following is an Event dialog. The appearance and procedures for setting events within each dialog box is similar. For a description of the commands available for each Gilson device, refer to page Event menu for generic protocol Note: When creating a generic protocol for a 116 Detector, you may need to insert a wait time between each command sent to the detector. To program the wait, use the Wait Time command in the System Event dialog. adding step To add the first step to a new list or add a step to the bottom of the list: 1 Make sure New Event appears in the upper right corner of the dialog box. If not, choose New. 2 Use the areas in the dialog box to select a command or routine and indicate any parameters. (If a routines file is loaded, it is selected in the Device/Routines File list box.) 14-6

299 protocols Note: Instead of entering a value in the parameter box for a command, you can indicate a variable name, for example, SampleVol or Wavelength#. See page 14-8 for more information. 3 Choose Insert. The command parameter can be the result of performing a mathematical function on two variables or a variable and number, or even two numbers. For example, AirGap + SampVol and SampVol * 5 are valid command parameters. You can add (+), subtract (-), multiply (*), and divide (/). 4 Repeat to add more steps. inserting step between steps To insert a step before the first step or between two existing steps: 1 Use the up or down arrow to locate the step before which the new step inserts. For example, to insert a step between Steps 3 and 4, display information for step 4 in the text boxes. 2 Use the areas in the dialog to select the command and indicate any parameters for the device. 3 Choose Insert. deleting step To remove a step: 1 Use the up or down arrow so the information for the step is displayed. 2 Choose Delete. modifying step To change options for a step: 1 Use the up or down arrow so the information for the step is displayed. 2 Use the areas in the dialog to make the necessary changes to the step. 3 Choose Change. 14-7

300 unipoint user's guide Variables To make your protocols more flexible, you can indicate a variable name instead of entering a value in a command parameter box for a step. A variable name can be any combination of letters and numbers and can contain special characters (_, as long as it is not the first character or a mathematical operator (+, -, *, /, %). It cannot include spaces. Valid variable names would be Wavelength# or Sample_Vol1. UniPoint does not differentiate between upper and lower case letters in a variable name. For example, it views the names Wavelength, wavelength, and WAVELENGTH as identifying the same value. The command parameter specified for a step can also be the result of performing a mathematical function on two variables or a variable and number. For example, AirGap + SampVol, or SampVol + 15 are valid command parameters. You can add (+), subtract (-), multiply (*), divide (/), and return the remainder (%). When you set up a step in a control method that executes the protocol, UniPoint prompts you for a value to assign to each variable in the protocol. You can indicate the value in the control method or wait and indicate the value in the operations list that uses the control method for a run. To see what variable names have been set for a protocol and to indicate lower and upper limits for and a default for each variable, do the following: 1 From the Event menu, choose Variables. 2 When the dialog box appears, choose the variable name in the list box. 3 Check the prompt that appears whenever the software references the variable. The prompt can be any combination of letters and numbers and can contain special characters and spaces. 4 To indicate minimum and maximum values for the variable, indicate the numbers in the Values Limits box. Separate the values with a colon, for example, 1:100. If the input is required, put an R before the minimum value, for example, R1:

301 protocols You can also indicate the variable name of SYRINGE for syringe volume or Z_ARM for Z_arm height as a limit. 5 To set a default value for the variable name, type the number in the Default Value box. 6 To remove an unused variable name, choose it in the list box and choose Delete. 7 Choose OK when finished. if you add, change, or delete variable names If you add, change, or delete variable names and the modified protocol is used in a control method, do the following: 1 Open the control method. 2 Locate and double-click on the event for the modified protocol. The parameter boxes at the bottom of the dialog update to reflect any variable changes made in the protocol. 3 Make sure the correct information is indicated in each parameter box. If you make changes, click on Change again to update the control method table. 4 Choose Done to exit the Event dialog. 5 Save and close the control method using the File menu. If any variables are no longer being used in the method, UniPoint asks you if it should remove them from the External Variables dialog (accessed via the Event menu). Choose Yes. 14-9

302 unipoint user's guide Saving the protocol file When you save a protocol for the first time, you see an additional dialog box before you see the Save As dialog. In the dialog, indicate a description for the protocol in the Description box. You can indicate any combination of upper and lower case letters in the text box. If you use an & in the description, the character following the & is underlined, for example, Rinse&Home. UniPoint shows the protocol's description whenever you load the protocol file into a control method. If you have not referenced the protocol in a control method, you can change the description even though the protocol file has been saved. Choose Save As in the File menu. When you see the Protocol dialog, type the new description and choose OK. When the Save As dialog appears, choose OK again and overwrite the current version of the protocol file

303 protocols Synchronizing injector protocol and control method So the start of data collection corresponds with the time of injection, coordinate the control method and the injector protocol. The following diagram shows the interaction between the control method and injector protocol during a run. As the diagram shows, once the control method gets system components ready, it starts the protocol and then puts itself, and the computer, into a wait state. While the control method waits, the protocol performs steps for preparing the sample. Once the protocol tells the injector to inject the sample, it issues a command to release the wait. The control method can then initiate data collection and issue other device control commands. Note: The synchronize command in the protocol can be located in a routine used by the protocol. where to find synchronize command The Synchronize command is in the command lists for the System and for the Injector under the Event menu of the Control Method or Protocol window

304 unipoint user's guide Gilson-supplied protocols Gilson supplies example protocol files on the UniPoint installation disks. Unless you changed the default installation path for the software, the files are in C:\GILSON\UNIPOINT\INJ_RP. Note: If you are using the 215 Liquid Handler/819 Valve Actuator as your sampling injector, example protocols are in the C:\GILSON\INJ_215 directory. For more information on the 215/ 819 protocols, refer to the Using the 215/819 for Injection and Using the 215/819 for Injection and Collection Technical Notes, available from your Gilson representative. Gilson recommends that you copy the protocol(s) to be used into your working directory. Keep the original protocols in case you need to refer to them. Following is a description of each protocol file. Open the protocol file to view its steps. diln.gip diln_rvr.gip inj_cenl.gip inj_parl.gip inj_totl.gip Dilutes sample using diluent from tube. Performs multiple aspirations and dispenses to mix diluent and sample. Variables used: DILUENT_LOCATION - diluent tube location DILUENT_VOLUME - diluent volume to aspirate and dispense into mix tube MIX_TUBE - mix tube location for diluent and sample TUBE - sample location SAMPLE_VOLUME - sample volume to aspirate and dispense into mix tube Dilutes sample using diluent from syringe pump or dilutor s reservoir. Variables used: DILUENT_VOLUME - diluent to dispense into tube TUBE - sample location Locates and aspirates a sample; uses centered loop fill injection technique to load and inject the sample; includes synchronization command to coordinate protocol and control method. Variables used: INJECT_VOLUME - sample volume TUBE - sample location PUSH_VOLUME - solvent volume from reservoir Locates and aspirates a sample; uses partial loop fill injection technique to load and inject the sample; includes synchronization command to coordinate protocol and control method. Variables used: INJECT_VOLUME - sample volume TUBE - sample location Locates and aspirates a sample; uses total loop fill injection technique to load and inject the sample; includes synchronization command to coordinate protocol and control method. Variables used: LOOP_VOLUME - size of injection loop TUBE - sample location

305 protocols mix.gip rinse.gip fc233par.gip fc233tot.gip Mixes by aspirating and dispensing sample volume. Variables used: MIX_TUBE - tube used for mixing MIX_VOLUME - sample volume to mix Cleans needle and injection port. (Used to control a 233 XL that is being used for injection and collection) Locates and aspirates a sample; uses partial loop fill injection technique to load and inject the sample; issues synchronization command to indicate the sample has been injected; rinses the injection port and the outside of the needle; issues a second synchronize command to let the control method know that the 233 XL can be issued fraction collector events. Variables used: INJECT_VOLUME - sample volume TUBE - sample location (Used to control a 233 XL that is being used for injection and collection) Locates and aspirates a sample; uses total loop fill injection technique to load and inject the sample; issues synchronization command to indicate the sample has been injected; rinses the injection port and the outside of the needle; issues a second synchronize command to let the control method know that the 233 XL can be issued fraction collector events. Variables used: LOOP_VOLUME - size of injection loop TUBE - sample location Note: For more information on FC233PAR.GIP and FC233TOT.GIP, obtain a copy of the Using the 233 XL for Injection and Collection Technical Note from your Gilson representative. Updating protocol configuration Before modifying the Gilson protocol file that you copied to your working directory, update its configuration information. You need to change the configuration so it specifies the injector and syringe pump or dilutor installed in your HPLC system. The Gilson example protocols were written for a 231XL injector and 401C dilutor. And, if you have a different sampling injector and syringe pump or dilutor, additional commands may be available. 1 Open the protocol file. 2 Choose System Configuration from the File menu. 3 When the System Configuration dialog appears, choose Browse. 4 Use the options in the Browse System Configuration dialog to select the configuration file for your HPLC system and then choose OK

306 unipoint user's guide 5 If a Configuration Comparison box appears, choose Yes to replace the protocol configuration with the updated configuration. When the System Configuration dialog appears again, the devices for your configuration appear in the Unit ID/Installed Devices list box. 6 Check the setup parameters for the injector and, if necessary, the dilutor: a) Choose the injector in the Device list box. b) Choose the Setup button. c) When the dialog box appears, modify the parameters as needed and choose OK. d) If necessary, repeat for the dilutor. 7 Choose Save. 8 When the Save As dialog appears, choose OK. When prompted, indicate that you want to replace the configuration. 9 Choose OK to exit the System Configuration dialog. 10 Choose Save then Close from the File menu. 11 Repeat the above for each protocol file. Checking protocol Before using a protocol in a programmed run, test it to see if it works. 1 Create or open a control method. 2 Load the protocol into the control method using the Injector dialog, accessed via the Device menu; see page Save and close control method. 4 Create an Operations window. 5 Choose Control Method from the Manual menu and verify that the correct control method is being used for manual control. If the method s name is shown, choose Cancel. If not, use the options in the Browse Control Method dialog to select the method and choose OK. 6 Choose Injector from the Manual menu. 7 Choose the protocol in the dialog and click on Send

307 15 Routines Routines consist of a set of instructions executed as part of a protocol. They can make creating protocols easier because the name of the routine identifies what it does, for example, Make Air Gap. Consequently when you set events for routines in a protocol, you can more quickly identify what is happening instead of reading through individual commands. There are two kinds of routines that can be created: injector routines to control an injector and its syringe pump or dilutor and generic routines to control any Gilson device connected along the GSIOC. One or more routines can be stored to the same file. Gilson has supplied routines for injector systems. You can modify these to meet your needs. See page for a description of the supplied routines. 15-1

308 unipoint user's guide Routines windows There are two Routines windows available in UniPoint: Injector Routines window supplies options for controlling an injector and its syringe pump or dilutor. Generic Routines window supplies options for controlling any other Gilson device. menu bar toolbar table pane annotation box status bar Injector Routines window Generic Routines window menu bar Choose available functions from the drop-down menus found beneath the items on the menu bar. Menu File Edit View Device Event Table Window Help Description Create, save, print, delete files or exit a window or UniPoint. Locate, make changes to, or transfer information. Select a routine so its steps can be viewed. Name installed devices and other resources used by the routines. Issue instructions to devices. Display or modify appearance of the table pane. Re-arrange windows or icons, or activate a window; display file annotation or toolbar. Learn about UniPoint. table pane Lists the steps currently set for a routine. The name of the routine is listed in the title bar for the document window. If you see the message **invalid command** in the table, the device for which a command was previously set is not listed in the configuration for the routine. Before executing the routine during a run, check the configuration using the System Configuration command in the routine's File menu. This message also occurs if a routines file is not available or listed in the Injector dialog or Generic Device dialog accessed via the Device menu. 15-2

309 routines Double-clicking on a row for an event automatically displays the dialog box used to set up the event. Using the right mouse button, you can double-click on a row that specifies an event for a routine and open the file that shows the instructions set in the routine. Double-clicking on the top (header) row displays a dialog for changing the format of the columns shown in the table. toolbar Provides quick access to some options available in the Injector or Generic Routines window. To activate a tool, just click on its icon. (Not all of the tools, below, are available in the Injector Routines window.) navigator tool open tool save tool print tool data channels tool contact tool injector tool detector tool fraction collector tool generic device tool system tool Displays Navigator. Displays Open dialog. Stores routine to disk. Displays Routine Name then Save As dialog. Displays Print dialog. Displays Data Channels Event dialog. Displays Contact Event dialog. Displays Injector Event dialog. Displays Detector Event dialog. Displays Fraction Collector Event dialog. Displays Generic Device Event dialog. Displays System Event dialog. To turn off/on the toolbar, choose its command from the Window menu. annotation box Displays configuration information for and parameters set in the routines file. To turn off/on the annotation, choose its command from the Window menu. To add your own comments, use the Notes command in the Edit menu. status bar Displays help message if a menu command is highlighted. 15-3

310 unipoint user's guide Overview of creating routines Following is an overview of how to set routines. 1 If running multiple configurations, ensure that the appropriate configuration file has been selected. To do this: a) Exit all open document windows. (The Navigator if it appears can remain on-screen.) b) Choose Default System Configuration from the File menu. c) When the dialog appears, check the name of the file indicated at the bottom of the dialog to see if the correct one is chosen. If not, use the Browse button to select the appropriate file. 2 Create the document window for the routines. You can do either of the following: Using the Navigator, choose Advanced to access the window for selecting the Injector or Generic Routines option. Then choose New. Or choose New from the File menu. When the dialog appears, choose More Documents to access the list box containing the selections for routines. Choose the routine category: Injector or Generic. 3 When the Routines window appears, use the Device menu to check or assign descriptions to, and specify options for, components. (See page 15-6.) If you have created routines files, you can use the Injector and Generic commands to load the information from another routines file into the routines file being created. 4 Use the Event menu to indicate each step for the routine. (See page 15-7.) Note: Instead of entering a value in the parameter box for a command, you can indicate a variable name, for example, SampleVol or Wavelength#. (See page ) You can also indicate a mathematical operator (+, -, *, /) in the parameter box. For example, when aspirating a volume you can indicate SampVol * 5 as a valid parameter. 5 When the steps for the routine are set, choose Save from the File menu. (See page ) 6 When the dialog appears: a) Type a description for the collection of routines in the Routines box, for example, Injector Routines. b) In the other text box, type the name to assign to the group of commands you just set up, for example, Make Air Gap if that is what they do. c) Choose OK. 15-4

311 routines 7 When the Save As dialog appears, indicate the name and path to which the routines file is saved and choose OK. 8 Choose New Routine from the Edit menu. This clears the table so you can set steps for the next routine. 9 Use the Event menu to indicate each command for the routine. 10 When finished indicating steps, choose Save from the File menu and assign a routine name. The file description can be modified only if you want to. 11 Repeat steps 8 to 10 for each additional routine to create. 12 When the last routine is created, save and name it as described in step 10. Then choose Close from the File menu. 15-5

312 unipoint user's guide Assigning descriptions Before you can issue commands to Gilson components, assign a userdefined description to them. To assign descriptions, use the command(s) under the Device menu. Device menu for injector routine Following is an example dialog for assigning description(s). The procedures for checking and modifying descriptions are the same as those for a control method. See Section 6 for more information. Device menu for generic routine Note: Gilson uses the special character * when assigning descriptions in the example routines files supplied with the software. This causes the software to forgo the device configuration checks it performs when a routines file is associated with a protocol file. Use this symbol with care in your files. Problems may occur at run time if device parameters do not match. For example, if the configuration for the routines file has a 231 XL injector and the configuration for the protocol has a 234 injector, the software does not warn you of any mismatches if the description for the 231 XL injector is Injector*. It warns you if the description is Injector. loading routines files Within a routines file, you can access the routines in another routines file and use them to set up a new routines file. 1 Choose the Injector or Generic command. The dialog box that appears includes a Routines Files Description area. 2 Choose the Browse button to locate the routines file and place its label in the Routines Files Description area. 3 Repeat step 2 for each routines file to use. When you choose Injector or Generic from the Event menu, you can insert a command that executes the instructions in a routine. 15-6

313 routines Issuing commands Each command issued when a routine is executed is referred to as a step. You can specify steps using the dialog boxes in the Event menu. Plus you can quickly duplicate, remove, or modify steps in the table using the command in the Edit menu. The Event dialog boxes are used to set the commands to send to defined components. To display an Event dialog box: Event menu for injector routine 1 Choose the device from the Event menu. Or, click on a tool in the toolbar. Or, double-click on an event in the table. Hint: While an Event dialog box is on-screen, you can quickly access the dialog for another device type by selecting its tool from the toolbar. Following is an Event dialog. The appearance and procedures for setting events within each dialog box is similar. For a description of the commands available for each Gilson device, refer to page Event menu for generic routine Note: When creating a generic routine for a 116 Detector, you may need to insert a wait time between each command being sent to the detector. To program the wait, use the Wait Time command for a System Event. adding step To add the first step to a new list or add a step to the bottom of the list: 1 Make sure New Event appears in the upper right corner of the dialog box. If not, choose New. 2 Use the areas in the dialog box to select a command or routine and indicate any parameters. 15-7

314 unipoint user's guide Note: Instead of entering a value in the parameter box for a command, you can indicate a variable name, for example, SampleVol or Wavelength#. See page for more information. 3 Choose Insert. The command parameter can be the result of performing a mathematical function on two variables or a variable and number, or even two numbers. For example, AirGap + SampVol and SampVol * 5 are valid command parameters. You can add (+), subtract (-), multiply (*), and divide (/). 4 Repeat to add more steps. inserting step between steps To insert a step before the first step or between two existing steps: 1 Use the up or down arrow to locate the step before which the new step inserts. For example, to insert a step between Steps 3 and 4, display information for step 4 in the text boxes. 2 Use the areas in the dialog to select the command and indicate any parameters for the device. 3 Choose Insert. deleting step To remove a step: 1 Use the up or down arrow so the information for the step is displayed. 2 Choose Delete. modifying step To change options for a step: 1 Use the up or down arrow so the information for the step is displayed. 2 Use the areas in the dialog to make the necessary changes to the step. 3 Choose Change. Viewing and modifying routines via the edit menu You can use the commands in the Edit menu to select a routine to view and then if necessary make changes to it. If you need help using Edit menu commands for deleting, copying, and pasting steps in a routine, see page The procedures for clearing the table to create a new routine, for selecting a routine to view, for deleting a routine, and for copying between two routines are given below. 15-8

315 routines creating new routine To clear the table so you can create a new routine, choose New Routine from the Edit menu. If the currently displayed routine has been saved, it is not deleted. It is just no longer displayed on-screen. You can re-display it using the procedures in viewing a routine, below. viewing a routine To view the steps in a routine that has been created but is not currently being displayed: 1 Choose Routines from the View menu. 2 In the Routine Names box, choose the routine s description in the list box and choose OK. (Or just double-click on the description.) The steps set for the routine appear in the table. 3 Repeat the previous step to review another routine. The Routine Names dialog is sizeable. Position the mouse pointer at the side or corner of the dialog box. Click and drag the box to the desired size. The dialog is also moveable. Click on the title bar and drag the box to the desired location. Choose Done to remove the Routine Names box. deleting a routine To delete a routine: 1 Choose Routines from the Edit menu. 2 In the Routine Names box, choose the routine s description in the list box and choose Delete. copying steps between routines To use the steps in one routine to generate a new routine: 1 View the source routine as described above. 2 In the table, highlight the steps to copy. (To highlight multiple lines, just drag the mouse pointer.) Then choose Copy from the Edit menu. 3 Choose New Routine from the Edit menu. Or if the routine already exists, display the destination routine as described above. 4 In the table, highlight the line before which the step insert and choose Paste from the Edit menu. If any variables are set in the copied lines, they are also copied to the modified routine. 15-9

316 unipoint user's guide Variables To make your routines more flexible, you can indicate a variable name instead of entering a value in a command parameter box for a step. A variable name can be any combination of letters and numbers and can contain special characters (_, as long as it is not the first character or a mathematical operator (+, -, *, /, %). It cannot include spaces. Valid variable names would be Wavelength# and Sample_Vol1. UniPoint does not differentiate between upper and lower case letters in a variable name. For example, it views the names Wavelength, wavelength, and WAVELENGTH as identifying the same value. The command parameter specified for a routine step can also be the result of performing a mathematical function on two variables or a variable and number. For example, AirGap + SampVol, or SampVol + 15 are valid command parameters. You can add (+), subtract (-), multiply (*), divide (/ ), and return the remainder (%). When you set up a step in a protocol that executes the routine, UniPoint prompts you for a value to assign to each variable in the routine. You can indicate the value in the protocol. Or, you can wait and indicate the value in the control method that uses the protocol or the operations list that uses the control method. To see what variable names have been set for a routine and to indicate lower and upper limits for and a default for each variable, do the following: 1 From the Event menu, choose Variables. 2 When the dialog box appears, choose the variable name in the list box. 3 Check the prompt that appears whenever the software references the variable. The prompt can be any combination of letters and numbers and can contain special characters and spaces. 4 To indicate minimum and maximum values for the variable, indicate the numbers in the Values Limits box. Separate the values with a colon, for example, 1:100. If the input is required, put an R before the minimum value, for example, R1:

317 routines You can also indicate the variable name of SYRINGE for syringe volume or Z_ARM for Z_arm height as a limit. 5 To set a default value for the variable name, type the value in the Default Value box. 6 To remove an unused variable name, choose it in the list box and choose Delete. 7 Choose OK when finished. if you add, change, or delete variable names If you add, change, or delete variable names and the modified routine is used in a protocol or another routines file, do the following: 1 Open the protocol or routines file. 2 Locate and double-click on the event for the modified routine. The parameter boxes at the bottom of the dialog update to reflect any variable changes made in the routine. 3 Make sure the correct information is indicated in each parameter box. If you make changes, click on Change again to update the protocol or routine table. 4 Choose Done to exit the Event dialog. 5 If you are updating a routines file, repeat steps 2-6 in each routine that uses the modified routine. 6 Save and close the protocol or routines file using the File menu. If any variables are no longer being used in the file, UniPoint asks you if it should remove them from the External Variables dialog (accessed via the Event menu). Choose Yes

318 unipoint user's guide Saving a routine and the routines file When you select the Save or Save As command from the File menu, you are prompted for two labels, as you can see in the dialog box below. The Routines File Description describes the collection of routines included in the routines file. You see this description whenever you load the routines file into a protocol or another routines file. Once this has been set for the file, there is no need to change it unless you come up with a better description while setting routines. (Use the rename feature with care especially if the routines file has been referenced in other protocol or routines files.) The Routine Name identifies the routine whose commands you just setup (or are about to set up) in the Routines window. If the Routines Name text box is blank, no name has been assigned to the routine; the title bar refers to it as New. If there is a name in the text box, you can rename it. (Once again, use the rename feature with care.) For each description, you can indicate any combination of upper and lower case letters and blank spaces in the text box. If you use an & in the description, the character following the & is underlined, for example, Rinse&Home. If the Routines window is "untitled" or if you selected the Save As command, you see the Save As dialog (shown on next page) after you exit the Routines dialog. Use the dialog to name the routines file

319 routines Gilson-supplied routines file Gilson supplies an example routines file INJ_ROUT.GIR on the UniPoint installation disks. Unless the default installation path for the software, it is copied to C:\GILSON\UNIPOINT\INJ_RP. Note: If using the 215 Liquid Handler/819 Valve Actuator as the injector, use the 215_ROUT.GIR file in the C:\GILSON\ INJ_215 directory. For more information, refer to the Using the 215/819 for Injection or Using the 215/819 for Injection and Collection Technical Note, available from your Gilson representative. Gilson recommends that you copy the routines file into your working directory. Since the routines file is referenced by Gilson-supplied protocols, do not change its name if using a Gilson-supplied example protocol. Keep the original routines file in case you need to refer to it. Following is a description of each routine. To view the commands in each routine, open the routines file. Then use the Routines dialog accessed via the Edit menu to select a routine to view. air gap aspirate dilution dilution/reservoir dispense home injection / centered loop fill injection / partial loop fill injection / total loop fill mix Aspirates air gap. Aspirates sample. Variable used: SAMPLE_VOLUME - volume to aspirate Aspirates diluent from tube and dispenses into mix tube. Variables used: DILUENT_LOCATION - diluent tube DILUENT_VOLUME - diluent volume to aspirate and dispense MIX_TUBE - mix tube location Aspirates and dispenses diluent, from syringe pump or dilutor reservoir, into sample tube. Variables used: DILUENT_VOLUME - volume to aspirate and dispense TUBE - sample location Dispenses sample volume. Variable used: SAMPLE_VOLUME - volume to aspirate Moves arm to left rear of tray; homes syringe pump or dilutor syringe; lowers needle into rinse station. Loads and injects sample using centered loop fill technique. Variables used: INJECT_VOLUME - sample volume PUSH_VOLUME - solvent volume from reservoir Loads and injects sample using partial loop fill technique. Variable used: INJECT_VOLUME - sample volume Loads and injects sample using total loop fill technique. Variable used: LOOP_VOLUME - size of injection loop Performs multiple aspirations and dispenses to mix. Variable used: MIX_VOLUME - volume to mix 15-13

320 unipoint user's guide reservoir rinse injection port rinse needle inside rinse needle outside sample location synchronize Aspirates solvent from syringe pump or dilutor s reservoir. Variable used: SOLVENT_VOLUME - solvent volume Moves needle to injector port and cleans injection port using solvent from syringe pump or dilutor s reservoir. Moves needle to rinse station and cleans it using solvent from syringe pump or dilutor s reservoir. Moves needle to rinse station and cleans it using solvent from syringe pump or dilutor s reservoir. Moves arm and lowers needle into sample tube. Variable used: TUBE - sample location Releases synchronize set in control method (used for injector/control method coordination). Updating routines configuration Before modifying the INJ_ROUT.GIR routines file that you copied to your working directory, update its configuration information. You need to change the configuration so it specifies the injector and syringe pump or dilutor installed in your HPLC system. The routines in INJ_ROUT.GIR were written for a 231XL injector and 401C dilutor. And, if you have a different injector or syringe pump or dilutor, additional commands may be available. 1 Open the routines file. 2 Choose System Configuration from the File menu. 3 When the System Configuration dialog appears, choose Browse. 4 Use the options in the Browse System Configuration dialog to select the configuration file for your HPLC system and then choose OK. 5 Check the setup parameters for the injector and, if necessary, the syringe pump or dilutor: a) Choose the injector in the Device list box. b) Choose the Setup button. c) When the dialog box appears, modify the parameters as needed and choose OK d) If necessary, repeat for the syringe pump or dilutor. 6 Choose Save. 7 When the Save As dialog appears, choose OK. When prompted, indicate that you want to replace the configuration. 8 Choose OK to exit the System Configuration dialog. 9 Choose Save then Close from the File menu

321 16 Tray Editor This section introduces the Tray Editor Software supplied with the Gilson UniPoint System Software. You use the Tray Editor to create a tray file that contains rack, zone and tube information for your Gilson injector or fraction collector. UniPoint then uses this information to access tubes during a run. Setting up a tray file consists of the following: Choosing the template for the work area of the auto-injector or fraction collector. (When you open the Tray Editor from UniPoint, the template is automatically selected and displayed.) Placing each rack into the appropriate location in the template. Identifying the zone names to assign to groups of tubes. (By default, UniPoint sets up the descriptions TUBES and FRACTIONS for you. You can use these names or specify different names.) Assigning a zone and number to each tube location used. This section describes how to do the above. However, first you learn how to start the Tray Editor and learn about the Tray Editor window. 16-1

322 unipoint user's guide Starting the tray editor You can start the Tray Editor using UniPoint. You access the Tray Editor by selecting its command button in the Injector and Fraction Collector dialogs of the Device menu. This menu is available in the Control Method, Protocol, and Routines windows. When the Tray Editor window appears, you see a work area containing a template for the work area of the device. See diagram on next page. Tray editor window Following is an example Tray Editor window with tray information already set up. menu bar toolbar template status bar menu bar Choose Tray Editor functions from the drop-down menus found beneath the seven items on the menu bar. Menu File Edit View Zone Options Window Help Description Create, open, save or print files; exit the Tray Editor. Add or remove zones, racks, and tubes. Change size of graphic; remove toolbar and status bar. Select a zone. Change tubing numbering or place rack in custom position. Re-arrange windows or icons, or activate a window. Learn about the Tray Editor. 16-2

323 tray editor template toolbar Displays a graphical representation of instrument s work area. It illustrates the position of racks and tubes. Consists of several tools that represent various functions of the Tray Editor. The table below describes each of the tools, from left to right. To activate a tool, just click on its icon. Tool New Open Save Print About Zone Left and Right Arrows Tube Numbering Description Creates a tray file. Recalls existing tray file. Stores tray file to disk. Generates a printout of the active tray file. Displays current software version and copyright information. Sets the names assigned to groups of tubes. Activates each zone in turn. The name of the active zone appears in the status bar located at the bottom of the window and described below. Identifies how blocks of tubes are numbered. The toolbar can be turned on and off from the View menu. tatus bar Shows the following information, from left to right: Field message X Location Y Location zone indicator Description Shows operational status of Tray Editor or a help message if a menu command is being highlighted. Shows the X axis position, in millimeters, of the mouse pointer. Shows the Y axis position, in millimeters, of the mouse pointer. Shows the name of the currently selected zone. If no zone is active, then No Zone Selected appears. The status bar can be turned on and off from the View menu. 16-3

324 unipoint user's guide Setting up a tray file The following sections take you through creating a tray file. As stated earlier, you do the following to set up a tray: Choose a template. Insert rack(s). Set up zone names. Assign zone name and numbers to tubes. Save the tray file. Choosing a template The first step in creating a tray file is to select a template file supplied with the Tray Editor. Unless their destination was changed when the Tray Editor was installed, these files are located at C:\GILSON\TRAY\RACKS. A template file identifies the work area of Gilson injectors and fraction collectors. Each template file contains rack locations on an instrument bed outline. The instrument bed outline represents the actual limitations of the instrument probe or dispense head. The area outside of this outline is inaccessible to the probe or dispense head. The name of each template file identifies the following: the model number of the device, for example, m203 is for the FC 203 Fraction Collector or m234 is for the 234 Autoinjector. if applicable, the version of the device, such as m203b for version B of the 203 Fraction Collector, m231a for the non-xl version of the 231 Auto-Sampling Injector, and m231x for an XL version of the 231 Auto- Sampling Injector. type of rack locator installed _an for narrow antlers, _aw for wide antlers _p for tray, fn for funnel adapter, or mt for microtiter plate adapter. When you open the Tray Editor from UniPoint, the template is automatically selected for the installed injector or fraction collector and displayed on-screen. To create a second tray file for the injector or fraction collector while in the Tray Editor, you must select a template. 1 Choose New from the File menu. Or, click on the New tool in the toolbar. 16-4

325 tray editor 2 When the dialog box appears, type or select the file to open. If the file is not listed in the File Name list box, do one or both of the following: In the Drives box, select a different drive. In the Directories box, select a different directory. 3 Choose OK after selecting a template file. The Tray Editor displays a document window with a graphical representation of the instrument s work area. (An example template is shown on page 16-2.) On-screen, the blue outline represents the area that can be accessed by the device. The gray rectangles identify locations for racks. Defining racks Once a template file has been selected, you identify the rack. Unless their destination was changed when UniPoint was installed, rack files are located at C:\GILSON\TRAY\RACKS. For rack files, the file name identifies the rack code and has the extension.grk, for example, CODE_028.GRK. Also be aware of the following: Some rack codes can be installed on a tray or on wide antlers. The file name with the T after the code number is for use with a tray, for example, CODE_30T.GRK. The other file is for use with wide antlers, for example, CODE_030.GRK. If using rack code 29, the file CODE_29L.GRK is for use with large Eppendorf vials and CODE_29S.GRK is for use with small Eppendorf vials. 16-5

326 unipoint user's guide identifying rack information To identify information for pre-determined rack locations, follow the procedures below. Note: Refer to page if using non-gilson racks or placing racks in non-standard location in the work area. 1 Click on one of the rack positions in the template to select it. A red outline appears whenever a rack position is selected. Then choose Add Rack from the Edit menu. Or, double-click on the rack location in the template. The Select rack to add dialog appears. The File Name list box displays valid rack files for the selected position. 2 In the File Name box, type or select the file to open. If the rack file is not listed in the File Name list box, do one or both of the following: In the Drives box, select a different drive. In the Directories box, select a different directory. 3 Click on OK. Note: If you see the Custom Placement dialog at this point, that feature has been turned on. Just click on OK. The Tray Editor positions information in the correct location. To turn off custom placement, choose its command from the Options menu. For more information on custom placement of racks, see page The template now displays tube locations that can be accessed for the rack. 4 Repeat the above until each rack has been defined. 16-6

327 tray editor deleting rack If a rack was just defined, you can choose Undo Last Add from the Edit menu. This command is only available immediately after a rack file has been opened. Once another action is performed, this command is dimmed. You can also delete a rack by double-clicking outside of a tube or insert. You see message box asking you to verify the deletion. Setting up and modifying zone names After identifying racks, you name the groups of tubes to process, for example Reagents, Samples, and so on. When you access the Tray Editor from UniPoint, the Tray Editor automatically assigns the default zone names, TUBES and FRACTIONS, and lists them in the Zone menu. You can use those zone names or different ones. Note: If you use the New command in the Tray Editor to create a new template, the Tray Editor assigns the default zone names, Zone 1-4. To the right of the zone name is its identifier (or shortcut key). When the template window is on-screen, pressing the letter (A - Z) on the keyboard enables you to activate the zone without having to use the Zone menu. When the zone is inactive, this is also the letter that appears in any tubes associated with the zone. The No Zone Selected command makes all zones inactive. Only the identifier (A - Z) appears in tubes assigned to a zone. To view the dialog box for managing zones, choose Zone Names from the Edit menu. Or, click on the Zones tool in the toolbar. You see the Edit Zones dialog that enables you to identify up to 26 zone names (identified by a letter between A and Z). Refer to the procedures below to make changes to, add, and remove zones. The new and updated zone names are then listed in the Zone menu. 16-7

328 unipoint user's guide changing a zone name To modify a zone name: 1 Select the zone to replace. 2 In the text box, type the new name. 3 Click on Replace to change the old name to the new name. adding a new name To identify another zone name: 1 In the text box, type the new name. 2 Click on Add to add the new name to the list. Plus, the Tray Editor also indicates the 1-letter identifier for quickly activating the zone. When a zone is inactive and tubes have been assigned to it, this letter appears in the tubes associated with the zone. New zones are added to the list in the order they are entered. To change the order, use the changing a zone name procedures, above. removing a zone name To delete a zone name: 1 Choose the zone to delete. The name of the zone appears in the text box. 2 Click on Delete to remove the zone from the list. Remaining zones move up in the order to replace the deleted zone. Assigning zone and number to tubes You can select tubes to put into different zones that you have named for your convenience. Numbering tubes establishes identification information for each one. The liquid handling program can then refer to a specific tube by zone name and sequence number. When adding tubes to and removing them from a zone, note the following: Tubes can be numbered individually, across single rows or columns, or in blocks. Tubes from more than one rack can be in the same zone. Tubes in the same zone can have different numbering patterns. 16-8

329 tray editor numbering tubes individually Tubes can be numbered one at a time without an apparent pattern. To number tubes individually: 1 Select a zone from the Zone menu. 2 Click on tubes in the order that you want them numbered in the zone. To remove a tube from a zone, click on the tube again. Higher numbered tubes are renumbered to maintain the sequence. numbering tubes in a single column or row Tubes can be numbered sequentially across a column or row. The numbering order is determined by the direction they are selected. For example, tubes that are selected from left to right display numbers in ascending order from left to right. If tubes are selected across racks, the numbering sequence is not interrupted. To number tubes in a column or row: 1 Select a zone from the Zone menu. 2 Position the mouse pointer just outside the first tube to assign to the zone. 3 While holding down the left mouse button, drag the mouse in the direction you want the tubes numbered. You can select a row of tubes across multiple racks. 4 When all of the tubes have been selected in the single row or column, release the mouse button. Tubes can be numbered from left to right, right to left, back to front, or front to back Re-selecting tubes remove them from the zone. Note: If you select multiple rows or columns, the Tray Editor thinks you are selecting a block of tubes and uses the default numbering pattern. This pattern is set using the Tube Numbering command in the Options menu. 16-9

330 unipoint user's guide numbering blocks of tubes Tubes can be numbered in a selected pattern across multiple columns and rows. The numbering order is determined by the pattern selected in the Numbering Patterns dialog. To number blocks of tubes: 1 Select a zone from the Zone menu. 2 Choose Tube Numbering from the Options menu. The Numbering Pattern dialog appears. 3 Select where numbering begins and the pattern to use. As you can see, numbering patterns can be rectangular or serpentine. By selecting each of the Starting corner option buttons and then looking at the pattern diagrams, you can see how the numbering of tubes is affected. There are 16 different numbering patterns available. (The numbering patterns shown in the dialog box are representative and can be expanded to include as many tubes as needed.) 4 Click on OK to close the dialog box and return to the Tray Editor window so you can select tubes. 5 Point the mouse just outside of one of the corner tubes to assign to the zone. 6 Holding down the left mouse button, drag the mouse until all of the tubes have been selected. You can select multiple racks. Release the mouse button. When you are numbering a block of tubes, the direction they are selected does not affect the pattern. When the selected block of tubes expands across racks, the numbering sequences are not interrupted and the pattern continues as if the rack outlines do not exist

331 tray editor 7 Repeat the above to assign numbers to additional blocks of tubes. Remember to change the zone and tube numbering scheme if necessary before selecting the tubes. changing tube numbers You can change the numbers assigned to tubes in a zone as described below: 1 Choose a zone name from the Zone menu to select and highlight all of the tubes associated with a zone. 2 Select a tube or group of tubes to renumber. This action removes the tubes from the current zone and removes the numbers as well. Gray filled circles appear for the tubes. Remaining tubes in that zone adjust their numbering so that no numbers are skipped. 3 Select the tubes again in the pattern that you want them numbered. removing tubes from zone To remove tubes from a zone: 1 Choose No Zone Selected from the Zone menu. 2 Then click on each tube or select a group of tubes by clicking and dragging to draw a box around the tubes to remove. Remaining tubes in the zone are renumbered so that no numbers are skipped. The removed tubes are displayed as gray-filled circles. Viewing techniques determining tube status The appearance of a tube determines its current status. Gray-filled - The tube does not belong to a zone and is consequently not numbered. Clicking on the tube assigns it to the currently selected zone. (If the No Zone Selected option is active; the state of the tube remains the same.) Blue-filled, numbered - The tube is in the currently selected zone. Clicking on the tube: Reverts the tube to an ungrouped and unnumbered state. A grayfilled circle results. Causes remaining tubes in the zone to adjust their numbering so that no number is skipped. Lettered - The tube is assigned to a zone that is represented by the letter. Clicking on the tube activates its zone so you can see the tube number assigned to the tube plus any other tubes in the zone. The name of the zone is then shown in the status bar

332 unipoint user's guide scrolling through zones Do any of the following to scroll through zones and see what tubes have been assigned to each one: 1 In the toolbar, click on the left or right arrow to move through the assigned zones, activating each in turn. Or, choose the name of the zone from the Zone menu. Repeat for each additional zone. Or, using the keyboard, press the 1-letter shortcut key assigned to the zone. getting tube information SHIFT + Pressing the SHIFT key while simultaneously clicking on a tube displays a box showing the current state of the tube (zone and number assignment) and the tube s physical dimensions. getting template and rack information Pressing the SHIFT key while simultaneously clicking outside a tube displays a box showing the name of the associated template and rack file. SHIFT

333 tray editor enlarging/reducing size of the template You can zoom in and out on the information displayed in the template by changing its size. To do this, choose Scaling from the View menu. The Scaling dialog appears. Some considerations when using the Scaling dialog are: Maximum scale factor is 400% and the minimum scale factor is 25%. Choose the Auto Scale check box to display the entire instrument bed in the opened window. (Note that manually setting scale factor turns off this option.) You can indicate the scale factor directly by highlighting the current scale factor in the text box and typing the new number or by using the scroll bar to select the factor. turning off/on toolbar To remove the toolbar from display, choose Toolbar from the View menu. To redisplay it, select the command again. turning off/on status bar To remove the status bar from display, choose Status bar from the View menu. To redisplay it, select the command again

334 unipoint user's guide Managing tray files saving a file To save a new file to disk for future retrieval: 1 From the File menu, choose Save or Save As. Or, click on the New tool in the toolbar. 2 When the File Save As dialog appears, use its options to name and select a storage location for the file. 3 Choose OK to save the file. To quickly save the contents of a named file to disk: 1 From the File menu, choose Save (rather than Save As). Or, click on the Save tool in the toolbar

335 tray editor opening a file To display the contents of an existing tray file: 1 From the File menu, choose Open to display the Open Tray File dialog. Or, click on the Open tool in the toolbar. 2 When the Open Tray File dialog appears, use its options to locate and select the tray file. 3 Choose OK to open the file. making a copy of a file To store the changes made to file to a new name while still keeping a previous version, do the following: 1 From the File menu, choose Save As. 2 When the File Save As dialog (shown on previous page) appears, use its options to name and select a storage location for the file. 3 Choose OK. deleting a file To remove one or more tray files, use the Windows File Manager. See your Microsoft Windows User s Guide for more information

336 unipoint user's guide Printing tray files previewing what is printed To see what is printed for a tray file: 1 Choose Print Preview from the File menu. The Print Preview window appears. 2 Use the command buttons at the top of the window to move between pages, manipulate what you see on screen, and send the pages to the printer. Note: Tray diagrams usually print better if landscape orientation has been selected. See specifying printer and setup options on the next page. The first page lists the zones that have been created. The second page is the tray diagram. Both pages identify the page number, the date and time, and the name of the tray file. 3 Choose Close to return to the Tray Editor window

337 tray editor specifying printer To select and if necessary set options for the printer to use when printing tray files: 1 From the File menu, choose Print Setup. 2 When the dialog appears, indicate the printer options. The available options depend on the installed printer. Refer to the documentation accompanying your printer for more information. Note: Tray diagrams usually print better if landscape orientation has been selected. See specifying printer and setup options on the next page. You install printers using Microsoft Windows. See the Microsoft Windows User s Guide for details. 3 Choose OK. printing To obtain a printout: 1 Choose the Print command from the File menu. 2 When the Print dialog appears, make sure the appropriate printer is indicated at the top of the dialog. To select a different printer, choose Setup. 3 Select the pages to print and indicate any additional options in the dialog available for your printer, for example, print quality. 4 Choose OK to start the printing process

338 unipoint user's guide Identifying custom racks and tubes indicating rack placement for custom tray If your instrument has a custom tray, you must define where each rack is. Custom Placement allows you to identify a rack anywhere in the instrument s work area. 1 Choose Custom Placement from the Options menu. When selected, a check mark appears next to the command. 2 Choose Add Rack from the Edit menu. 3 When the Select Rack to Add dialog appears, choose from the list of available racks and click on OK. 4 When the Placement Information dialog appears, indicate the location and orientation of the rack; then click on OK. X location. The distance (in millimeters) to the center of the rack along the X axis. (See diagram to the left.) Y location. The distance (in millimeters) to the center of the rack along the Y axis. (See diagram to the left.) Orientation. How the rack is positioned on the tray. Vertical is the default position for a rack installed on the tray, such as shown in the diagram to the left. Horizontal indicates that the rack is positioned from left to right along the front face of the instrument. The diagram on the next page shows how different values in the Degrees text box affect how the rack is displayed. For reference, it identifies tube 1 for a Gilson rack in a standard vertical position. As the diagram illustrates, 0 degrees is a Vertical orientation and 270 degrees is a Horizontal orientation. 5 Repeat steps 2-4 for each rack. 6 Choose Custom Placement again from the Options menu so that command is no longer active

339 tray editor indicating tube placement for custom rack If you are using a non-gilson rack or customized Gilson rack, use the following procedures to identify the size, location, and number of tubes that the rack can hold. 1 Click on a rack location to select it. 2 Choose Add Vessels from the Edit menu. 3 When the dialog box appears, indicate tube information and click on OK. Distances can be entered to the nearest tenth of a millimeter. Home position is located at the left rear of the instrument s work area

340 unipoint user's guide X Origin. The distance from Home position to the center of the array along the X axis. Y Origin. The distance from Home position to the center of the array. X Count. The number of vessels in the rack along its X axis. Y Count. The number of vessels in the rack along its Y axis. X Delta. The distance between the centers of each tube in the X direction. Y Delta. The distance between the centers of each tube in the Y direction. Diameter. The tube s diameter. Top. The distance to the top of the tube in the rack. Bottom. The distance to the bottom of the tube in the rack. 4 Click on OK to exit the dialog. Locations for the identified tubes appear in the rack. 5 Repeat the above for any additional racks that require custom tube information. Quitting the tray editor To quit the program. 1 Choose Exit from the File menu. Or, double-click on the control menu box at the top left corner of the window. The UniPoint dialog, from which you accessed the Tray Editor, appears

341 17 Extract, Import, and Export Features This section describes how to: retrieve archived information from data, report, and calibration files import channel data from a Gilson 160 spectral data file or from a text file for display in a Results window export channel data and report information into a text file 17-1

342 unipoint user's guide Extracting archived information A data file contains more than just the information collected for data channels. It also includes the operations list, control method(s), and analysis method(s) used during the run. UniPoint contains an archive extract feature for getting to this information. You can also use this feature to recreate the analysis method used to produce a report or calibration file. 1 From the File menu, choose Archive Extract. 2 In the File Name box, type or select the data, report, or calibration file. If the file is not listed in the box: In the Drives box, select a different drive. In the Directories box, select a different directory. 3 Select the option button for the document type to retrieve. 4 If multiple files for the document type are available in the file, select the name of the file, whose information is extracted, in the Available File list box. 5 In the Destination File box, specify the path and file name into which the information are stored. To store the file to its original name and location, click on the name in the Available Files list box. The selected information automatically appears in the Destination File text box. You can use the Browse button to locate a different file that is overwritten with the extracted information. 6 Choose Extract. 7 Choose Done when finished extracting information. 8 Use the Open command in the File menu to locate and display the information for the file(s) just extracted. 17-2

343 extract/import/export Importing data Viewing Gilson 160 chromatogram If you have a Gilson 160 Diode Array Detector, you import channel data from a spectral data file (.??D extension) and display its trace in an already opened Results window. 1 From the File menu of the Results window, choose the Import command and display its cascading menu. Then choose 160 DAD Chromatogram. 2 When the Import DAD Chromatogram dialog appears, use the upper part of the dialog box to locate and select the file. 3 Use the lower part of the dialog box to specify extract and reference wavelength information. Refer to field descriptions below. Extract Wavelength. Type the wavelength of the chromatogram to display. The range of wavelengths available for the file are listed to the right of the text box. Reference Selection. Use this area to indicate parameters for the reference wavelength. The reference wavelength is used to correct for background noise and instabilities in the detector. The absorbance at the extract wavelength is calculated using the reference wavelength. See the Software Reference section in the Gilson 160 Diode Array User s Guide for the chromatogram calculation. For the reference wavelength, choose a wavelength that is close to the extract wavelength and in a region of non-absorbance. If you select the Wavelength Only option, a new reference value is calculated at each time point and is used in the chromatogram calculation. 17-3

344 unipoint user's guide If you select the Wavelength and Time option, the reference value at the specified time point is used in the chromatogram calculation. As a recommendation, use a time point near the beginning of the run, for example, 0.1 min. Duration. How long spectral data was acquired. Date and Time. When the data in the file was acquired. 4 Choose OK to display the chromatogram. The legend in the Results window identifies the trace. 17-4

345 extract/import/export Viewing chromatogram from non-gilson detector If you have a non-gilson detector and can save the detector s output in an ASCII format, you can view the data in the Results window of System Controller. Each line of data within the file must include a time and a voltage. The time and voltage can be separated by a tab, comma, or space. Each line must end with a carriage return. To display a chromatogram for the data: 1 From the File menu, choose the Import command and display its cascading menu. Then choose ASCII Chromatogram. 2 When the dialog box appears, indicate the appropriate information. Import File. The complete path to and name of the file. You can use the Browse button to locate the file if necessary. Delimiter. How the time and voltage values are separated. 3 Choose Import. Then choose Done to exit the dialog box. The selected trace information appears in the Results window. The legend identifies the trace. 17-5

346 unipoint user's guide Exporting data and reports The Export commands within the Results window enable you to convert data channel and report information into a text format for use in a spreadsheet program. Exporting chromatogram data You can convert data files into a text format that can be viewed and manipulated in a spreadsheet program such as Microsoft Excel. These converted data files contain channel information. Each line of data within a channel file includes a time and a voltage. The time and voltage are separated by a tab or comma depending on the delimiter you selected before exporting data. Each line is ended with a carriage return. To generate an export file containing raw data: 1 From the File menu, choose the Export command and display its cascading menu. Then choose Data. 2 Indicate information in the dialog and choose Export. Export File. Indicate the name of the export file to create. To replace an existing file with new information, click in this text box and then use the Browse button to locate the file. Data Channel. If multiple channels are displayed, choose the channel information to convert. Num. Data Pts. The number of data points in the file. Extract Interval. Identifies how often UniPoint extracts a data point and places it in the export file. For example, if the data file has 7000 data points and the extract interval is set to 100, the exported file has 70 data points. UniPoint skips the first 99 data points and places the 100 th data point in the export file. It then skips the next 99 data points and places the 200 th data point in the export file. The software continues this procedure until all 7000 data points are processed. 17-6

347 extract/import/export Delimiter. How the columns of text generated by the software are separated. The software can separate each column by tab, comma, or blank space. viewing raw data in spreadsheet program You can view Gilson raw data ASCII files in a spreadsheet program, such as Microsoft Excel. Use the spreadsheet program to manipulate and graph data. Following is a description of how to create a graph of a data channel file using Excel. 1 Create an ASCII text file of a Gilson data file using the Export Data command as described above. 2 Iconize or exit UniPoint. 3 From the Program Manager, start the Excel program (EXCEL.EXE). An empty spreadsheet appears. 4 Choose Open from Excel s File menu. 5 Once the Open dialog box appears, use the list boxes and text box to choose the data channel file. When the correct file name appears in the File Name box, click on OK. The spreadsheet for the file appears. Column A lists the time, and column B lists the corresponding voltage. 6 Highlight column B. To do this, click the cross mouse pointer next to the B column heading. 7 Choose New from the File menu. 8 When the dialog box appears, select Chart. Then click on OK to generate a plot for the data. 9 Use the Excel menu and toolbar items to make any necessary modifications to the plot. 17-7

348 unipoint user's guide Exporting report information You can convert report information into a text format that can be viewed and manipulated in spreadsheet program. Each line of the exported information includes the text currently shown in the table, separated by tabs, plus the path, file name, and description of the sample. A carriage return marks the end of each line. If you don t want a column of information exported, remove the column using the Format Columns commands within the Table menu. creating an export file To produce an export file for the information shown in the table: 1 Ensure that information shown in the table is what you want exported. 2 From the File menu, choose the Export command and display its cascading menu. Then choose Report. 3 Type the name of the export file to create in the Export File box. Use the.txt extension. 4 Use the list box to select each sample whose report information is exported. 5 To add the exported information to the end of the specified file, choose the Append check box. This option enables you to place report information for different samples in the same export file. 6 Choose Export to generate the file. appending to existing file You can add information to an existing text file. This feature enables you to place report information for multiple samples in the same export file. 1 Make sure the information shown in the table is what you want exported. 2 Choose Export Report from the File menu. 17-8

349 extract/import/export 3 Use the Browse button to locate an existing export file. 4 Choose the Append check box. 5 Choose Export to add the information to the end of the file. viewing report information in Excel You can view Gilson text files in a spreadsheet program such as Microsoft Excel. Use Excel to manipulate the information in the file. 1 Create the text file for the report(s) as described above. 2 Iconize or exit UniPoint. 3 From the Program Manager, start the Excel program (EXCEL.EXE). An empty spreadsheet appears. 4 Choose Open from Excel s File menu. 5 Once the Open dialog box appears, use the list boxes and text box to choose the export file. When the correct file name appears in the File Name box, click on OK. The spreadsheet for the file appears. 6 Use the Excel menu items to make any necessary modifications to the spreadsheet, such as increasing the size of the sample ID column so that the entire sample ID can be seen. 17-9

350 unipoint user's guide Exporting calibration information You can convert calibration information into a text format that can be viewed and manipulated in spreadsheet program such as Microsoft Excel. Each line of the exported information includes the text currently shown in the table, separated by tabs, plus the path and file name of the calibration. A carriage return marks the end of each line. There are no headings only exported information. If you do not want a column of information exported, remove the column using the Format Columns command in the Table menu. creating an export file To produce an export file for the calibration information shown in the table of a Calibration window: 1 Ensure that columns shown in the table pane are what you want exported. You can remove columns from display and from export using the Format Columns command in the Table menu. 2 From the File menu, choose the Export command. 3 Type the name of the export file to create in the Export File box. Use the.txt extension. 4 Use the Calibrations list box to select the calibration table(s) whose peak information is exported. 5 Use the Peak Selection area to indicate if information for all standard peaks or for the peak currently selected in the Calibration window is exported. 6 If the file listed in the Export File box already exists, choose the Append box to add exported peak information to the end of the file. If Append is not selected, the file is overwritten with the exported information. 7 Choose Export to generate the file. Refer to page 17-8 for information on viewing the file in Microsoft Excel

351 Appendix A System Configuration In order to issue commands to Gilson devices connected to the HPLC system, UniPoint must know about those devices. The first time UniPoint was started, a message box appeared indicating that the software was going to scan the Gilson communications channel (GSIOC) for Gilson devices. If the scan was performed and setup parameters were indicated for located devices, a default configuration file DEFAULT.CFG was created. Multi-system users. If you are controlling two or more HPLC systems with UniPoint, create separate directories for each system, for example, SYSTEM1 and SYSTEM2. Store the configuration file and all method, results, etc., files in the directory for that system. See page 1-8 for more information. A-1

352 unipoint user's guide Working with configuration files The configuration file not only identifies the names of Gilson devices connected to the HPLC system but also identifies any resources that a device can contribute to system operation, such as inputs, outputs, and analog channels. Additionally, if you have Gilson 730 Sampler Manager Software, you indicate that in the configuration file also. System configuration information is stored in control methods, protocols, and routines because those file types contain commands for controlling instruments. If you are running multiple HPLC systems, you need to check the name of the configuration being used before creating any of those file types. Reviewing configuration file A configuration file remains the default until another one is selected. To see what devices UniPoint thinks are currently turned on and connected to the computer, do the following: 1 If necessary, close all open document windows. (The Navigator can remain open.) 2 Choose Default System Configuration from the File menu. The Default System Configuration dialog appears. Its list box shows the names of Gilson devices installed in your HPLC system. 3 Verify that all listed devices are correct for your HPLC system. Do one of the following: If all devices are listed, choose OK to exit the dialog. If only the computer system is listed, see Scanning the GSIOC on page A-6 to install devices quickly. If a device is missing or you need to change a device, see Modifying the configuration file on page A-3. If UniPoint controls multiple HPLC systems and the appropriate configuration is not shown, see Searching for configuration file on page A-6. A-2

353 system configuration Modifying the configuration file If a new device is added to the system or if you want to remove a device from the system configuration, you can do so. Note: Changing the configuration after control method, protocol, and routines file have been created may cause a message to appear when you open one of those files. Refer to Configuration checks on page A-7. 1 Access the Default System Configuration dialog as described on page A-2. 2 Refer to the appropriate side bar below, depending on whether you want to add, delete, or replace a device. adding device To add a device: 1 Choose Install. The dialog displays additional options. 2 Select the device in the Known Devices list box. 3 If necessary, indicate (or modify) the unit ID for the device. For most devices, UniPoint inserts the default unit ID for the device. If a default ID is not indicated, refer to the user s guide for the device for information on checking and setting its unit ID. 4 Choose Add. The information for the device is inserted into the Unit ID/Installed Devices list box. If the device requires additional information, a Setup dialog appears. Enter information for the device and choose OK. In most cases, the Setup dialog supplies options to assist you in specifying device parameters. Note: For a 232 XL or 233 XL Injector, the setup parameters request the location of the right rinse station. If the rinse station is located above the right valve, choose Right. If the rinse station is located to the left of the right valve, choose Center. A-3

354 unipoint user's guide deleting device If a device is no longer in your system s hardware configuration, you can remove it from the configuration file. To remove a device from the configuration file: 1 Choose Install, if it hasn t already been selected. 2 Select the device in the Unit ID/Installed Devices list box. 3 Choose Remove to delete the device from the Unit ID/Installed Devices list box. replacing device To replace a device with a different device: 1 Choose Install. The dialog displays additional information. 2 Select the new device in the Known Devices list box. 3 If necessary, indicate (or modify) the unit ID for the device. For most devices, UniPoint inserts the default unit ID for the device. 4 Select the device being replaced in the Unit ID/Installed Devices list box. 5 Choose Replace. If the replacement device requires setup parameters you need to indicate those before change is made. saving changes To save changes to the configuration file: 1 Choose Save As to display a dialog box for saving the updated information. In most cases, you want to replace the old configuration file with the updated information. Searching for configuration file If you are controlling multiple HPLC systems, you may need to select a different configuration file and load its information into the computer s memory. 1 Access the Default System Configuration dialog as described on page A-2. The name of the configuration file whose information is being shown appears at the bottom of the dialog. Its associated devices appear in the Unit ID/Installed Devices list box. A-4

355 system configuration 2 If the correct file name is not shown, choose Browse. The Browse System Configuration dialog appears. 3 In the File Name box, type or select the configuration file you want to open. If the file is not listed in the box, do one or more of the following: In the Drives box, select a different drive. In the Directories box, select a different directory. 4 Choose OK when the desired name appears in the File Name box. The System Configuration dialog re-appears and the Unit ID/Installed Devices list box contains information for the selected file. Scanning the GSIOC for devices You can have UniPoint search for Gilson devices that are turned on and connected to the Gilson Serial Input Channel (GSIOC). This simplifies the setup of the configuration file. 1 Access the Default System Configuration dialog as described on page A-2. 2 Choose Install. The dialog displays additional information. 3 Choose Scan. 4 When the message box appears, verify that all instruments are turned on and then choose OK. UniPoint scans for unit IDs from 63 to 0. If a device requires additional setup parameters, a dialog box appears requesting the setup information. Note: If the software locates a 402 Syringe Pump during the scan, it selects the 402 Left Syringe Pump description and places it in the Unit ID/Installed Devices list box. If the pump is configured differently, replace the 402 Left Syringe Pump description with the correct configuration. Choose 402 Dual Syringe Pump, 402 Dual Syringe Pump w/ Tee, or 402 Right Syringe Pump. If you have a 122 Fluorometer connected to a 606 GSIOC to RS- 232 Converter, the software selects the 606 GSIOC to RS-232 Converter description and places it in the Unit ID/Installed Devices list box. Replace the 606 GSIOC to RS-232 Converter description with 122 Fluorometer Converter. A-5

356 unipoint user's guide Note: If you are using a 233 XL or 215 for both injection and collection, the Unit ID/Installed Devices list box must include the device as an Auto Injector and a Fraction Collector. If you scanned the GSIOC, the scan identifies the device as an Auto Injector. You must add the device as a Fraction Collector to the Unit ID/Installed Devices list box. When you add the device as a Fraction Collector, use the same unit ID as the Auto Injector device. For a 232 XL or 233 XL Injector, the setup options request the location of the right rinse station. If the rinse station is located above the right valve, choose Right. If the rinse station is located to the left of the right valve, choose Center. Note: When UniPoint locates a VALVEMATE during the scan, it selects the VALVEMATE (2 Positions) device and prompts you for valve information. If a multi-position valve is installed, you should click on OK when the Device Setup dialog appears and continue the GSIOC scan. After the scan completes, replace the VALVEMATE (2 Positions) device with the VALVEMATE (4-8 Positions) device and indicate the requested valve information. When you create a control method, protocol, or routines file, UniPoint assigns the default description VALVEMATE followed by its unit ID to the installed VALVEMATE. 6 Choose OK to exit the Default System Configuration dialog and save any changes to the current file. Specifying system units In the configuration file, you can view and modify the pressure and if applicable the temperature units used by devices, such as pumps, pressure regulators, and temperature controllers. 1 Access the Default System Configuration dialog as described on page A-2. 2 Choose Units. 3 When the Units Selection dialog appears, choose the appropriate units for pressure and temperature. 4 Choose OK. 5 Choose OK to exit the System Configuration dialog and save any changes to the current file. Or, choose Save to copy the configuration file to a new name (see Saving changes to configuration file if you need more information). A-6

357 system configuration Saving changes to the configuration file To save the changes to the file: 1 Choose the Save As button in the Default System Configuration dialog. 2 When the Save As dialog appears, use its options to assign a name and select a storage location for the configuration file. Configuration checks 3 If a configuration file already exists for the given name, a message box appears. Choose OK to replace the existing file or choose Cancel to stop the replacement. When a control method, protocol, or routine file is created, UniPoint stores the following configuration information in it: Path and name of the configuration file Unit IDs and names of devices in the configuration file The software obtains this information from the configuration file indicated as the default. See page A-2 for procedures on identifying the software s default configuration file. When the control method, protocol, or routines file is opened, UniPoint checks the information in the file against the information in the configuration file stored on disk. To ensure compatibility, UniPoint also checks the configuration information in protocol file when its loaded into a control method and the information in a routine files its loaded into a protocol file. When comparing configuration information, UniPoint checks to see if the corresponding device names and unit IDs can be found in both files. A-7

358 unipoint user's guide configuration cannot be found If you open a file and UniPoint cannot locate the configuration file used to create it, UniPoint displays <internal> in the System Configuration dialog accessed via the File menu. You can use the Browse button to locate the configuration file if it was moved to a new location. Or, you can use the Save button to store the configuration to a file so it can be located the next time the file is opened. configuration comparison You see a Configuration Comparison box if the configuration stored in the method, protocol, or routines file does not match the current HPLC configuration. You also see a Configuration Comparison box if you browse for a different configuration, from the System Configuration dialog, and the selected configuration does not match the configuration for the file. The list boxes, in the Configuration Comparison box, display the configuration information so you can see the differences. The flow chart on the next page identifies different messages boxes that may appear depending on what your action is during UniPoint s configuration comparison procedure. When you associate a routines file with a protocol or a protocol with a control method, UniPoint also checks the configuration information stored in the files to ensure they are compatible. If the control method does not include a device specified in the protocol or the protocol does not include a device specified in the routines file, you see the following. If the control method or protocol includes a different model number of the same type of a device or the same device type with a different unit, you see the Functional Device Exchange box, shown in the flow chart on the next page. A-8

359

360 Appendix B Sample Tracking Sample tracking enables you to see the injector tray location from which a sample came and/or the fraction collector location(s) into which its eluent was collected. The tray information along with the chromatogram and sample list can be viewed in the Results window. Results window Injector tray Fraction collector tray Samples dialog Following are details on pre-run option setup and post-run viewing for sample tracking. You must have a Gilson GSIOC-compatible injector and/or fraction collector to use the sample tracking capability. B-1

361 unipoint user's guide Before the run injector protocol/routines file Before the run, tell UniPoint which tube position to associate with the injected sample. You do this by selecting the Track Sample check box for the Move to Sample Location command, accessed via the Event menu s Injector command. Note: If the Track Sample check box is selected more than once for a sample, UniPoint uses the last tube position marked for sample tracking. If using a Gilson-supplied injection protocol, modify the sample location routine in the routines file as described below. This change will automatically be used by each protocol that uses the routines file: 1 Open the routines file, INJ_ROUT.GIR. At UniPoint installation, this file is placed in C:\GILSON\UNIPOINT\INJ_RP. 2 Choose Routines from the View menu and select the sample location routine from the list box. Then choose View. 3 In the table pane, double-click on the step for the Move Arm to Sample Location command. B-2 4 When the Injector Event dialog appears, choose the Track Sample check box and choose Change. Then choose Done to exit the dialog.

362 sample tracking 5 In the table pane, notice that <Tracked> now appears in the step for the Move to Sample Location command. 6 Save the change to the routines file by choosing Save from the File menu. If using your own protocol, decide which tube location to associate with the sample. If the protocol does sample preparation steps, you can choose the initial location for the sample before any mixing or dilution steps or choose the location from which the prepared sample was aspirated for injection. control method In the control method, do the following. 1 Use the Device menu to set device descriptions and options for the injector, fraction collector, pumps, data channels, and other devices. For the injector, check that a description is assigned to the injector and its dilutor or syringe pump, and create or select the protocol file and tray file. You can access the Tray Editor via the Injector dialog. For the fraction collector: Check that a description is assigned to the fraction collector, and create or select the tray file. You can access the Tray Editor via the Fraction Collector dialog. If collecting peaks, choose the Options button to access the Fraction Collector Options dialog. Then, choose the channel to monitor for fraction collection and set the mv full scale value to 10 mv. The mv full scale value specified in this dialog box must be the same as that set for the channel in the Strip Chart Options dialog, described below. You can also use the Fraction Collector Options dialog to indicate a delay volume. UniPoint s on-line help provides information on how to calculate delay volume based on the length and diameter of tubing. B-3

363 unipoint user's guide If collecting peaks, choose the Options button to access the Strip Chart Options dialog. In that dialog, choose the channel to monitor for fraction collection and set its maximum full scale value to 10 mv. The mv full scale value specified in this dialog box must be the same as that set for the channel in the Fraction Collector Options dialog, described above. 2 Use the Event menu to specify the timed instructions to send to the injector, fraction collector, pump, and other devices, and to start and stop data channel collection. For fraction collection, you can set events to collect by time, volume, or peak. Refer to the following examples for injector and fraction collector control when creating your control method. The injector is controlled via a Gilson-supplied injection protocol. Variable names are in all capital letters (for example, TUBE). Example 1. The following example uses the partial loop fill protocol to control the injector. The fraction collector events collect by time. Time Device(s) Command % acetic acid / acetonitrile 0.30(ml/min): 86.50% 0.1% acetic acid, 13.50% acetonitrile 0.02 injection = partial loop fill <start> TUBE, INJECT_VOLUME 0.03 Fraction Collector Lock/Unlock Front Panel Lock 0.05 Fraction Collector Set Time per Tube TIME_PER_FRACTION 0.10 Fraction Collector Set Fraction Site FRACTION_SITE 0.19 UV/VIS Detector Autozero Channels 0.20 System Controller Synchronize 0.21 Data Channels Start 2.50 Fraction Collector Start Collection 6.00 Fraction Collector Stop Collection 7.00 Data Channels Stop If you want to collect by volume, set the following command at 0.05 min. Time Device(s) Command 0.05 Fraction Collector Set Volume per Tube VOL_PER_FRACTION B-4

364 sample tracking Example 2. The following control method listing contains fraction collector events for peak collection by slope. It controls the injector using the center loop fill protocol. Time Device(s) Command % acetic acid / acetonitrile 0.30(ml/min): 86.50% 0.1% acetic acid, 13.50% acetonitrile 0.02 injection = center loop fill <start> TUBE, INJECT_VOLUME, Fraction Collector Lock/Unlock Front Panel Lock 0.05 Fraction Collector Collect Positive Peaks Yes 0.11 Fraction Collector Set Peak Width & Peak Sensitivity PEAK_WIDTH, PEAK_SENSITIVITY 0.15 Fraction Collector Set Fraction Site FRACTION_SITE 0.19 UV/VIS Detector Autozero Channels 0.20 System Controller Synchronize 0.21 Data Channels Start 2.50 Fraction Collector Start Collection 6.00 Fraction Collector Stop Collection 7.00 Data Channels Stop analysis method In the analysis method, you can add a Fraction Site(s) column to the report. This column identifies the tube location(s) into which a sample s eluent was collected. 1 Use the Analysis Method window to set analysis conditions as required by your application. See Section 7 in the UniPoint User s Guide for details. 2 From the Report menu, choose Column Formats. 3 Add the Fraction Site(s) column to the unknown report format. To do this: a) Select the Unknown Report option button. b) In the Column Contents list box, click beneath the last column to add a column there. Or, to insert the column between two columns, click on the column before which you want the new column to be placed. c) Choose Fraction Site(s) in the Contents list box. d) Choose Insert. e) Choose OK to exit the dialog. 4 Repeat the above to add the column to the calibrator, unknown summary, and calibrator summary report layouts. B-5

365 unipoint user's guide After the run To track the sample, open the data or report file. Then use the commands in the View menu of the Results window to display the injector tray and/or fraction collector tray, or to select a different sample. If an injector tray or fraction collector tray is not associated with the sample, its command is gray in the View menu. An example Results window is shown on page B-1. The Display All command automatically sizes and shows the Results window, Samples dialog, injector tray (if available), and fraction collector tray (if available). To view information for a different sample, double-click on its description in the Samples dialog. You can also double-click on a tube in the injector tray or fraction collector tray and view information for the corresponding sample. A message appears if there is not a sample associated with a selected tube location. You can also view the fraction site(s) associated with one or more areas of the chromatogram. When you use the following procedures, the area between selected fraction collector marks is white while non-selected areas are gray. The corresponding fraction site(s) in the Fraction Location window are red and the other sites are cyan (light blue). To view the fraction site(s) for: Do the following: single point on the plot 1 Click on the cross-hairs tool ( ). 2 Move the mouse pointer to a position between fraction collector begin ( ) and end ( ) marks on the plot. 3 Click and release the left mouse button. area on the plot multiple points or areas on the plot 1 Click on the cross-hairs tool. 2 Move the mouse pointer to the plot. 3 Click the left mouse button and drag to enclose the fraction collector begin and end marks within a box. Release the mouse button. 1 Click on the cross-hairs tool. 2 While pressing the Ctrl key, select fraction collector begin and end marks as described above. Then release the Ctrl key. To turn off the cross-hairs tool, click on the arrow tool ( ). To remove the gray background, click on the original view tool ( ). To enlarge or reduce the size of the tray in the injector tray or fraction collector tray window, click in the window using the right mouse button. You see a Scale menu for selecting sizing options. B-6

366 Appendix C File Extensions Following are the extensions of files that can be created or opened by you within UniPoint. Extension File Type Description.GCT control method Specifies device control information to be used during a run..gan analysis method Defines the conditions used to analyze acquired data..gop operations list Lists step-by-step instructions for the run-time actions of the HPLC system..gdt data Contains the raw data acquired during a run, plus information on how the data was collected..gr? report Contains report information for analyzed samples. The question mark is a number between 1 and 9..GB? calibration Contains the calibration plot(s) generated for each standard peak during a run or batch re-analysis. The question mark is a number between 1 and 9..GAR re-analysis list Is a list of information for samples injected during a run; provides for quick re-analysis of data.glg log Is a record of the events that took place during a run..gwk workspace Stores the arrangement of document windows as they appeared on-screen..gip injector protocol Contains program-like instructions.ggp generic protocol to be executed by Gilson device(s)..gir injector routine Consist of individual commands that.ggr generic routine have been grouped together; used within protocols..cfg configuration Identifies the components of the HPLC system. C-1

367 Appendix D Password Protection In UniPoint, you can protect document types from being modified and saved to the same name unless the user indicates the appropriate password. In the operations and re-analysis files, you can indicate that columns for a password protected file can be modified even if the user did not enter the correct password. D-1

368 unipoint user's guide Setting up password protection When UniPoint is installed, no password has been indicated. To set a password and choose document windows to password protect, do the following: 1 Close all open document windows using the Close All command in the Window menu. If it appears, the Navigator may remain onscreen. 2 Choose Password Protection in the File menu. 3 When the New Password dialog appears, type the password in the box and choose OK. 4 Verify the password you just entered by retyping it and choosing OK. (If the password does not match, you see a message box. Repeat the process of indicating the password and then verifying it.) 5 When the Password Protection dialog appears, select the document type in the list box and choose the Password Required check box. 6 Repeat the previous step for each document type to password protect. 7 Choose OK. D-2

369 password protection Indicating new password To indicate a new password: 1 Close all open document windows using the Close All command in the Window menu. If it appears, the Navigator may remain onscreen. 2 Choose Password Protection in the File menu. 3 When Password Verification dialog appears, type the current password and choose OK. 4 When the Password Protection dialog (shown on previous page) appears, choose New Password. 5 When the New Password dialog (shown on previous page) appears, type the password in the box and choose OK. 6 Verify the password you just entered by retyping it and choosing OK. (If the password does not match you see a message indicating that. You then repeat the process of indicating the password and then verifying it.) 7 When the Password Protection dialog appears, choose OK to exit. Removing password protection To remove password protection from a document type: 1 Close all open document windows using the Close All command in the Window menu. If it appears, the Navigator may be left on-screen. 2 Choose Password Protection in the File menu. 3 When prompted, type the current password. The Password Protection dialog appears. 4 Select the document type in the list box and remove the X from the Password Required check box. 5 Choose OK. D-3

370 unipoint user's guide To remove password protection completely: 1 Access the Password Protection dialog as described above. 2 Choose New Password. 3 When the dialog appears, make sure the text box is blank and choose OK twice. Unlocking columns in operations or re-analysis list You can remove password protection from columns in an operations or re-analysis file. This enables a user that does not have password authority to modify information in the file without entering a password. The user cannot save the changes to the existing file but can save them to a new file. unlocking columns in a file 1 Open the operations or re-analysis file. Indicate the correct password when prompted. 2 From the Table menu, choose Format Columns. Or, double-click on one of the column headings of the on-screen table. 3 Select the column in the column heading list box. 4 Remove the X from the Password Protect check box. 5 Repeat steps 3 and 4 for each column to unlock. 6 Choose Done to exit the dialog box. 7 Save the changes to the file before exiting the document window. the next time the file is opened If the user indicates the correct password the next time the operations or re-analysis file is opened, the user can modify information in any of the columns. If the user does not indicate the correct password when the file is opened, the user can only modify information in unlocked columns. Entry boxes for password protected columns are grayed in dialog box(es). D-4

371 Appendix E Merging Security Keys The information in this appendix applies only to users who run more than one kind of Gilson key-protected software on a single computer. If a cable (other than the mouse cable) prevents access to the security key, turn off the computer, detach the cable, restart the computer and Microsoft Windows. If a device error occurs at startup, ignore the message; press any key to continue. Then follow these key merge instructions. To merge the security codes from an existing key into a new key: 1 Double-click on the Key Utility icon in the Gilson Applications box. 2 When the Gilson Key Utility window appears, choose Tutorial from the Keys menu to learn more about Gilson security keys. To advance through the tutorial, point and click on the Ok button in each dialog box. 3 In the box labeled Security Key, point and click on the name(s) of the program(s) in the existing key. An X appears in the adjacent square(s). 4 Point and click on the right Move arrow to move the codes into temporary memory. Do not shut the computer off or restart (reboot) the computer when codes are in temporary storage! 5 Remove the key from the 506C System Interface and replace it with the UniPoint key. 6 Point and click on the name(s) of the program(s) in the temporary box. An X appears in the adjacent square(s). 7 Point and click on the left Move arrow to move the codes into the new key. E-1

372 unipoint user's guide 8 Check that the program name(s) are now listed in the Security Key box. 9 Point and double-click on the control-menu box at the upper left corner of the Key Utility window to quit the program. If you do not quit the Key Utility program correctly, essential key information is lost at shutdown. 10 If you do not have any key information in temporary memory, you may turn off your computer. If you removed an interfering cable, reconnect it now. 11 Restart your computer and UniPoint. E-2

373 Appendix F Calculations amount For calibrators, the peak amount indicated in the peak table. For unknowns reported in calibration report types, the amount of component is determined using the calibration plot generated for calibrators. If you specified nominal and actual amounts for an unknown, UniPoint automatically adjusts the amount of material in each calculated peak and reports instead the amount that would have been found if the sample had contained the nominal amount of unknown: For unknown samples in internal standard reports, UniPoint automatically adjusts for material that was lost during sample preparation and injection. The software uses the following formula: Adjusted Amount A = Amount A x (IS Actual Amount IS Observed Amount) where: Adjusted Amount A Amount A Is the adjusted amount calculated for component A in the unknown sample. For the component peak, this amount is appears under the Amount (int std) heading in the unknown report. Is the amount of component A in the unknown sample. UniPoint obtains this amount using the calibration table generated for calibrator injections for component A. To report this amount, add the Amount column heading to the unknown report format. See Report format review and modification in Section 7. F-1

374 unipoint user's guide IS Actual Amount IS Observed Amount Is the amount of internal standard added to the unknown sample. You entered this amount in the Peak Table dialog or in the operations list if the Internal Standard Actual Amount check box was set the Peak Table dialog of the analysis method. For the internal standard peak, this amount appears under the Amount (int std) heading in unknown report. Is the amount of internal standard peak observed in the unknown injection. UniPoint obtains this amount using the calibration table generated for calibrator injections for the internal standard. If you specified the actual internal standard amount for an unknown, UniPoint adjusts reported peak amounts: If you specified actual amount, actual internal standard amount, and nominal amount for an internal standard report type, UniPoint calculates the reported peak amount using the above equation. It then inserts that amount for the calculated peak amount in the equation used if actual and nominal amounts were indicated. amount % Percentage of the peak in relation to the total amount of all named peaks. area % F-2

375 chromatography calculations asymmetry Factor that identifies the shape of the peak. If this value is 1, the peak is symmetric. If the peak is skewed to the right (tailing peak), this value is greater than 1. If the peak is skewed to the left (fronting peak), this value is less than 1. back resolution Resolution of a peak in relation to next peak. back slope Slope at half height of peak s descending edge. capacity factor Measures the degree of retention. coefficient of determination coefficient of variation correlation coefficient A measure of the mutual relationship between two variables. F-3

376 unipoint user's guide end value The mv (or user units) at the end of the peak. front resolution Resolution of a peak in relation to previous peak. front slope Slope at half height of peak s ascending edge. height Distance from the baseline to the maximum of the peak. height % mean where x is a measurement (such as area, height, amount, and so on) and n is the number of measurements. peak start time Elapsed time between the point of injection and the start of the peak. peak end time Elapsed time between the point of injection and the end of the peak. peak width at half height Peak Width at Half Height = Time at Half Height of Descending Edge - Time at Half Height of Ascending Edge F-4

377 chromatography calculations peak width at 10% Peak Width at 10% = Time at 10% Height of Descending Edge - Time at 10% Height of Ascending Edge plates This value can be used to determine column efficiency. % concentration retention time Elapsed time between the point of injection and the maximum of the peak. standard deviation where n is the number samples in which the peak was integrated and x is the area (or height) determined each time the peak was integrated. standard error of estimate start value The mv (or user units) at the start of the peak. tailing factor F-5

378 Appendix G Peak Table Retention time windows Description Reference and non-reference peaks in calibration and unknown samples are located by matching actual retention times to retention times listed in the analysis method's peak table. To compensate for possible retention time drift, the software defines a time window around each expected peak. It finds an expected peak if that peak lies within the retention time window. You can modify the Absolute Error and/or Relative Error parameters to control the width of retention time windows. The default settings (Abs. Error = 0.10 min, Rel. Error = 5.0%) are appropriate for many separations. Absolute error is a constant amount of time flanking a peak s nominal retention time. The software automatically searches for eluted peaks at every named retention time plus or minus this amount of time. Relative error is a percentage of peak retention time. Relative error, and, therefore, the width of retention time windows, increase with increasing retention time. Because late-eluting peaks are often broader than early peaks, the software adds this correction factor to improve the likelihood of locating late-eluting peaks. You can calculate the beginning and end of any peak s time window using the following formulas: Beginning: End: Ret. Time - [Abs. Error - (Ret. Time x Rel. Error)] Ret. Time + (Abs. Error + Ret. Time x Rel. Error) G-1

379 unipoint user's guide Setting time window parameters It is easy to understand how absolute error contributes to the width of retention time windows since it is simply a constant amount of time that brackets every named retention time. The contribution of relative error is harder to grasp because it varies with retention time. The following diagram and table show what happens when absolute error is held constant at 0.1 minutes and the relative error is varied (1%, 5% or 10% of retention time). The time windows were calculated as described above. Notice that the retention time of Peak 3 (9.94 minutes) was very close to the upper limit (9.98 minutes) of the windows at 1% relative error. In this example, relative error should be set higher than 1% to avoid missing peaks. Peak 1 Peak 2 Peak 3 Nominal Ret. Time Actual Ret. Time Window (1% rel. error) Window (5% rel. error) Window (10% rel. error) Effect of Relative Error on Width of Retention Time Windows G-2

380 peak table Resolving overlapping windows Retention time windows must be wide enough to permit detection of every peak yet narrow enough to minimize windows overlapping. Overlapping can occur if windows are too wide or if peaks are too close together. Since the software cannot know whether a peak belongs to the earlier or later window in an overlapping pair, it uses the following algorithm during data analysis to revise the limits of both windows and to establish a new boundary between them. The following examples are presented in the order of least to most amount of overlap. Type 1. When the beginning of Window 2 occurs after the midpoint of Window 1, a new boundary is established at the midpoint of the overlapping region. Type 2. When the beginning of Window 2 occurs before the midpoint of Window 1, a new boundary is established midway between the midpoint and end of Window 1. Type 3. When the midpoint of Window 2 occurs at any time during Window 1, a new boundary is established midway between the center of Window 1 and the center of Window 2. G-3

381 unipoint user's guide Using the peak table during data analysis When UniPoint begins to analyze data collected during a run, it looks for the reference peaks and non-reference peaks that you identified in the peak table. It knows where to look because you set the peak retention times and retention time window parameters. After finding the peaks, UniPoint checks the retention times of those peaks and updates its internal peak table so it knows when to expect those peaks in the next step. If the current method is generates a calibration type report, the software uses the amounts listed in the peak table. It then uses information from the calibration table to report results about the data collected during the run. After a calibration run, UniPoint automatically saves the calibration table to file. reasons for missing peaks The software might fail to identify reference or non-reference peaks if: the peak width and peak sensitivity were incompatible with chromatogram peaks the wrong sample was injected changing chromatographic conditions (flow rate, mobile phase composition, pressure, temperature) or leaks in the system resulted in inconsistent retention times an insufficient number of or a poor choice of reference peaks were named the absolute error and/or relative error need to be adjusted. G-4

382 peak table Locating reference peaks When you create a peak table, you can specify retention times for one or more reference peaks. The software looks for peaks named in the peak table whose actual retention times fall within the reference peaks time windows. Use reference peaks with cautiously. An ideal reference peak is a large, well separated, isolated peak with a consistent retention time. If, during data analysis, it finds a peak in a retention time window, it presumes it found the desired reference peak. As the software identifies reference peaks, it constructs an internal graph of actual retention times versus expected retention times. If two or more peaks fall within the same reference peak time window, it chooses the largest one as the reference peak. If two peaks in a window are equally tall, the earliest peak is chosen as the reference peak. G-5

383 unipoint user's guide Locating non-reference peaks Using the internal graph, described on the previous page, the software updates the expected retention times (and retention time windows) for all non-reference peaks named in the Analysis Peak Table. It then looks for non-reference peaks named on the peak table whose actual retention times fall within a corrected retention time window. If the software finds such a peak, it presumes it is the desired nonreference peak. If two or more non-reference peaks fall within the same window, it chooses the earliest peak whose retention time is closest to the window s center. G-6

384 peak table Updating peak retention times Over the course of multi-loop runs, retention times could drift so much that peaks would elute outside their original time windows. To ensure that the software always knows the most likely retention time of each named peak, the software continuously updates the retention time windows of reference and non-reference peaks on its internal peak table. Note: You can turn off peak tracking in the analysis method. See page In the first loop through any run, the software expects that named peaks have the retention time specified in the analysis method's peak table. In each subsequent loop, it adjusts the expected retention time according to information it gathers during the run. The new midpoint of each peak s retention time window is calculated as a weighted sum of each peak s previously determined window midpoint (75%) plus its retention time in the current loop (25%). A weighted sum is necessary to minimize the influence of a single bad loop in a multiloop run. For example, after several loops, the midpoint of a peak s retention time window is at 3 minutes. In the next loop, that peak s retention time was 3.2 minutes. On the software s internal peak table, the midpoint of the retention time window is adjusted to 3.05 minutes. (3 minutes x 0.75) + (3.2 minutes x 0.25) = 3.05 minutes This updating only occurs if all reference peaks are found in the current loop. If all reference peaks are found, the retention time window of each reference and non-reference peak found during that loop is updated as described above. G-7

385 unipoint user's guide Generating a calibration table While creating an analysis method, you listed in the peak table an amount for each standard peak at each calibration level. When you ran that method, you injected calibrator samples for each level and optionally unknown samples. All repeats at calibration level 1 All repeats at calibration level 2 (if used) All repeats at additional calibration levels (if used) Unknown samples When analyzing a sample, the software determines the peak area (or height) for each named component. For each peak, the software generates a calibration curve to determine the amount of that component in each unknown sample. A component s calibration curve plots the average peak area (or height) vs. amount injected at each level. You can choose between the following types of calibration curves: Point to Point. UniPoint connects the mean amount at each calibration level using a linear segment. For a one-level calibration plot, UniPoint generates a linear segment between the origin (0 area, 0 amount) and the mean peak area at the calibration level. Point to Point through Zero. UniPoint generates a linear segment between the origin (0 area, 0 amount) and the mean peak area at the first calibration level. It then connects the mean amount at each additional calibration level using a linear segment. Linear. Using a best fit method, UniPoint constructs a line that best represents the set of means. This method minimizes the sum of the squares of the error distance between each mean and the line. Quadratic. UniPoint generates a smooth curve that best represents the set of means. The curve may have one bend. Cubic. UniPoint generates a smooth curve that best represents the set of means. The curve may have two bends. To determine the amount of that component in an unknown sample, the software determines where that component s peak area intersects the calibration curve. The figure on the next page shows how the software determines the amount for an unknown. A point to point calibration curve is shown, but the same procedure is used for a linear regression, quadratic, or cubic calibration curve. G-8

386 peak table Multi-Level Calibration G-9

387 Appendix H 715 Data Translator The 715 Data Translator converts data files created by Gilson 715 Software to a format that can be read by UniPoint. starting and using the data translator To start and use the 715 Data Translator Software: 1 Double-click on its icon in the Gilson Applications box on the Windows desktop. The 715 Data Translator window appears. 2 In the UniPoint Data File box, type the path and name of the file to which the converted data information is stored. To replace an existing file with the converted information, click in this text box and then use the Browse button to locate the file. It is not necessary to enter the.gdt extension; the software automatically assigns that extension. Do not indicate an extension other than.gdt. If you do not indicate a complete path, the new file is stored to the same path as the 715 file(s) being converted. 3 Use the Drives and Directories list boxes to locate the 715 files to convert. 4 Use the File Name list box to select each file. In most cases, you want to select all data files generated for a 715 run and store their information into one UniPoint file. 5 Choose Translate when all files are selected. 6 Choose Done to exit the 715 Data Translator Software. H-1

388 unipoint user's guide opening converted data file in UniPoint To open and analyze sample information in a converted data file: 1 Start UniPoint by double-clicking its icon on the Program Manager. 2 Use the Navigator or Open command in the File menu to open the converted data file. The chromatogram plot for the first sample appears in a Results window. See Section 11 for information for manipulating data in the Results window. 3 To analyze the data, use the Navigator or New command in the File menu to display the Analysis window for a new analysis method. Use the options in the Analysis window to set analysis and reporting conditions for the data file. See Section 7. To name the data channel(s) without setting a control method, use the Edit option in the Data Channel Scales dialog. See pages 7-10 through The legend in the Results window identifies the data channel(s) for the sample. 4 To generate a report file for the data file, use the Re-Analysis window. See Section 13. H-2

389 Appendix I GSIOC Utility The GSIOC Utility allows you to issue commands to Gilson GSIOC devices outside of UniPoint. Gilson Customer Service may ask you to use this utility to verify that a device is connected correctly to the computer. How you use the GSIOC Utility varies depending on whether your computer has Microsoft Windows 95/NT or Windows 3.1. Refer to the appropriate section below. Using the GSIOC utility within Windows 95/NT The GSIOC Utility enables you to check communication between your computer and a Gilson device. For communication to occur, the Gilson device must be connected via an RS-232 connection to the computer or connected via a GSIOC connection to a Gilson interface device that is connected to the computer. starting the GSIOC utility To start the GSIOC Utility: 1 Double-click on GSIOC Utility icon in the Gilson Applications box on the Windows desktop. The GSIOC Utility window appears. I-1

390 unipoint user's guide reviewing the port, IRQ, and baud information In the GSIOC Utility window, review the COM port, IRQ, and baud information. If any information is incorrect or missing, close the GSIOC Utility and use the GSIOC Configuration Utility to update the information. Refer to Appendix K. listing GSIOC devices Using the GSIOC Utility, you can determine the devices currently connected to the computer. 1 Click on Scan! The Unit ID box displays the current GSIOC devices and their Unit IDs. If all Gilson devices are not listed, ensure that the proper RS-232 or GSIOC connection exist between the computer and Gilson devices. sending commands Using the GSIOC Utility, you can send commands to Gilson devices. Each device has a set of commands that it understands. A complete list of GSIOC commands for any device is given in its user s guide. 1 Type or select the unit ID assigned to the device in the Unit ID box. If you don t know the Unit ID, click on Scan! to reveal a list box with the GSIOC devices and their Unit IDs. 2 In the Command box, type the command string. Commands consist of strings of no more than 40 characters that specify an instruction to the specified device. 3 Click on the appropriate command button to select a command type according to the function of the desired command. The command is issued when you select the command type. Buffered commands send instructions to a device. These commands are executed one at a time. Immediate commands request status information from a device. These commands are executed immediately, temporarily interrupting other commands in progress. 4 Monitor the device s response to your command in the Response area of the box. The response to a successfully completed buffered command is a period (.). Immediate status responses also appear in this area. The user s guide for a Gilson device describes the valid responses to immediate commands. exiting GSIOC utility To exit the software, click on. I-2

391 GSIOC utility Using the GSIOC utility within Windows 3.1 The GSIOC Utility, GSUTIL.EXE, enables you to set the baud rate and communications port for the GSIOC and send commands to GSIOC devices. When you exit this utility, any changes to the communications port and baud rate are automatically made to the CONFIG.SYS file so you do not have to edit that file. (See Appendix J for more information on manually editing the CONFIG.SYS file.) run GSIOC utility To start the GSIOC Utility: 1 If necessary, exit UniPoint by double-clicking on the control-menu box in the upper left corner. 2 Double-click on the GSIOC Utility icon in the Gilson Applications box. The GSIOC Utility window appears. selecting communication port The computer needs to know the communications port to which the interface module is connected. 1 Choose the option button of the correct port. selecting baud rate To communicate with devices: 1 Choose the option button of the correct baud rate for the communications port. Note: If you cannot get responses from GSIOC devices using a baud rate of 19200, try However, Gilson recommends a baud rate of listing GSIOC devices Using the GSIOC utility, you can determine the devices currently connected to the GSIOC. 1 Click on Show List. I-3

392 unipoint user's guide A list box listing the current GSIOC devices and their Unit ID appears. 2 Use the scroll bar to display additional devices. 3 Click on OK to remove the list box from the screen. send command Using this utility, you can send commands to GSIOC devices. Each device has a set of commands that it understands. Each command is classified as buffered or immediate. A complete list of GSIOC commands for any device is given in its User s Guide. 1 Position the focus (flashing vertical line) in the Unit box, if it is not already there. 2 Type the unit ID assigned to the device in the Unit ID box. If you don t know the Unit ID, click on Show List to reveal a list box with the GSIOC devices and their Unit IDs. 3 In the Command box, type the command string. Commands consist of strings of no more than 40 characters that specify an instruction to the specified device. 4 Click on the appropriate command button to select a command type according to the function of the desired command. The command is issued when you select the command type. Buffered commands send instructions to a device. These commands are executed one at a time. Immediate commands request status information from a device. These commands are executed immediately, temporarily interrupting other commands in progress. Each GSIOC device s User s Guide tells you the command type of each command. 5 Monitor the unit s response to your command in the Response area of the box. The response to a successfully completed buffered command is a period (.). Immediate status responses also appear in this area. Each GSIOC device s User s Guide describes the valid responses to immediate commands. I-4

393 GSIOC utility exit GSIOC utility To exit the GSIOC Utility, double click on the control-menu box ( ). Any changes to the baud rate and communications port are automatically made to the CONFIG.SYS file so you do not have to edit the CONFIG.SYS file. I-5

394 Appendix J GILSON.SYS Device Driver File Note: Refer to the information in this appendix only if your computer has Windows 3.1. The alterations described on the following pages are technical and are not recommended to the novice computer user. If you see device driver errors that you do not understand or cannot resolve, call Gilson before modifying the CONFIG.SYS file. Consult your DOS manual for a detailed description of the CONFIG.SYS file. The GILSON.SYS device driver file was installed when you installed UniPoint. It enables the software to communicate with Gilson devices connected to the 506C System Interface via the Gilson Serial Input/ Output Channel (GSIOC). It also enables the software to locate the security key. When you installed UniPoint, the device driver information is also added to the CONFIG.SYS file of the computer. The software automatically adapts this information to your computer s hardware. Following is a description of the GILSON.SYS device driver line in the CONFIG.SYS file. J-1

395 unipoint user's guide device driver line in CONFIG.SYS The complete syntax of the GILSON.SYS device driver line in the CONFIG.SYS file is shown below. Parameters enclosed in brackets are optional. If you include the optional parameters, do not type the brackets as shown! DEVICE = [d:] [\path\] GILSON.SYS [/Pport] [/Brate] [/Ktxxxx] [/N] where: d: and \path\ tell the computer which drive and directory contain the GILSON.SYS file. When UniPoint is installed, the default path to the GILSON.SYS file is set to C:\GILSON\SYS. /Pport identifies the number of the communications port to which the 506C System Interface is connected. For port, indicate 1, 2, 3, or 4 default is 1. /Brate sets the baud rate (300, 600, 1200, 2400, 4800, 9600, or the default is 9600 for the GILSON.SYS driver. See baud rate, on page J- 3, for more information. /Ktxxxx identifies the type (t) and address (xxxx) of the security key. For type (t), indicate G, for 506C security key. For address (xxxx), indicate the GSIOC address of the 506C's key port, in hexadecimal, usually 3F. /N tells the computer to complete the startup process uninterrupted, even if it encounters errors. J-2

396 device driver file Example: The GSIOC device driver is located in the \SYS directory of the computer and the key is installed in a 506C System Interface. You want to control Gilson devices through Com2: at baud. But you do not want the computer to pause during start-up if it encounters an error. The device driver line in CONFIG.SYS should read: DEVICE = \GILSON\SYS\GILSON.SYS /P2 /B19200 /KG3F /N In the above line, 3F is the hexadecimal address of the key on the 506C System Interface. baud rate The GILSON.SYS device driver line in your computer s CONFIG.SYS file must be set to the same baud rate as the interface module. The default baud rate of the GILSON.SYS device driver is During installation of Gilson software, the GILSON.SYS baud rate is automatically set to In other cases, you should manually override the GILSON.SYS default baud rate to match that of the interface module. To change the baud rate to 19200, follow these instructions: 1 Load the CONFIG.SYS file into your text editor or word processor (for example, Windows Notepad). CONFIG.SYS is usually located in the root directory of your computer s fixed disk (typically C:\). 2 Edit the line that reads: DEVICE = C:\GILSON\SYS\GILSON.SYS to read: DEVICE = C:\GILSON\SYS\GILSON.SYS /B Then save this change to the CONFIG.SYS file. J-3

397 Appendix K GSIOC Configuration Utility Note: Refer to the information in this appendix only if your computer has Windows 95. The GSIOC Configuration Utility enables you to modify COM (serial communications) port, IRQ (interrupt request), and baud rate information. You need to use this utility if you connect the liquid handler to a different COM port after installing the Sampler Software. Or, you can use this utility if incorrect information appears in the GSIOC Utility window; see Appendix I. 1 To start the GSIOC Configuration Utility, double-click on the GSIOC Configuration icon in the Gilson Applications box on the Windows desktop. The Gsioc Configuration window appears. 2 In the Port box, indicate the computer s serial communications port (COM) port to which the Gilson interface device (such as the liquid handler or 506C System Interface) is connected. 3 If necessary, indicate an interrupt request value in the Irq box. An interrupt request is a signal to a computer s microprocessor that it should stop doing whatever it is currently doing and pay attention to the cause of the interrupt. After the microprocessor has serviced the interrupt, it goes back to doing whatever it was doing before the interrupt occurred. For a Gilson device, an interrupt request occurs when a character arrives from the device or when the device disconnects. K-1