Agilent 1100/1200 HPLC ChemStation Operation Course Number H4033A Laboratory Manual
Agilent 1100/1200 HPLC ChemStation Operation Course Number H4033A Laboratory Manual ChemStation B.
Notice The information contained in this document is subject to change without notice. Agilent Technologies makes no warranty of any kind with regard to this material, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent Technologies shall not be liable for errors contained herein or for incidental, or consequential damages in connection with the furnishing, performance, or use of this material.
Table Of Contents LAB EXERCISE: INTRODUCTION TO THE HPLC CHEMSTATION..... 1 IN THIS LABORATORY YOU WILL: ....................................................................... 2 ACCESSING THE HPLC CHEMSTATION ................................................................ 3 CONFIGURATION EDITOR ..................................................................................... 5 SCHEDULER .........................................................................................................
LAB EXERCISE: SPECTRAL LIBRARIES .................................................. 43 IN THIS LABORATORY YOU WILL: ..................................................................... 44 BUILDING A LIBRARY ........................................................................................ 45 LIBRARY SEARCHING ......................................................................................... 47 LAB EXERCISE: OVERVIEW AND DIAGNOSTICS .................................
LAB EXERCISE: RUNNING A SEQUENCE................................................ 99 IN THIS LABORATORY YOU WILL: ................................................................... 100 BUILDING A SEQUENCE .................................................................................... 101 SEQUENCE SUMMARY REPORTS ....................................................................... 104 STARTING THE SEQUENCE ................................................................................
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Lab Exercise: Introduction to the HPLC ChemStation
Lab Exercise: Introduction to the HPLC ChemStation In this Laboratory You Will: In this Laboratory You Will: • Access the HPLC ChemStation software and understand the purpose of each View. • Use the ChemStation Configuration Editor. • Learn about the Scheduler. • Learn how to maintain the HPLC ChemStation computer. • Become familiar with some Windows features (optional).
Lab Exercise: Introduction to the HPLC ChemStation Accessing the HPLC ChemStation Accessing the HPLC ChemStation 1) Find the Start button in the bottom left hand corner of your screen. Click on the Start button. Select the menu choice All Programs. You will find a list of the software programs installed on this computer arranged in alphabetical order. To launch the ChemStation for LC 3D Systems, find and select the Agilent ChemStation menu item.
Lab Exercise: Introduction to the HPLC ChemStation Accessing the HPLC ChemStation 6) You can also change views by utilizing the Navigation Buttons. Switch Views Here Minimize, Maximize Close 7) Click on the small triangle at the top of the ChemStation Explorer. Click again. Notice that you can sort your files. 8) Click on the Configure buttons icon at the bottom of the Navigation Pane – you can use this to add or remove buttons.
Lab Exercise: Introduction to the HPLC ChemStation Configuration Editor Configuration Editor You customer engineer configured your instrument during installation. This process includes configuring the instrument devices and setting the LAN IP address so that the software can communicate with the instrument.
Lab Exercise: Introduction to the HPLC ChemStation Scheduler Scheduler The Scheduler may be used to make your ChemStation automatically execute commands on a one time, daily, daily weekday, or weekly basis. You may schedule events such as controlling a valve, loading a method or sequence, starting a sequence, or initiating a blank run.
Lab Exercise: Introduction to the HPLC ChemStation Scheduler 7) After you have witnessed the logbook display, return to the Scheduler to check the Result column. 8) Close the Scheduler. Note: The Scheduler can be useful for turning on the detector lamp prior to your arrival each day to warm-up. The command is “Lampall On”. Do not include the parentheses.
Lab Exercise: Introduction to the HPLC ChemStation Maintaining the Windows 2000 Workstation Maintaining the Windows 2000 Workstation (Skip to Maintaining the Windows XP Workstation if you have Windows XP) Cleaning up Temporary Files After the ChemStation has been used for some time, temporary files may accumulate in the directory specified by the TEMP Variable. These files are generally left open when Windows is abnormally terminated. The files generated have a .tmp extension.
Lab Exercise: Introduction to the HPLC ChemStation Maintaining the Windows 2000 Workstation 3) Click on the Tools tab. Find the Error-Checking tool and click Check Now.... 4) Select to Automatically fix file system errors and Scan for and attempt recovery of bad sectors. 5) This process can take several minutes. If you don’t wish to do this in lab, Cancel, otherwise select Start. Disk Defragmentation A defragmentation utility is included with Windows 2000.
Lab Exercise: Introduction to the HPLC ChemStation Maintaining the Windows XP Workstation Maintaining the Windows XP Workstation (Skip this section if you completed Maintaining the Windows 2000 Workstation) Cleaning up Temporary Files After the ChemStation has been used for some time, temporary files may accumulate in the directory specified by the TEMP Variable. These files are generally left open when Windows is abnormally terminated. The files generated have a .tmp extension.
Lab Exercise: Introduction to the HPLC ChemStation Maintaining the Windows XP Workstation 3) Click on the Tools tab. Find the Error-Checking tool and click Check Now.... 4) Select to Automatically fix file system errors and Scan for and attempt recovery of bad sectors. 5) This process can take several minutes. If you don’t wish to do this in lab, Cancel, otherwise select Start. Disk Defragmentation A defragmentation utility is included with Windows XP.
Lab Exercise: Introduction to the HPLC ChemStation Maintaining the Windows XP Workstation complete, you will be prompted for a blank, formatted floppy disk. Several files are written to the disk to complete the process.
Lab Exercise: Introduction to the HPLC ChemStation Windows Features (Windows 2000 or XP) Windows Features (Windows 2000 or XP) Optional Section Mastering WINDOWS could take a week long class in itself. This section of the laboratory is simply a brief familiarization of some of the important aspects frequently utilized in conjunction with ChemStations. If you are already familiar with Windows, skip this section. Windows Explorer The Windows Explorer is a powerful file manager.
Lab Exercise: Introduction to the HPLC ChemStation Windows Features (Windows 2000 or XP) 5) Check to make certain that you have copied the chosen file into drive A: by expanding the 3 1/2 Floppy (A:) item. 6) To delete a file or folder (when you delete a folder, all the contents of the folder will be deleted along with the folder itself) right click on the folder or file in the left or right pane. From the File menu select Delete. Try this now with one of the data files in the Data\Demo folder.
Lab Exercise: Introduction to the HPLC ChemStation Windows Features (Windows 2000 or XP) Clipboard You may use the clipboard to capture the entire screen, an entire window or any selected material within a document or graphic using the cut or copy commands found in Windows based programs. The image is temporarily stored until you paste it into the same or another application. For instance, say that you were creating a written HPLC method.
Lab Exercise: Introduction to the HPLC ChemStation Windows Features (Windows 2000 or XP) If these actions do not clear your problem, you would have to reboot the computer.
Lab Exercise: Acquisition Methods
Lab Exercise: Acquisition Methods In this Laboratory You Will: In this Laboratory You Will: • Prepare your instrument for acquisition. • Set-up an acquisition method. • Run an acquisition method. • Take a snapshot of the data.
Lab Exercise: Acquisition Methods Preparation Preparation Before starting, make certain that the you have the following: • A sample vial labeled, sample, which contains the Test Mix, Part # 0108068704 (diluted). • HPLC grade water in channel A and HPLC grade acetonitrile in channel B. • A 4.0 x 100 mm, C-18 column installed in the column compartment. • An Agilent 1100/1200 with all modules powered on. • An HPLC ChemStation, B.03.xx, equipped with a spectral module.
Lab Exercise: Acquisition Methods Preparing the HPLC Preparing the HPLC 1) Enter the Method and Run Control view of the Agilent Online ChemStation. 2) From the View menu, select Instrument Actuals, the System Diagram and Sampling Diagram. If a check mark exists beside the item, it is already on. 3) Load the default method, DEF_LC.M as a starting point for method creation (Method, New Method) or double-click on DEF_LC.M in the ChemStation Explorer. 4) Under the Instrument menu, select More DAD, Control.
Lab Exercise: Acquisition Methods Priming the Agilent 1100/1200 Solvent Delivery System Priming the Agilent 1100/1200 Solvent Delivery System You should prime the pump when you change mobile phases, when the instrument has been sitting without flow for long periods of time, when you have performed maintenance on the HPLC, or when you experience periodic perturbations in the baseline. 1) If you are equipped with a vacuum degasser, make certain the degasser is on.
Lab Exercise: Acquisition Methods Priming the Agilent 1100/1200 Solvent Delivery System 6) Wait until a steady stream of solvent comes out of the purge valve waste tube. 7) Repeat steps 3 through 6 for the other channels of the pump. Change to the composition for your next run, %B=70. 8) Turn off the pump. 9) Close the purge valve. Set the required composition and flow rate for your next application. In this case: Flow 1.5 mL/min and %B 70. 10) Turn on the pump and allow the column to equilibrate.
Lab Exercise: Acquisition Methods Creating an Acquisition Method Creating an Acquisition Method 1) From the Method menu select Edit Entire Method... or select the Edit entire Method tool. 2) Select only the Method Information and Instrument/Acquisition sections for editing. OK this window. 3) The Method Information dialog box now appears. You may enter any information here that pertains to the method. Enter a description now. OK the window.
Lab Exercise: Acquisition Methods Practice Entering Gradients Practice Entering Gradients 1) Select the Insert button from the Time Table group. Program a linear gradient to 100%B over 4.0 minutes. In the Time column, type 4. In the %B column type 100. 2) Go to the Display dropbox. Select Solvents. 3) Return to the Timetable display. Click on the 1 next to the timetable line, then cut. Unfortunately, you do not need a gradient for this analysis, OK the Pump Parameters window.
Lab Exercise: Acquisition Methods Practice Entering Gradients Question: How is the threshold setting used? ________________________________________________________________ 4) Set the column compartment temperature to 40°C. Make certain that the right compartment has the same temperature as the left compartment. OK the window. 5) From the Method menu, select Save Method As.... Name the method, Class.m. The ChemStation will not let you overwrite DEF_LC.
Lab Exercise: Acquisition Methods Practice Entering Gradients 8) Add DAD A to Selected Signals. Fill in an x-axis range of 10 minutes. Select the draw zero line box. 9) Click on the DAD A Signal in the Selected Signals box then set the y-axis range to 100 mAU and the offset to 10%. OK the dialog box. 10) From the Method menu, select the Run Time Checklist. Select Data Acquisition only. Save a copy of the Method with the data. OK the dialog box. A report will not automatically print with this method.
Lab Exercise: Acquisition Methods Snapshot Snapshot 1) Once the first major peak has eluted, open an Offline Session of the Agilent ChemStation. 2) Go to the Data Analysis view of the Offline Session. 3) Go to the File menu and select, Snapshot. The current chromatogram appears in the chromatogram window of the Offline Session. 4) Click on the Data button in the ChemStation Explorer and find your subdirectory. 5) Double-click on the Single Runs and find the SNAPSHOT.D in the navigation table.
Lab Exercise: Acquisition Methods Snapshot 28
Lab Exercise: Qualitative Data Analysis Ideally, use the data file generated from the isocratic test mix in the previous laboratory exercise. If this is not available, use demo\005-0104.d and demo\demodad.d.
Lab Exercise: Qualitative Data Analysis In this Laboratory You Will: In this Laboratory You Will: 1) Use signal options to display data. 2) Annotate chromatograms. 3) Use the Window Functions. 4) Use the peak purity algorithm to examine the purity of chromatographic peaks. 5) Overlay and view UV spectra. 6) Use Isoabsorbance and 3D plotting.
Lab Exercise: Qualitative Data Analysis Graphics - Signal Options Graphics - Signal Options 1) Enter the Data Analysis view. 2) Load the method democal2.m. Either double-click on democal2.m in the method ChemStation Explorer or select Method > Load Method. 3) In Data Analysis, just below the Navigation Table on the left, there are five tools for five types of data analysis tasks: Integration, Calibration, Signal, Purify and Spectrum.
Lab Exercise: Qualitative Data Analysis Graphics - Signal Options 11) A panel with 3 tabs will appear. The Signals tab should be highlighted and all signals will be initially selected. Click off one of the signals. 12) Select the Inst. Curves tab. Click to make sure there is a check in the High Pressure checkbox. Click the General Info tab to see what information is available. 13) Click the right mouse button in the row containing the datafile. Choose Load Selected Signals.
Lab Exercise: Qualitative Data Analysis Graphics - Annotation Graphics - Annotation 1) Using the Signal(s) Displayed drop box, select DAD1A only. 2) Select the Graphics too. 3) From the Graphics menu, select New Annotation or select the Add Annotation tool. Position the cursor in the center-top of the chromatogram and click the left mouse button. 4) When the Text Annotation box appears, type in Test Mix. Select Options. You may select a font and font size.
Lab Exercise: Qualitative Data Analysis Graphics - Annotation 7) To print this window, pull down the File menu and select Print, Selected Window. Now Print the annotated chromatogram. 8) Practice with the Delete Annotation and Move Annotation menu items found under the Graphics menu or use the corresponding tools. 9) When finished with these tools, you can turn off the Graphics Tool bar by clicking the graphics tool again.
Lab Exercise: Qualitative Data Analysis Window Functions Window Functions 1) Use the Signal(s) Displayed drop box to view All Loaded Signals. 2) From the View menu, select Window Functions, List Content. 3) Position the cursor in the margin left of the Pump1, Pressure entry. Click using the left mouse button. This line should now be highlighted. 4) Press the Delete Obj. button.
Lab Exercise: Qualitative Data Analysis Peak Purity Peak Purity The Peak Purity algorithm can help you determine whether a chromatographic peak is comprised of one or more components. In quality control, impurities hidden behind the peak of interest can falsify results. In research, a hidden component left undetected might lead to a loss of essential information. 1) In the Methods ChemStation Explorer, find DEF_LC.M and load the method by double-clicking on the name.
Lab Exercise: Qualitative Data Analysis Peak Purity Major Peaks Peak 1 Peak 2 Peak 3 Retention Time Calculated Threshold Purity Factor Spectral Options – Wavelength Range Spectral Options – Spectra per Peak: Threshold Does the ChemStation indicate purity or impurity? In your opinion, does the spectral overlay indicate impurity? In your opinion, does the relationship between the similarity and threshold curves indicate impurity? Do the number of diamonds in the red band indicate impurity? Does the rela
Lab Exercise: Qualitative Data Analysis Automated Peak Purity Automated Peak Purity The Short + Spectrum, Detail + Spectrum, and Full report styles will produce automated peak purity analyses of integrated chromatographic peaks. 1) Make certain that you are in the Data Analysis view and demo\demodad.d is loaded. Load the method, purity.m. 2) To print an automated peak purity report, open the Report menu and select Specify Report. 3) Change the Report style to Short + Spectrum.
Lab Exercise: Qualitative Data Analysis Displaying Spectra Displaying Spectra 1) With demo\demodad.d still loaded, use the signals drop box to select only one signal for the window. If necessary, enlarge the chromatogram window by grabbing the bottom of the window and dragging to view the peaks more easily. 2) Under the Spectra menu, select Spectra Options or select the Spectral Task tool, then the Edit Spectra Display and Processing Options tool.
Lab Exercise: Qualitative Data Analysis Isoabsorbance Plot Isoabsorbance Plot In this section, you will try to optimize the data acquisition sample signal with the emphasis on obtaining the best signal to noise ratio for one chromatographic peak. Note that to use the Isoabsorbance plot, data must have been acquired with the spectra stored in the All format 1) Reload Signal B of DEMODAD.D. Use the Select Spectrum at Peak Apex tool to display the spectrum of the peak at 4.85 minutes.
Lab Exercise: Qualitative Data Analysis Isoabsorbance Plot selected in Data Analysis, that of the peak at 4.85 minutes. Convince yourself that this is the case by moving the vertical cursor to a time of 4.85 minutes. 6) Now point the cursor at the horizontal line across your Isoabsorbance plot. As you move this cursor up and down, watch the chromatogram in the lower section of the screen. The black chromatogram changes as you change the sample signal wavelength. 7) Change the cursor mode to Signal.
Lab Exercise: Qualitative Data Analysis 3D Plot 3D Plot 1) From the Spectra menu, select 3D Plot. Maximize the window. 2) Rotate the 3D plot. Notice that you can use the cursor on the wavelength axis to change the wavelength and view the resulting chromatogram. Use the cursor on the time axis to show a spectrum at different times. 3) Use the Data View button at the lower left to change the time range or wavelength range. 4) Explore the other options. 5) Close the 3D plot.
Lab Exercise: Spectral Libraries
Lab Exercise: Spectral Libraries In this Laboratory You Will: In this Laboratory You Will: • Build a spectral library. • Perform a library search. You will need Class.M and Class.D created in the acquisition laboratory. If you don’t have either of these files, use Isocra.m and one of the demo sequence files that was created with this method.
Lab Exercise: Spectral Libraries Building a Library Building a Library 1) From the View menu, select Data Analysis. 2) Load Class.D created in the acquisition exercise. (File, Load Signal). Load the acquisition method as well, Class.M. 3) Your first task will be to create a library file. Under the Spectra menu, select Library, then New Library. A new panel appears. Give the library a unique name or call it Class.UVL. OK this window. 4) The Edit Library Header box appears.
Lab Exercise: Spectral Libraries Building a Library 11) Click on Name and type in Dimethylphthalate. (Do not press Enter, as doing so will complete the entry.) 12) Other fields are present that you can use to customize your entries. Find the Sample entry and fill in Test Mix. 13) Scroll down until you find Slit Width. This parameter may be found on the Set up DAD Signals screen under the Instrument menu in the Method and Run Control view. Fill in the applicable width: 1, 2, 4, 8, or 16 nm.
Lab Exercise: Spectral Libraries Library Searching Library Searching In this section you will test your library with the same data file. Later we will use the library with new files in an automated fashion. Check that the library file you want to search from is currently loaded (look in the Library tool bar). Normally, if you were going to perform a library search, you would have to open the library first. Open Library is found under the Spectra, Library menus.
Lab Exercise: Spectral Libraries Library Searching 48
Lab Exercise: Overview and Diagnostics
Lab Exercise: Overview and Diagnostics In this Laboratory You Will: In this Laboratory You Will: • Learn to correctly identify the hardware elements and flow path of the 1100/1200 Series Modules. • Learn how to track errors and use the logbook • Learn how the ChemStation can help you diagnose problems • Learn how to set EMF Limits • Become familiar with the Maintenance and Repair CD-ROM and some maintenance procedures.
Lab Exercise: Overview and Diagnostics The Agilent 1200 Flow Path The Agilent 1200 Flow Path Please locate the items listed below with your instructor and fill in the table provided.
Lab Exercise: Overview and Diagnostics Error Messages and the Logbook Error Messages and the Logbook When an error (blockage, component failure, etc.) occurs on one of the Agilent 1200 Series modules, the tool in the Method and Run Control view that represents that component will turn red and the Run Status window will display a Not Ready message. Information about the error may be found in the bubble above the module tool. To obtain more information, open the Logbook.
Lab Exercise: Overview and Diagnostics Diagnosis View Diagnosis View The 1200 Series ChemStation is equipped with a view to help you: 1) diagnose systemic problems such as poor retention time precision, 2) perform diagnostic tests, such as the lamp intensity test, 3) record information pertaining to instrument maintenance; and 4) provide you with a means to determine when instrument maintenance should be performed, Early Maintenance Feedback (EMF). This view is called Diagnosis.
Lab Exercise: Overview and Diagnostics Diagnosis View Note: Exceeding an EMF limit will not shut down your system or abort any method or sequence. It simply changes the appearance of the EMF indicator. When the EMF indicator is yellow, the user should go to the Diagnosis view, click on the EMF tool and examine the EMF Memo Pad to find out what preventive maintenance is due by comparing the Limit and the Actual values. 4) Return to the Diagnosis view.
Lab Exercise: Overview and Diagnostics Diagnosis View Maintenance Logbook, and Date Changes Logbook. Examine each one of these entries. 5) Click on the open book tool and examine the menu choices available. Take a look at the Solvent Fillings... option. This panel will help you keep track of the amount of solvent in a reservoir. OK the panel. 6) Now, click on the Thermometer, then Update Variables Display. Now select, Pump Temp History.
Lab Exercise: Overview and Diagnostics Diagnosis View 7) Click on the Start button located next to the Leak Test option. The Leak Test is displayed. Click on the Explain button to find out more about the test. At this point in your troubleshooting process, you could Start the Leak Test, analyze the results, and then perform the required maintenance. 8) Close the Leak Test and Leak Test Information. You will get a chance to run an instrument test later.
Lab Exercise: Overview and Diagnostics Diagnosis View maintenance CD-ROM and then learn to perform user maintenance procedures on the 1200 modules. 1) Insert the HPLC Maintenance and Repair CD into the CD-ROM drive. (The CD may already be there.) 2) When the contents of the CD-ROM are displayed, double-click the file start.htm. Alternatively, you can get started by clicking the Windows Start button, selecting Run…, then Browse to find the CD-ROM drive. View Files of type: All Files, select start.
Lab Exercise: Overview and Diagnostics Diagnosis View Other important information found on the Maintenance and Repair CD includes Part and Materials, where you can find diagrams of instrument assemblies and part numbers, and information such as Optimizing Performance for each of the modules. Browse the contents of the CD as time allows and familiarize yourself with its contents.
Lab Exercise: Overview and Diagnostics Routine Maintenance Routine Maintenance Using the CD-ROM, perform the instrument maintenance listed below. If you have any questions during this part of the laboratory, please contact your instructor. Please reuse old parts for the purposes of this laboratory unless otherwise instructed.
Lab Exercise: Overview and Diagnostics Instrument Tests Instrument Tests The tests have been saved for the end of the laboratory in order to make certain the instrument is still functioning well. 1) In the Diagnosis view, select Diagnosis, then Tests.... 2) Perform the following tests using the step-by-step instructions provided on the ChemStation: • Pressure Test • Leak Test • Injector Steps • DAD Self-Test.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series In this Laboratory You Will: In this Laboratory You Will: • Learn to correctly identify the hardware elements and flow path of the Agilent 1200 SL Series modules. • Learn how to track errors and use the logbook. • Learn how the LC Diagnostic Tool can help you diagnose problems. • Learn how to set EMF Limits. • Become familiar with the Maintenance and Repair CD-ROM . • Become familiar with some maintenance procedures and tests.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series The Agilent 1200 - SL Flow Path The Agilent 1200 - SL Flow Path Please locate the items listed below with your instructor and fill in the table provided.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Error Messages and the Logbook Error Messages and the Logbook When an error (blockage, component failure, etc.) occurs on one of the Agilent 1200 Series modules, the tool in the Method and Run Control view that represents that component will turn red and the Run Status window will display a Not Ready message. Information about the error may be found in the bubble above the module tool. To obtain more information, open the Logbook.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Agilent LC Diagnostic Tool Agilent LC Diagnostic Tool The Agilent 1200 Series HPLC- SL modules are shipped with a tool to help you: 1) troubleshoot problems such as high pressure errors, 2) perform diagnostic tests, such as the pressure test, 3) perform calibrations, 4) provide you with a means to determine when instrument maintenance should be performed, Early Maintenance Feedback (EMF), and 5) provide instrument status reports.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Agilent LC Diagnostic Tool this case you may want to set the flow rate to 2-3 mL/min so that the seal wear counter will change more quickly. 6) Return to the Method and Run Control view. Notice that the check mark on the EMF indicator is currently green. Under the Instrument menu, select System On or turn the pump on at the System Diagram.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Agilent LC Diagnostic Tool 2) The Test Selection panel is displayed. Select the detector in the drop-down box. A list of tests available for the detector is shown. 3) Click on Intensity Test. A description of the test is shown by clicking on the More… button. The Start button is found below for beginning the test. 4) Explore the other available tests, but do not run any just now. You will run some of these tests later.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Agilent LC Diagnostic Tool part of the repair procedure. For practice, select the procedure for Exchanging the Pump Seals and Seal Wear-in Procedure. 5) The instructions for this procedure will appear on the right side of the program window. The information provided includes: When required, Tools required, Parts required, Preparation and Completion Steps, and the step by step directions.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Instrument Status Report Instrument Status Report You can print a status report for your own use or for a Service Engineer. 1) Select the Instrument Status Report tool. 2) Fill in the required information and Generate Report. 3) Examine the report details. 4) Press the Back button to return to the main menu.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Routine Maintenance Routine Maintenance Using the CD-ROM, perform the instrument maintenance listed below. If you have any questions during this part of the laboratory, please contact your instructor. Please reuse old parts for the purposes of this laboratory unless otherwise instructed.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Instrument Tests Instrument Tests The tests have been saved for the end of the laboratory in order to make certain the instrument is still functioning well. 1) Open the LC Diagnostic tool go to the Tests section and Tools section (injector steps). 2) Perform the following tests using the step-by-step instructions provided on the ChemStation: • Pressure Test • Pump Test • Injector Steps • Intensity Test.
Lab Exercise: Overview and Diagnostics for the Agilent-SL Series Instrument Tests 72
Lab Exercise: Integration
Lab Exercise: Integration In this Laboratory You Will: In this Laboratory You Will: • Perform autointegration. • Integrate using the Events Table. • Add Timed Events. • Use Manual Events.
Lab Exercise: Integration Integration Preparation Integration Preparation ChemStation revisions B.01 and higher have a new, more efficient and accurate integrator than previous revisions. During this lab exercise, practice integration using the data file, Integration.d. 1) Enter the Data Analysis view. Using the Load Method Tool, load the method, DEF_LC.M. 2) From the File menu, select Load Signal…. Under the Data folder, find the file Integration.d. Load the data file.
Lab Exercise: Integration Auto Integration Auto Integration One of the easiest ways to obtain initial integration events is through Auto Integration. Auto Integrate determines initial values for the slope sensitivity, peak width and area reject. First, however, you will display the default events. 1) Click on the Integration tool, then the Edit/Set Integration Events Table tool or from the Integration menu, select Integration Events.
Lab Exercise: Integration Auto Integration 5) Using the Events Table drop box, select signal DAD1 B Specific. 6) To Auto Integrate, select the Integration tool then the Auto Integrate tool or select the Integration menu, then Auto Integrate. When asked if you want to save the changed events table, reply No. 7) View the events and chromatogram that were created for Signal B.
Lab Exercise: Integration Integrate Using the Events Table Integrate Using the Events Table Now, you will set up integration events for the Integration.d chromatogram using the approach discussed in lecture. 1) Click on the Integration tool, then the Edit Integration Events tool. 2) Using the signal drop box, select DAD1B Specific. 3) Set the Slope Sensitivity to 50. 4) Estimate the peak width from the initial integration. Use the smallest peak width from a real chromatographic peak, not noise.
Lab Exercise: Integration Adding Timed Events Adding Timed Events At times, the initial events are not able to properly construct baselines or place tic marks for integration. Timed events may be added to improve integration. In this section, you will learn how to add timed integration events to your method. 1) Open the Select an event drop box. 2) To practice with timed events, select the timed event, Integration. 3) Move the line that appears in the chromatogram to time=0 and click.
Lab Exercise: Integration Adding Timed Events 6) Practice adding other timed events. Note, you can also add and remove timed events with the tools shown below. Practice this now. 7) When finished, delete all timed events by selecting the event then clicking the remove event tool shown above. 8) Exit the Integration Events using the Exit and save events to method tool. NOTE: Once the appropriate events are found, they must be saved as part of the method.
Lab Exercise: Integration Using Manual Events Using Manual Events Manual integration allows you to integrate specific peaks or groups of peaks with complete control. For example, you may set the integration start and stop points of any chromatographic peak yourself. This type of integration is convenient, but may not be applicable from run to run. It also may not be as reproducible from operator to operator. 1) Select the Manual Integration: Remove integrated Peak(s) from the Integration Results tool.
Lab Exercise: Integration Using Manual Events 1) Select Copy Manual Events to Method in the Integration menu. 2) Select the Edit/Set Integration Events Table tool to display the integration events. 3) Integrate the chromatogram using the Integrate Current Chromatograms tool. 4) Notice that the manual integration events have not been applied. 5) Click in the Manual Events box and re-integrate the chromatogram (this box is located integration events section of the window on the left side).
Lab Exercise: Quantification
Lab Exercise: Quantification In this Laboratory You Will: In this Laboratory You Will: • Run three external standards to create a calibration table. • Select appropriate integration parameters and save them to your method. • Build a three level calibration table. • Test the calibration table • Print a basic report.
Lab Exercise: Quantification Preparations Preparations Obtain the low, medium and high standards. Insert them into vial positions 1, 2 and 3 of the autosampler. Turn on the detector lamp. Prime your solvent delivery system. Priming the Agilent 1100 or 1200 Solvent Delivery System 1) If you are equipped with a vacuum degasser, make certain the degasser is on. 2) Ensure that the outlet tube is connected from the purge valve to the waste container.
Lab Exercise: Quantification Preparations 5) To turn the solvent delivery system on, you may use the menu items: Instrument, More Pump, Control; or access the tool shown in the diagram below. Do this now. 6) Wait until a steady stream of solvent comes out of the purge valve waste tube. 7) Repeat steps 3 through 6 for the other channels of the pump. Complete the priming process by setting the composition to %B=65 and pump at 5 mL/min. 8) Change the flow rate to 1.5 mL/min and then close the purge valve.
Lab Exercise: Quantification Creating the Acquisition Method Creating the Acquisition Method 1) Load the default method, DEF_LC.M as a starting point for method creation (Method, New Method) or double-click on DEF_LC.M in the ChemStation Explorer. From the Method menu select Edit Entire Method.… Select only the Instrument/Acquisition section for editing. OK this dialog box. 2) Fill in the following parameters for the solvent delivery system: OK the dialog box.
Lab Exercise: Quantification Creating the Acquisition Method Note: If a VWD detector is being utilized, select the same wavelength as signal A. 5) Set the column compartment temperature to 40°C (both left and right). OK the dialog box. 6) From the Method menu, select Save Method As.... Name the method Quant.m. 7) Type in a short comment for the Method History and OK. 8) Turn on the pump and equilibrate the HPLC column for several minutes with 30% water/70% acetonitrile.
Lab Exercise: Quantification Data Acquisition-Standards Data Acquisition-Standards In this example, you will run each sample individually, not as a sequence. Of course, it is possible to run the three standards in sequence mode. 1) From the RunControl menu, select Sample Info.... The same panel may be accessed from the tool: Fill in the information for the low level standard and OK the dialog box.
Lab Exercise: Quantification Data Acquisition-Standards 2) Check that your baseline and pressure are stable. 3) Start the run either from the Start tool or from the RunControl menu, then Run Method. 4) Now acquire data for the medium standard and the high standard as well. Call the data files, med.d and high.d, respectively. When you have finished, turn off the pump.
Lab Exercise: Quantification Integration Integration In this section you will optimize the integration events for the analysis and save them to the method. 1) Go into the Data Analysis view. 2) From the View menu, select Preferences. 3) Select the Signal Options tab. Make certain that integrate after load is the only box checked. 4) Find your data analysis directory in the ChemStation Explorer. Double-click on the Single Runs to place the files in the Navigation Table. Find the data file, Low.
Lab Exercise: Quantification Integration 9) Check the integration for signal B as well. Make certain that both signals are mapped to the DAD Default Events Table by selecting the tool, or if you choose, create different events for each signal. 10) Exit and save the events to the method using the Exit and save events to method tool. Then, resave the method, Quant.m.
Lab Exercise: Quantification Setting up Signal Details Setting up Signal Details The Signal Details dialog box describes which signals will be evaluated during a method run. Signals that are not in the list box will not be integrated or reported if all defined signals can be found in the current data file. Whenever the defined signals cannot be found the system loads all available signals from the data file and tries to generate the report. 1) From the Calibration menu, select Signal Details….
Lab Exercise: Quantification Building a Calibration Table Building a Calibration Table You have already loaded and integrated the lowest level standard. The first step toward building your calibration table will be to select the calibration settings. 1) In the Calibration menu, select Calibration Settings... 2) Fill in the Title of the Calibration Table, e.g., Class. 3) You will Use Sample Data From Data File. 4) Type in µg/mL for Amount Units. 5) The Default RT Windows should be set at 5%.
Lab Exercise: Quantification Building a Calibration Table 12) Now find the last major peak at approximately 3.3 minutes. The compound name is o-terphenyl and the amount is 2.4 µg /mL. Select the signal that provides the most abundant area counts. You will also make this a reference peak. 13) OK this initial Calibration Table to remove lines with zero amounts. This step will make building your table easier. 14) From the Calibration menu, select Calibration Table to redisplay the Calibration Table.
Lab Exercise: Quantification Setting up Low and High Amount Limits for Calibration Standards Setting up Low and High Amount Limits for Calibration Standards You may specify low and high amount limits in the calibration table. If the calculated amount is outside the limits, a warning appears in the report. The amount limits can be used as qualifiers to confirm the peak identification. Compounds that are outside of the amount limits, are not used to recalibrate the Calibration Table.
Lab Exercise: Quantification Setting up Qualifiers (Diode Array only) Setting up Qualifiers (Diode Array only) A qualifier peak is used to confirm that the main peak within the retention time window belongs to the expected compound. It is assumed that the peaks of one compound have a constant response ratio at two different wavelengths. The qualifier peak response is a percentage of the main peak response.
Lab Exercise: Quantification Testing the Calibration Table Testing the Calibration Table 1) Load the medium standard's data file, med.d. Go to the Report menu, then Specify Report... or select the Specify Report Calculation and Print Style tool. 2) Make the following selections: Destination = Printer and Screen Calculate = ESTD Based on = Area Report Style = Detail Select chromatogram output. 3) OK the report specifications and save this to your method.
Lab Exercise: Running a Sequence
Lab Exercise: Running a Sequence In this Laboratory You Will: In this Laboratory You Will: • Build a sequence with automated recalibration of the existing calibration table. • Learn how to pause and restart the sequence. • Print a Sequence Summary report. • Access the Sequence Logbook. Sequence operation allows you to perform unattended analysis of more than one sample, using more than one method.
Lab Exercise: Running a Sequence Building a Sequence Building a Sequence 1) Go to the Method and Run Control view. 2) Under the Sequence menu, select New Sequence. This menu selection loads the default sequence template. You may also access this option by clicking on the sequence tray setup tool located on the left hand side of the screen. 3) Under the Sequence menu, find Sequence Parameters…. 4) Fill in the Operator Name. Create a subdirectory using your last name. Do not change the Path.
Lab Exercise: Running a Sequence Building a Sequence 7) Now enter the Sequence Table from the Sequence menu or from the sequence tray setup tool. You would like to do the following: a) Replace all existing calibration table response factors with new response factors at all three calibration standard levels. b) There after, perform cyclic recalibrations every 2 sample injections averaging the response factors and retention times.
Lab Exercise: Running a Sequence Building a Sequence 8) OK the Sequence Table after completing the entries and save the sequence either through the Save Current Sequence tool or through the Sequence menu. 9) Select Sequence>Partial Sequence to view the injection order that will be followed by your Sequence Table. Cancel out.
Lab Exercise: Running a Sequence Sequence Summary Reports Sequence Summary Reports The ChemStation can print a variety of standard reports for individual sample analyses as directed in the Specify Report panel. These styles are stored as part of the method and are printed at the end of each analysis if desired. Sequence summary reporting is an additional, optional way of reporting.
Lab Exercise: Running a Sequence Starting the Sequence Starting the Sequence 1) Make certain that Quant.M is currently loaded. 2) View the RunTime Checklist (under the Method menu). Make certain that both Data Acquisition and Data Analysis are selected. In previous exercises, data acquisition was the only item selected. 3) Turn on the pump, column thermostat, and detector. Allow the column to equilibrate.
Lab Exercise: Running a Sequence Pausing a Sequence Pausing a Sequence 1) Pausing a sequence to run a priority sample is quite easily accomplished. Pull down the Run Control menu and select Pause Sequence. The current sequence will pause after the current method has completed. You can make changes in the sequence for those samples that have not yet been injected. 2) To resume the sequence, select Resume Sequence from the Run Control menu. The sequence continues with the next run.
Lab Exercise: Running a Sequence Post Sequence Post Sequence 1) Examine the printed reports. How did your sequence go? 2) If you have errors during your sequence, it may be helpful to print the Sequence Logbook. To do this, go to the View menu. Select Logbook, then Sequence Logbook. To print this logbook, select Print Logbook after you have displayed the logbook on the screen. The logbook can be displayed with the Sequence Logbook tool, as well.
Lab Exercise: Running a Sequence Post Sequence 108
Lab Exercise: Sequence Review and Reprocessing
Lab Exercise: Sequence Review and Reprocessing In this Laboratory You Will: In this Laboratory You Will: • Load a sequence into the sequence navigation table. • Reprocess a sequence after changing the folder method. • Reprocess a sequence after updating DA methods. • Use Batch Review to review a sequence. You will need the following data: • Batch.
Lab Exercise: Sequence Review and Reprocessing Reprocess with Sequence Folder Method Reprocess with Sequence Folder Method You will reprocess a sequence after updating the Folder Method. This sequence has two problems. First, calibrated compounds fell outside the method’s retention time windows, and therefore, no amounts were calculated. The retention time windows are set in the Calibration Settings.
Lab Exercise: Sequence Review and Reprocessing Reprocess with Sequence Folder Method You will update the Sequence Folder Method with new retention time windows and drop the 4th level calibration data point, then reprocess the entire sequence. Note that only the method (.M) and sequence file (.S) in the sequence folder will change. The master method stored in Chem32\1\Methods and the sequence template stored in Chem32\1\Sequence will not change. 1) Go to Data Analysis.
Lab Exercise: Sequence Review and Reprocessing Reprocess with Sequence Folder Method 4) First, let’s change the retention time windows so that the amounts will be calculated. 5) Go to the Calibration menu and then Calibration Settings. 6) Change the Default RT Windows as below. OK the dialog box.
Lab Exercise: Sequence Review and Reprocessing Reprocess with Sequence Folder Method 7) Now, take a look at the calibration curve. Select the Calibration menu and then Calibration Table. 8) Click next to the 4th level row to highlight the line and press the Delete button. 9) Remove all level 4 rows from the table. 10) Notice that the Correlation is now improved. 11) Save the sequence folder method. Always check to make certain you are in the correct directory path.
Lab Exercise: Sequence Review and Reprocessing Reprocess with Sequence Folder Method The Sequence folder .S file is currently loaded. If you want to check this, go to Method and Run Control and check the path. 13) Open the Sequence Output dialog box by clicking on the tool shown below. 14) Deselect Print individual reports for each run as well. OK the dialog box. 15) Select the Sequence Summary tool shown below. Make certain only 5 and 9 are selected. Make section 9, Summary, is a Compound Summary.
Lab Exercise: Sequence Review and Reprocessing Reprocess with Sequence Folder Method 16) To reprocess the sequence, select the Start sequence reprocessing tool outlined below. The sequence will reprocess. You will notice that the status bar turns blue to indicate the sequence is reprocessing. The method and a Compound Summary should print at the end of the sequence. Examine the method. You should see the record of your action in the Change History.
Lab Exercise: Sequence Review and Reprocessing Reprocess with DA Methods Reprocess with DA Methods In this section, you will see how to change the individual DA methods. You will add manual integration events to two runs and reprocess. 1) Go to the View menu, then Preferences… >Signal Options. 2) Make certain that Integrate after load is selected under Load Signal Options and Individual Method from Data File is selected under Method used for Review of Sequence Data. OK the dialog box.
Lab Exercise: Sequence Review and Reprocessing Reprocess with DA Methods 4) You can review the data to check integration by stepping through the files manually or automatically. To step through the files manually, press the tool below . 5) To step through the files automatically, press the button outlined below. 6) To stop, press the Pause button. Practice navigation for a moment. 7) Now we will change individual DA Methods. Load 001-0101.D. 8) Press the Inegration button.
Lab Exercise: Sequence Review and Reprocessing Reprocess with DA Methods 12) Resave the DA method. Make certain you have saved the desired changes to the correct method by checking the path. Insert a comment for the method history. 13) Perform the same exercise on 003-0301.D and save its DA method. In order to use the DA methods for these data files instead of the sequence folder method during reprocessing, you must change the methods in the Sequence Table.
Lab Exercise: Sequence Review and Reprocessing Reprocess with DA Methods 19) Now, reprocess the sequence. Take a look at the resulting Compound Summary page and compare it to the one at the end of this lab to make certain your changes took affect.
Lab Exercise: Sequence Review and Reprocessing Batch Review Batch Review If you generate a large number of samples on a daily basis and would like a convenient way to view the results and check the quality of integration then Batch Review may help. Batch Review allows you to step through calibration standards, control samples, and unknown samples adjusting the integration parameters and reviewing the results of sequence runs. 1) In the Data Analysis view, select Demo\Batch. Find the .
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Lab Exercise: Advanced Reporting
Lab Exercise: Advanced Reporting In this Laboratory You Will: In this Laboratory You Will: Preview the following report styles: • GLP • Performance (System Suitability) • Automated Peak Purity You will also learn how to add your own header to report styles.
Lab Exercise: Advanced Reporting Adding a Report Header Adding a Report Header You may add an individualized header to many of the report styles including: Detail, Header + Short, GLP + Short, GLP + Detail, and Full. The directions presented below will guide you through this process. 1) Go to the Data Analysis view. Load the method, Advrep.M. Try using the Load Method tool. 2) From the Start button, select All Programs, then Accessories. 3) Open Notepad.
Lab Exercise: Advanced Reporting Adding a Report Header Note: The GLP report style is not in any way connected to the Save GLP data selection found in the Run Time Checklist. It is simply a report style that includes a printout of the logbook, firmware, serial numbers and some other information.
Lab Exercise: Advanced Reporting Performance Reports (System Suitability) Performance Reports (System Suitability) Many chromatographers utilize the Performance report styles to help evaluate the analysis system before and during routine analyses. The information generated for each calibrated peak includes the retention time, k', symmetry, peak width, plate number, resolution, signal-to-noise ratio, and compound name. You may also determine the noise and drift using a Performance + Noise report style.
Lab Exercise: Advanced Reporting Performance Reports (System Suitability) Select System Suitability from the Report menu, followed by Edit Noise Ranges. 10) Examine prior chromatograms and select several time ranges for the noise calculation. The ranges should be greater than one minute in duration in a region where there are no chromatographic peaks. Fill in the table and OK the dialog box. 11) Now, go to the Report menu and select Specify Report or use the Specify Report tool.
Lab Exercise: Advanced Reporting Automated Peak Purity Automated Peak Purity The Short + Spectrum, Detail + Spectrum, and Full report styles will produce automated peak purity analyses of calibrated chromatographic peaks. 1) Make certain Quant.M is still loaded. Make certain the last data file you collected is still loaded, Syss.d. 2) To print an automated peak purity report, open the Report menu and select Specify Report or use the Specify Report tool. 3) Change the Report style to Short + Spectrum.
Lab Exercise: Advanced Reporting Automated Spectral Library Search Automated Spectral Library Search Make certain that Quant.M and Syss.D are still loaded. Quantification based solely on retention time correlation can lead to erroneous identification if: a) Compounds other than the calibrated ones appear in the specified retention time window. b) More than one peak elutes in the specified retention time window.
Lab Exercise: Advanced Reporting Automated Spectral Library Search to the calibration table for an identically named entry. If one is found, the data are used to calculate and report the amount. 6) OK this dialog box. Open the library you intend to use in the report and check that the names of the calibrated peaks are identical with the library names. In other words, check your calibration table as well as the entries in the library. 7) Under Specify Report, select Library Search. OK the dialog box.
Lab Exercise: Advanced Reporting Automated Spectral Library Search 132
Lab Exercise: Customized Report Design A Customized Report Layout view is included in the ChemStation for users who want to define the exact content of their reports. This laboratory will help you develop a customized report. For further information, study the customized report files that are included with the software.
Lab Exercise: Customized Report Design In this Laboratory You Will: In this Laboratory You Will: • Build your own customized report template. • Save the template as part of the standard report options. You will need the Demo File, 005-0101.D and Democal1.M.
Lab Exercise: Customized Report Design Preparations Preparations 1) Enter the Report Layout view. Load the method Democal1.m (Double-click on the file name in ChemStation Explorer, or choose File > Load, Method). 2) Load Signals from the file Demo\005-0101.d. using File > Load > Signals.
Lab Exercise: Customized Report Design Building a Customized Report Building a Customized Report 1) Under the File menu, select New Template. First, add a header and footer to the report style. The header would be reproduced on each page of the report at the top while any footer would appear on each page at the bottom. 2) Next to the Header designation you will see two gray horizontal borders which represent the section start and stop points.
Lab Exercise: Customized Report Design Adding a General Section Adding a General Section 1) Click on the blue triangle. The color will change to red. 2) Select Edit, New Section, General. 3) Expand the General section by dragging the bottom border down. 4) Click on the Text Tool and position a rubber-band box at the top. Select Constant Text, then OK. Type in any address. NOTE: Use the CTRL and Enter keys simultaneously to move to the next line within a text box.
Lab Exercise: Customized Report Design Inserting Tables Inserting Tables 1) Below the chromatogram, expand the size of the general white space. 2) Select the Table Tool, and outline a space approximately 80 x 20. 3) Select Cal. Peaks Sorted by Ret. Time. The Setup Cal. Peaks sorted by Ret. Time table should appear. From the Printed Columns box select Response Factor then <. Note that you have removed this item from the Printed Columns list. Check that the table layout lies within the bounding box length.
Lab Exercise: Customized Report Design Adding a Calibration Curve Adding a Calibration Curve 1) Click on the blue triangle found between the General section and the Footer. The triangle will change from blue to red. 2) Pull down the Edit menu and select New Section, Cal. Compounds. 3) Drag the Cal. Compounds borders to create free white space in this new section. 4) Click on the Graphics Tool. 5) Draw in a rubber band box for the calibration table.
Lab Exercise: Customized Report Design Finishing Up Finishing Up 1) From the File menu, select Save Template As. Give the template a unique name. 2) Now test the template. Use the Report Preview tool to display the report. Review the report and make any necessary modifications. Resave the template as needed. You may print the report now. 3) You can add your report to the list of report styles by selecting Add to Report Styles under the File menu. Try this now.
Lab Exercise: Commands
Lab Exercise: Commands In this LaboratoryYou Will: In this LaboratoryYou Will: • Become familiar with some of the more common commands on the HPLC ChemStation. • Understand the use of variables. • Understand the concept of data storage in registers and tables. • Become familiar with some of the commands used to access/manipulate data. During this laboratory exercise you will be using the HPLC ChemStation equipped with version B.02.xx software. The data file, demodad.
Lab Exercise: Commands Retrieving Information about Commands Retrieving Information about Commands Several resources are available to help you utilize the ChemStation commands. These resources include Online Help and the Show command. Start the exercise in the Data Analysis view. 1) Check to make certain that the command line is displayed at the bottom of the ChemStation software window. If the line is not available, select View then Command Line. 2) Type Show on the command line.
Lab Exercise: Commands Tracking Errors and Using some Common Commands Tracking Errors and Using some Common Commands A window can be displayed which will help you track software errors while your are using commands or writing/using macros. The window is accessed with the ListMessages command. 1) On the command line, type ListMessages On. Notice that a white message box is now displayed. The box can be sized differently by dragging the edges.
Lab Exercise: Commands Registers, Objects, and Tables Registers, Objects, and Tables A register is a place to store data such as chromatograms and signals. The ChemStation uses pre-defined registers such as ChromReg for chromatograms and SpecReg for spectra. The chromatograms and spectra are objects in the registers. If you load a data file containing signals A, B, and C; the ChromReg register will contain three objects.
Lab Exercise: Commands Registers, Objects, and Tables Objects Objects are the data you work with such as chromatograms or spectra. Each object has a header which contains a description of the object. For instance, if the object is a chromatogram, the header contains the injection time and date, the operator name and vial number. Objects will also have a data block. The data block is the actual x and y data points that describe the chromatogram or spectrum. Objects may also comprise tables.
Lab Exercise: Commands Registers, Objects, and Tables 4) To integrate the chromatogram, use the command IntegrateObj. Type: IntegrateObj ChromReg[1] Draw 2,ChromReg[1] 5) Access scalar variables(numbers) from the object header using ObjHdrVal.
Lab Exercise: Commands Registers, Objects, and Tables Try typing: Item$=ObjHdrName$(ChromReg[1],3) Obtype=ObjHdrType(ChromReg[1],item$) Print "Item 3 in object 1 is called",Item$,"and is a",Obtype,"variable" Note: The answer to your print appears above the command line. 0 is a string variable. Data Matrix To complete the next section, you will need to load the method, democal1.m, load the data file, demo\005_0101.d, and print a report. Do these tasks now.
Lab Exercise: Commands Registers, Objects, and Tables Tables 1) You have already integrated the chromatogram with the IntegratObj command. When each signal is integrated, the integration results are stored in a table called Intresults. One IntResults table is created for each integrated signal and the table is stored in the same object as the corresponding signal. When you quantify the peaks, the integration tables in ChromReg are identified and quantified.
Lab Exercise: Commands Registers, Objects, and Tables to obtain a single numeric element from a table. Type: pk_width=tabval(ChromReg[1],"intresults",2,"width") Print pk_width You obtained the peakwidth of the second integrated chromatographic peak. 3) Use the commands to obtain the name of the 4th compound from the quantification results table "compound" found in the ChromRes register. To get this information, you must print a report first.
Lab Exercise: Commands Registers, Objects, and Tables Viewing Tables Often, you will need to display the contents of a table to understand what row or column to access. EdTab is a useful command. 1) Type: EdTab 3,ChromRes,Peak where 3 is the window number, ChromRes is the Register and Peak is the table. Scroll to view all entries.
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Lab Exercise: Macro Writing
Lab Exercise: Macro Writing In this Laboratory You Will: In this Laboratory You Will: • Write and execute a macro. • Learn how to find errors in a macro. • Add a macro to your method and execute that macro.
Lab Exercise: Macro Writing Writing and Executing a Macro Writing and Executing a Macro During this first exercise, you will simply follow the directions to create and execute a simple macro. 1) Minimize the ChemStation. From the Start button, select All Programs then Accessories, and Notepad. 2) Type in the following simple macro: Name Class File “c:\chem32\1\data\demo\005-0101.
Lab Exercise: Macro Writing Writing and Executing a Macro Macro class.mac,go The macro should begin executing. If a problem is found, correct the macro and re-execute. Note: If the ChemStation cannot open the file, class.mac, go to the directory and check the file extension. Notepad may put a .txt after the .mac extension by default. Rename the file to class.mac.
Lab Exercise: Macro Writing Write Your Own Macro Write Your Own Macro Write a macro to do the following. Use your own registers and windows 2-10: • Get a file from your sequence. • Obtain signal B. • Get another file from your sequence. • Draw the overlaid signal to a full page window. • Send to the printer with the message: "Overlaid Chromatographic Signals". Hint: You will need to access the _Config register.
Lab Exercise: Macro Writing Possible Answer Possible Answer name overlaid file "c:\chem32\1\data\demo\005-0101.d" if regsize (myreg) >0 delreg myreg endif loadsignal dad,a, ,myreg file "c:\chem32\1\data\demo\005-0102.d” loadsignal dad,a, ,myreg draw 2,myreg settabtext _config [1] ,window,3,"destination","printer" settabval _config [1],window,3,defwyhigh,0.6 settabval _config [1],window,3,defwylow,0.
Appendix: Getting Started with New ChemStation Workflow G2170-90041
Agilent ChemStation Getting Started with New ChemStation Workflow A
Notices © Agilent Technologies, Inc. 2006, 2007 Warranty No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws. The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions.
In This Guide… In analytical laboratories, chromatography data need to be acquired efficiently in a short time. Clarifying ambiguous results can be time-consuming, and may result in high administrative costs. Since ChemStation Revision B.02.01, data storage and data browsing capabilities have been improved to enable fast review and reprocessing of result data. In this manual, the efficient use of the new data storage and retrieval functions in ChemStation B.02.01/B.03.
4 Data Organization
Contents 1 ChemStation Data Structure 7 ChemStation prior to B.02.01 8 ChemStation B.02.01/B.03.
Contents 6 Data Organization
Agilent ChemStation Getting Started with New ChemStation Workflow 1 ChemStation Data Structure ChemStation prior to B.02.01 8 ChemStation B.02.01/B.03.01 9 This chapter gives an overview of the differences between the data structure used in ChemStation revisions prior to B.02.01 and the new data structure in revision B.02.01 and its successors.
1 ChemStation Data Structure ChemStation prior to B.02.01 ChemStation prior to B.02.01 In ChemStation revisions prior to B.02.01, sequences, methods and the generated data files and results were stored in fixed, specified and separated locations. For example, methods were referenced by name in a sequence and it was the user’s responsibility to maintain the integrity of methods, sequences, and data files. Because of this, the long-term archiving of data, and reproduction of results was a tedious task.
ChemStation Data Structure ChemStation B.02.01/B.03.01 1 ChemStation B.02.01/B.03.01 In order to strengthen the association between data files and methods, the following new data organization scheme has been implemented with ChemStation B.02.01/B.03.01. When used with the ChemStation, the Agilent Enterprise Content Manager (ECM) also makes use of the new data concept, since the complete data set (sequence/methods/data files) can now be transferred (archived) to ECM as one entity.
1 ChemStation Data Structure ChemStation B.02.01/B.03.01 Similarly, the sequences in the folder Chem32\1\sequence serve as sequence templates that can be used to rerun (but not reprocess) a sequence several times. The data storage pattern varies depending on whether single run data or sequence data is acquired: 1 When a sequence is executed, a new folder is automatically created (“sequence container”) with a unique name in the specified subdirectory. When a single sample is run, the data file (*.
ChemStation Data Structure ChemStation B.02.01/B.03.01 1 The following chapters explain the impact of this structure on typical workflows in more detail. The corresponding settings in the ChemStation dialogs are also shown.
1 12 ChemStation Data Structure ChemStation B.02.01/B.03.
Agilent ChemStation Getting Started with New ChemStation Workflow 2 Data Acquisition Data Acquisition 14 Data Acquisition in a Sequence 15 Data Acquisition of Single Runs 17 This chapter explains how the new data structure influences the workflow for the acquisition of data for sequences and single runs.
2 Data Acquisition Data Acquisition Data Acquisition Starting with ChemStation B.02.01, flexible data storage for single runs and sequences allows you to specify various saving locations without reconfiguration. The Paths tab in the Preferences dialog box in the View menu gives you the opportunity to add multiple paths in addition to the default path C:\chem32\x\DATA (where x is the instrument number).
Data Acquisition Data Acquisition Figure 3 2 Data Path Selection Data Acquisition in a Sequence In order to run a sequence, appropriate pre-defined methods must be available. These are the master methods as outlined above. Typically, master methods and sequence templates are worked on in the Method and Run Control view of the ChemStation. For this reason, in Method and Run Control view, the ChemStation Explorer provides access to master methods and sequence templates.
2 Data Acquisition Data Acquisition The sequence template references these methods in the sequence table. As explained previously, when a sequence is run with sequence template .S, and the master method .M is used, a new folder is created that contains all resulting files from the sequence run (“sequence container”).
Data Acquisition Data Acquisition 2 the container. All necessary files are now available for future data review and reprocessing, without changes that were applied to the master method or sequence template for other sequence runs. During acquisition, the data files are stored to the sequence container. Within each data file (*.D), two additional methods, ACQ.M and DA.M, are saved for this specific run.
2 18 Data Acquisition Data Acquisition Data Organization
Agilent ChemStation Getting Started with New ChemStation Workflow 3 Data Analysis Data Analysis 20 Data Analysis: Data Review 23 The ChemStation User Interface during Data Review 29 Data Analysis: Reprocessing data 31 This chapter outlines the data analysis and review options that are available, and explains how consideration of the data structure affects your choice of options.
3 Data Analysis Data Analysis Data Analysis Once the data have been acquired, they can be analyzed in ChemStation Data Analysis view. When selecting the Data tab of the ChemStation Explorer, you can load all the runs of a sequence or all single runs in a specific folder by double-clicking the corresponding symbol. The corresponding data set is then available in the Navigation Table.
Data Analysis Data Analysis 3 With this toolset, you can jump to the beginning or end of the Navigation Table, step on to next or previous run, automatically step through the runs, and stop automatic stepping. A different way to analyze your data is to “Reprocess” a complete sequence. During this process, all runs are reanalyzed in the sequence context, i.e. the calibration tables of the sequence methods are updated in the case of calibration runs, multipliers, amounts etc.
3 Data Analysis Data Analysis Figure 8 22 Sequence Parameters in Method and Run Control view of ChemStation B.02.
Data Analysis Data Analysis: Data Review 3 Data Analysis: Data Review Data Review means analyzing on a run-per-run base. ChemStation allows you to specify default actions that are performed automatically when a data file is loaded from the Navigation Table. These include data analysis tasks like integrating the chromatogram directly after loading, and also specifying the method that is to be loaded.
3 Data Analysis Data Analysis: Data Review The remainder of this section specifies which method is loaded during data review when a run is loaded from the Navigation Table. They only apply to data review, but not to reprocessing.
Data Analysis Data Analysis: Data Review Figure 10 3 Keep the Current Method for Data Review You load the master method, most conveniently from the Method tab of the ChemStation Explorer. Now, you optimize the integration parameters and use the runs to construct the calibration table. All future acquisitions using this method will apply these optimized data analysis parameters.
3 Data Analysis Data Analysis: Data Review Figure 11 Load the Sequence Method for Data Review This means that each time you load a run from the Navigation Table, the sequence method corresponding to the run’s sequence line is loaded. Once the data review has been completed, it may be necessary to reprocess the complete sequence in order to apply the method changes not only on a run-per-run base, but also in the complete sequence context (including calibration table updates etc.).
Data Analysis Data Analysis: Data Review 3 or last reprocessing. Additionally, it is possible to perform run-specific changes and save them in the individual data analysis method of the run. This is especially useful for manual integration events. Figure 12 Load the Individual Method from Data File for Data Review Using the individual data analysis method (DA.M) a convenient means is available to store manual integration events to a specific data file only.
3 Data Analysis Data Analysis: Data Review Figure 13 Copy Manual Integration Events to a Method When the manual events have been saved to the individual method (instead of e.g. the corresponding master method), it is probably most useful always to apply those events when the chromatogram is integrated: 3 Check the “Manual Events” option in the Integration Events dialog (see Figure 14). Figure 14 Apply Manual Integration Events each time the chromatogram is integrated 4 Save the method.
Data Analysis The ChemStation User Interface during Data Review 3 The ChemStation User Interface during Data Review The ChemStation user interface provides a number of features to facilitate working with the different methods available for data analysis (Figure 15). Figure 15 User Interface in Data Analysis • The method modification status is displayed in Data Analysis view, so you can easily follow if there are unsaved method changes.
3 Data Analysis The ChemStation User Interface during Data Review Figure 16 Update the Master or Sequence Method This feature is available in the following situations: Table 2 Availability of the Update … Method Functionality Loaded Method Available Options Individual data analysis method (DA.
Data Analysis Data Analysis: Reprocessing data 3 Data Analysis: Reprocessing data In contrast to data review, sequence reprocessing means that all the runs of a sequence are reanalyzed in the sequence context, i.e. including calibration table updates, parameters changes in the sequence table, additions of new methods to the sequence, etc.
3 Data Analysis Data Analysis: Reprocessing data Figure 18 Browse to the Master Methods directory in the Sequence Table • In the sequence table, it is not possible to add or remove lines. • In the Sequence Parameters dialog, only the operator name, the sequence comment, and the usage of sequence table information can be changed. All other fields have to be set during data acquisition or do not apply to reprocessing.
Data Analysis Data Analysis: Reprocessing data Figure 19 Data Organization 3 Sequence Parameters in Data Analysis 33
3 34 Data Analysis Data Analysis: Reprocessing data Data Organization
Agilent ChemStation Getting Started with New ChemStation Workflow 4 Workflow with Unique Folder Creation switched off This chapter provides information on working with Unique Folder Creation switched off which allows you to store data as in ChemStation revisions B.01.03 or earlier. This mode does not take full advantage of the latest data review and reprocessing functionality in ChemStation.
4 Workflow with Unique Folder Creation switched off Working with Unique Folder Creation on or off? Working with Unique Folder Creation on or off? The new data concept as outlined in the previous chapters provides a number of advantages: • Sequence data are not overwritten. Each sequence acquisition stores the resulting data files in its own sequence container with unique name. • With the sequence container concept, the data are stored with all necessary information needed for data analysis, i.e.
Workflow with Unique Folder Creation switched off Workflow with “Unique Folder Creation” switched off 4 Workflow with “Unique Folder Creation” switched off In order to allow working with a data storage concept as in ChemStation revisions before B.02.01, the Sequence tab of the Preferences dialog box a Data Storage section. Here you can choose between “Unique Folder Creation ON” and “Unique Folder Creation OFF” (Figure 20). Per default, “Unique Folder Creation ON” is selected.
4 Workflow with Unique Folder Creation switched off Workflow with “Unique Folder Creation” switched off NOTE We recommend to decide between the two modes at the beginning of your work and not to switch between them. Switching Unique Folder Creation off is not supported with the ChemStation ECM Integration or ChemStore/Security Pack installed.
Workflow with Unique Folder Creation switched off Workflow with “Unique Folder Creation” switched off 4 • There are no data file specific methods ACQ.M and DA.M stored. Saving information about the original acquisition is only possible by including this information in the report or by selecting “Save Method with Data” from the method’s runtime checklist (Figure 21). With this option the acquisition method will be stored as RUN.M in each data file.
4 Workflow with Unique Folder Creation switched off Workflow with “Unique Folder Creation” switched off The enhanced ChemStation user interface as introduced with ChemStation B.02.01 is also available when Unique Folder Creation is switched off. However, there are functions you can not take advantage of in this mode. The same limitations also apply to any run acquired with ChemStation prior to B.02.01.
Workflow with Unique Folder Creation switched off Workflow with “Unique Folder Creation” switched off 4 • With the method usage options “Use method from data file” and “Use sequence method” (see “Data Analysis: Data Review” on page 23), a warning message will be displayed each time a run is double-clicked in the Navigation Table that the individual method/sequence method does not exist. As outlined above, these methods are not stored with the data.
4 42 Workflow with Unique Folder Creation switched off Workflow with “Unique Folder Creation” switched off Data Organization
www.agilent.com In This Book With revision B.02.01 of ChemStation, data review and data reprocessing capabilities have been significantly improved to enable fast review of result data. The new data storage functions in ChemStation help to efficiently organize sequence data and methods.
