Aqueous GPC Instructions - Detailed

Aqueous GPC Instructions - Detailed Waste Bottle Columns PL Datastream Injection Port GPC Computer UV Detector GPC Pump Eluent Bottle The Aqueous GPC Set Up RI Detector Important The points below must be observed: Please check and fill in the GPC queue sheet before you use this instrument. Fill in the log book every time you use this instrument. The GPC pump must never be allowed to run dry, and must not be left running overnight. All sample solutions must be filtered before being injected into the GPC, using 0.2 µm nylon filters. Samples must form a stable solution in the analysis conditions used, to prevent solid deposits forming in the GPC columns and blocking them. The UV Detector must be turned off when not in use to preserve the life of the lamp. Make sure the eluent is properly degassed to prevent air bubbles forming in the filter and being pumped through the column. Either degas the eluent every few days or make up fresh eluent. Flush the piston and piston seal areas at the end of each day with at least 50 ml deionised water, using a syringe or wash bottle connected to the flush tube. Peter Iddon Aqueous GPC (Detailed).doc Page 1 of 8

Eluent and Sample Preparation Eluents/solvents must be degassed before use. Aqueous solutions should be stirred under vacuum for 1 hour. Co-solvents (e.g. methanol) are best degassed in bulk, added to the degassed aqueous solution, and then the mixture degassed for an additional 10 minutes. Samples are normally prepared at a concentration of 5 to 20 mg polymer per ml eluent. It may be necessary to adjust the ph of a sample solution to match that of the eluent. All samples must be filtered through a 0.2 µm nylon filter before injection into the GPC. Samples must form a stable solution in the conditions used. Turning on the Aqueous GPC Pump Display Pump Purge Valve The Pump Controls 1. Make sure the waste bottle is empty, and that there is enough eluent in the eluent bottle to fully cover the filter for the whole time the pump will be running. 2. Turn on the GPC pump (at the back on the left) and PL Datastream (at the back). The RI detector should already be switched on (it should not be switched off). 3. Turn on the GPC pump by pressing FLOW CTRL, then YES on the pump control panel. The current flow rate and pressure will be displayed on the pump display. 4. If necessary alter the flow rate by pressing FLOW, then entering in the new flow rate using the number keys, then pressing ENTER (pump control panel). 5. The GPC should be purged for 1 hour at the start of each day it is used. Once the pump has been started, press the PURGE button on the front of RI detector (a green light will show). Press this button again at the end of the hour to turn the purge off. 6. Turn on the computer, and the 7. Turn on the UV Detector if required. 8. Open the Datastream Monitor program by either clicking on the desktop shortcut icon or by going through the Start menu (Start > Programs > PL Cirrus > Datastream Monitor). 9. Click on the Channel menu and select Add Channel. 10. Select PL Datastream on COM2 and RI (and also UV if required). Click on OK. 11. A real-time display of the detector output will now be displayed on the monitor. Peter Iddon Aqueous GPC (Detailed).doc Page 2 of 8

Running Samples 1. Fill in the Aqueous GPC Log Book, entering in the details for the current run. 2. Open the GPC Online program by either clicking on the desktop shortcut icon or by going through the Start menu (Start > Programs > PL Cirrus > GPC Online). 3. Either select an existing workbook from the Recent list, or select Other and click on OK to browse the workbook folders. If you need to create a new workbook, follow the instructions in the Creating a New Workbook section. 4. Click on OK to verify that the PL Datastream is ready for data collection. 5. If using both the RI and UV detectors, you will have to use two separate workbooks (one for each detector). 6. Click on the Runlist icon on the left side of the screen. 7. Click on the Add Sample toolbar button or select it from the Runlist menu. Keep on adding samples until there are enough entries for all the samples to be run (more sample entries can be added later if necessary). 8. Enter information into the following columns (the important columns are highlighted in bold): Batch Name The program will use the date as a batch name if you leave this blank. Sample Name Necessary to remember which chromatogram is which! File Name The program will create its own if you leave this blank. Sample Type Choose Unknown for sample analysis or Narrow Std. for calibrations. Method Choose the method (containing the calibration) to use for data analysis. If you have chosen Narrow Std. as the sample type a new calibration will be added to the method you choose. Cal. Version Will be Use Latest for unknown samples, or Create for calibrations. K Mark-Howink-Sakurada equation coefficient. Alpha Mark-Howink-Sakurada equation coefficient. Once a sample had started running its details will be saved (and cannot be changed), but sample type, method, K and Alpha can be varied during reanalysis using GPC Offline. 9. Check the baseline is flat using the Datastream Monitor program. If is not flat, read the Common Problems section. 10. Follow the instructions in the Sample Injection section below. 11. When the data collection time exceeds the time specified in the method being used, or collection is manually stopped, GPC Online will automatically stop collecting data. It will then be ready for the next sample, if one exists. Chromatograms are saved to file automatically. 12. To analyse the chromatograms follow the instructions in the Data Analysis (GPC Online) section. 13. The data collection will begin again automatically when the next sample is injected. Sample Injection The Sample Injection Port in the Left and Right Positions Peter Iddon Aqueous GPC (Detailed).doc Page 3 of 8

1. Make sure that the PURGE button on the RI detector is switched off (no green light). 2. Press the AUTO ZERO button on the RI detector. 3. Zero the UV detector if using it. 4. Make sure the sample injection port is turned to the left position (see diagram above). 5. For the first sample, rinse the sample injection port with about 1 ml eluent (making sure there are no air bubbles in the syringe). The eluent passes into the small bottle behind the sample injection port empty this if it becomes full. 6. For the first sample, rinse the 100 µl micro-syringe 3 times with distilled water. 7. Rinse and fill the micro-syringe with the sample solution, until there are no air bubbles visible. 8. Inject 100 µl of the sample solution into the sample injection port (being careful not to introduce air bubbles into the system). Do not remove the syringe. 9. Holding the syringe in the sample injection port, turn the sample injection port to the right position (see diagram above). Continue to hold the syringe in the port. After the sample has been flushed from the sample injection port, turn the sample injection port back to the left position, then remove the syringe (the time taken to flush the sample from the sample injection port will depend on the pump speed; allow about 12 seconds [0.20 minutes] at 1 mlmin -1 and about 24 seconds [0.40 minutes] at 0.5 mlmin -1 ). 10. When the sample injection port is moved to the right position, the data recording is started automatically. The Datastream Monitor output will change from blue to red, and will show the time since the sample was injected. 11. Rinse the 100 µl micro-syringe 3 times with distilled water or eluent, and leave with the barrel out. 12. Rinse the sample injection port with about 1 ml eluent (making sure there are no air bubbles in the syringe). Creating a New Calibration Calibrations are specific to individual workbooks, but identical calibrations can be created in other workbooks (using the same data) by importing the calibration chromatograms. Multiple calibrations can be created from the same chromatograms if necessary. 1. To create a calibration from narrow standards as you inject them, follow the instructions in the Running Samples section and then the Data Analysis (GPC Online) section. 2. Alternatively, calibrations can be created from previously run standards. Follow the instructions in the Analysis Using GPC Offline section to do this. Chromatograms run in other workbooks can be imported by clicking on Import Chromatograms in the File menu. Data Analysis (GPC Online) Samples can be analysed immediately after they are run using GPC Online, or analysed later using GPC Offline. 1. Once a chromatogram has been recorded, it can be analysed. 2. Click on the Analysis icon on the left side of the screen. 3. If there is no chromatogram waiting to be analysed, click on the Next Analysis toolbar button, or select it from the Processing menu. Depending on the options chosen in the method being used for analysis, the peaks may be assigned and results calculated automatically, or it may be done manually. 4. If analysing Unknown samples, peaks are assigned by clicking on the Detect Peak & Baseline toolbar button, or by selecting it from the Analysis menu. Click and hold the mouse pointer on one side of the peak to be analysed (on the baseline), then drag the baseline across to the other side of the peak. Peter Iddon Aqueous GPC (Detailed).doc Page 4 of 8

If analysing Narrow Standard samples for column calibrations: Peak maximums are assigned by clicking on the [Detect] Peak Max toolbar button or by selecting it from the Analysis menu, then clicking anywhere inside the peak. Once peak maximums are assigned, click on the Add to Calibration toolbar button or select it from the Processing menu. In the Assign Peak Molecular Weights window enter in the M p values for the standards used, next to the corresponding peak value (highest molecular weight standard will elute first). Then click on OK. 5. Peak parameters can be edited by clicking on the Move Markers [and Zoom] toolbar button, or selecting it from the Analysis menu. Move the mouse pointer and click under the baseline to move the baseline (pointer will change into a B) or move the mouse pointer and click above the baseline to move the peak limits (pointer will change into a P). Peaks can also be deleted by clicking on the Delete Peak toolbar button or by selecting it from the Analysis menu, then clicking anywhere inside the peak. 6. Once the peak assignments are satisfactory, click on the Calculate Results toolbar button, or select it from the Processing menu. 7. Results can be viewed by clicking on the Results icon on the left side of the screen. 8. Results files are not saved automatically. To save results, select Save Results from the Results menu. Chromatograms are saved automatically when the results are calculated, along with any peak/baseline assignments. 9. Results can be printed by selecting Print from the File menu (when in the Results view). 10. Once chromatograms are analysed, the analysis cannot be repeated you must reanalyse the file using GPC Offline (see the Analysis Using GPC Offline section below). Analysis Using GPC Offline Samples can be analysed at a later date using GPC Offline. To get exactly the same results as last time: If the sample peaks have been assigned previously, then the peak assignments will have been saved with the chromatograms, so the reanalysis of the peaks without altering any of the peak parameters (baseline and peak limits) will give the same results as the last time they were calculated (as long as the correct column calibration file for the date the samples were run is also used). 1. Open the GPC Offline program by either clicking on the desktop shortcut icon or by going through the Start menu (Start > Programs > PL Cirrus > GPC Offline). 2. Either select an existing workbook from the Recent list, or select Other and click on OK to browse the workbook folders. If you need to create a new workbook, follow the instructions in the Creating a New Workbook section. 3. Click on the Runlist icon on the left side of the screen. 4. Select the chromatogram files to be analysed from the middle file window. Double clicking on a batch folder (named from the Batch Name property) adds all the chromatograms with that batch name. 5. It is possible to import files from other workbooks. This option can be found in the File menu (File > Import Chromatograms > Cirrus ). These files are then stored in the workbook in the Imported batch folder. 6. Check the information into the following columns is correct and amend as necessary (the first three columns cannot be altered; the other important columns are highlighted in bold): Batch Name The default is the date the samples were run (unless changed by user). Sample Name The default is the date and time the samples were run (unless changed by user). File Name The default is the date and injection number (unless changed by user). Sample Type Choose Unknown for sample analysis or Narrow Std. for calibrations. Peter Iddon Aqueous GPC (Detailed).doc Page 5 of 8

Method Choose the method (containing the calibration) to use for data analysis. If you have chosen Narrow Std. as the sample type a new calibration will be added to the method you choose. Cal. Version Will be Use Latest for unknown samples, or Create for calibrations. You can choose to analyse files using older calibrations if you wish. K Mark-Howink-Sakurada equation coefficient. Alpha Mark-Howink-Sakurada equation coefficient. Any altered information will be for the current analysis only, no original information in the chromatogram file will be changed. 7. Now follow the instructions in the Data Analysis (GPC Online) section above. Creating a New Workbook Use a new workbook when using different run conditions or a different polymer. It would be best if users had their own folder in the Cirrus Workbooks folder (shortcut icon on desktop) into which they could put their different workbooks. It is possible to move workbook folders after they are created if necessary. 1. Open the GPC Online program by either clicking on the desktop shortcut icon or by going through the Start menu (Start > Programs > PL Cirrus > GPC Online). 2. Select Create a New Workbook and click on OK. 3. Click on the Default workbook icon, and click on OK. 4. Enter in a name for the new workbook next to Name. The save location of the workbook can be altered by clicking on browse (i.e. it can be placed inside your own folder). Information can be entered under Experimental Title and Comments in this step or the next step. Click on OK. 5. The workbook options window will now be displayed, showing five tabs. Enter in the following details by clicking on each tab (the important information is highlighted in bold): Workbook Enter information under Experimental Title and Comments if not already done so. Conditions A warning will appear when you first select this tab just click on OK. It only warns that Channel 1 is selected by default. Click on Edit in the Detectors section and for the Channel Number select RI(2) (for the RI detector) or UV(1) (for the UV detector). Make sure PL Datastream Input Voltage is set to 1.28 V and PL Datastream Mode is set to Input Data. Enter in details in the Pump and Column Set sections if desired. Data Collection Change Run Length to that required for your experiments. Other details here can be ignored. Instruments Instrument details can be entered if desired. Options Options here can be ignored. When all the details have been entered correctly, click on OK. This information can be edited at a later date. 6. When asked, click on Yes to ready the PL Datastream for data collection (the PL Datastream can be made ready by pressing F8 or selecting the option from the Collection menu if you press no by mistake). 7. Click on OK to verify that the PL Datastream is ready for data collection. 8. Edit or create the methods by following the instructions in the Editing or Creating a New Method section below. Peter Iddon Aqueous GPC (Detailed).doc Page 6 of 8

Editing or Creating a New Method You can either use one of the default methods for your calibration, edit an existing method, or create an entirely new one. Editing the default method Manual Analysis is recommended. 1. Click on the Method Browser icon on the left of the screen. 2. Select Edit Method or New Method from the Method menu. 3. The workbook options window will now be displayed, showing six tabs. Enter in the following details by clicking on each tab (the important information is highlighted in bold): General Name the method. Do Data Processing should be ticked. Time Between Chromatograms is the time delay between the completion of one analysis and the start of another (most relevant when automatic processing is used). Column Calibration Generate Using Narrow Standard Calibrants should be selected. Choose the default order of the polynomial to use for calibration. Select whether the calibration will have automatic or manual processing (Manual Processing without Automatic Peak Detection is recommended). Sample Analysis Select whether the sample analysis will have automatic or manual processing (Manual Processing without Automatic Peak Detection is recommended). Perform GPC Calculations should be ticked. Choose the Calculation Method (See Help for more information). Peak Detection Can change the limits and threshold options for automatic peak detection, if it is being used. Reporting Options here can be ignored. User Programs Options here can be ignored. 4. When all the details have been entered correctly, click on OK. This information can be edited at a later date, although editing the default polynomial order doesn t change the calibrations that have already been done. Changing the Column(s) and/or Eluent When putting on two columns in series, the higher molecular weight range column goes first. Before removing a column, purge it thoroughly with degassed double distilled water. Make sure no air gets into the column or tubing when removing a column from the GPC. When putting a new column onto the GPC, make sure no air gets into the tubing or columns. Purge it thoroughly with the eluent (until a stable baseline is seen with the Datastream Monitor program). Make sure the flow rate and pressure are not too high for the columns used. When changing the eluent, make sure the columns are properly purged with the new eluent. Shutting Down 1. Make sure the micro-syringe has been rinsed out with distilled water, it is empty, and the barrel left fully out after use. 2. Make sure the 2 ml syringe used to rinse out the sample injection port has been rinsed with distilled water. 3. Press FLOW CTRL, and then YES. 4. Shut down the computer. Peter Iddon Aqueous GPC (Detailed).doc Page 7 of 8

5. Switch off the computer, GPC pump, PL DataStream, and the RI detector. 6. The UV detector must be turned off to preserve the life of the lamp. 7. Flush the piston and piston seal area with at least 50 ml deionised water. 8. If you have finished analysing samples, please tick the box on the GPC queue sheet. More help with Cirrus software is needed Workbook does not exist error occurs Small & sharp negative peaks appear occasionally Air bubbles have formed in the pump or tubing PRESSURE FAULT is showing on the pump display Large numbers of air bubbles are forming in the eluent filter The baseline is not flat Extra peaks appear too early or too late The wrong order polynomial has been used for the calibration No data appears to have been collected after sample injection Common Problems For more detail on using the Cirrus software, either refer to the instruction manual, or press F1 at any time while using GPC Online or GPC Offline to show help information relevant to the current task. All the information in the instruction manual can be accessed through the Help menu. If a program says that a workbook does not exist when you click on one from the Recent list, try browsing for the workbook by clicking on the File menu and selecting Open. This can happen if workbook folders are moved or reorganised. This is normal, and happens when the PL Datastream is checked by the software, such as when GPC Online is opened. If air bubbles have formed in the pump or tubing, the system can be purged by first opening the pump purge valve (shown on the diagram of the pump controls). Press PURGE, then ENTER on the pump control panel. The pump will now pump at a high rate, and the eluent will emerge from the outlet just under the valve. To end the purge, press ESC on the pump control panel, and close the pump purge valve. This is usually due to air bubbles forming or being trapped in the pump head. Follow the instructions in the previous paragraph, then press ESC on the pump control panel to cancel the error message. If bubbles are continuously forming in the eluent filter, either degas the eluent again or make up fresh eluent. If this does not work, clean the solvent filter in 6M nitric acid in a sonic bath for 10 minutes (rinse very thoroughly afterwards). Check that the PURGE button on the RI detector is switched off. Make sure the detectors are zeroed before every injection. If the GPC has been on for a long time, it may be necessary to purge the RI detector for about 5 minutes. If extra peaks or a poor baseline appears after the run has finished, or before any sample is expected to elute though the columns, there may be retained polymer, monomer, solvent or salt. Either allow extra time in the method for all the components to elute properly, or change the eluent conditions to reduce adsorption of components to the columns. The polynomial order has to be set before the calibration is created. Change the setting (as described in the section Editing or Creating a New Method), then create a new calibration using GPC Offline (using the same data). Check that the PL DataStream has been turned on. Check that the PL DataStream is ready for data collection (by pressing F8 or selecting the option from the Collection menu). Last Updated: 1 July 2005 Peter Iddon Aqueous GPC (Detailed).doc Page 8 of 8