GPC User�s Booklet

This handout will give you the minimum information necessary to use the Waters GPC system.� If you have further questions or if you experience any problems, please contact Jim Windak at 647-2847 (jwindak@umich.edu).

A.� Description of the Instrument

�� This instrument has a reservoir of clean, filtered and helium-purged THF, which is pumped by a constant volume pump through a sampling loop to three GPC columns for separation.� The columns are an HT-4, HT-3, and HT-2.� The HT-4 gives optimal separation only up to 600,000 MW.� However, we have calibrated the system up to 1,000,000 MW.� The column effluent then passes through a UV detector (set at 254nm) and a refractive index detector before exiting to a waste container.� In the standby mode, the effluent is returned to the source reservoir.

�� Data is acquired and manipulated with Waters Millennium 32 software on a Dell GX1 Optiplex computer.

B.� Standby Configuration

�� When you first come in to use the GPC system, you should find it in the standby mode.� This means the pump should be set to a flow rate of 0.1 ml per minute, the system pressure should be 40 psi, and the system effluent tube should be inserted back into the THF source flask.� If these conditions are not met, do not use the instrument, and inform one of the key operators so that the proper steps can be taken.

C.� Set up to Run

Before running the GPC, you should first check the solvent reservoir to make sure that there is enough solvent available for the number of samples that you want to analyze. If the level is less than 1000 ml or if you intend to run a lot of samples, please contact one of the key operators listed above. The filling procedure is somewhat more complicated than might first be assumed.

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Please Note: If solvent is needed for sample preparation, take it out of the pump before you increase the solvent flow rate.� If you try to remove solvent from the pump while the pump is at operating pressure, damage to the columns will result from the sudden surge in pressure.

In order to withdraw solvent for sample preparation, the 30ml gas tight syringe is inserted into the Prime/Purge port and the black knob is turned counter-clockwise to open the valve.� Withdraw the syringe plunger to fill it with solvent, then tighten the black knob before removing the syringe.� Repeat as necessary, in order to get enough solvent for your sample preparation.

To set up for operation, you must increase the solvent flow to 1.0 ml/minute very slowly.� Increase it in small steps of 0.1 ml/min, and wait at least 20 seconds between each step.�� When the solvent flow rate reaches 1.0ml/min. the solvent pressure should be approximately 450 psi.�

�� Next, push on the purge button on the front of the Optilab refractive index detector.� This allows the reference cell to fill with solvent.� Keep it in on for a few minutes until the reading stabilizes.� Then push the purge button again to turn it off.� Next, push the zero button and wait for several minutes until a reading of� �000� comes up on the display.

Also zero the UV detector using the offset knob.

�At this point the computer should be set up for data acquisition and baseline monitoring before the sample can be injected.

D. Data Acquisition

Normally, the computer will already be on when you sit down at the instrument. You need only move the mouse to bring the computer out of sleep mode, which takes 20-30 seconds.

After it comes up, you should see the login screen.� Enter your username & password, and click on OK.� Then the Millennium 32 software should startup automatically.� When Millennium is done loading, enter your username & password to log into Millennium.

The drop-down menu lets you select a project to use.� To run samples, double click the �Run Samples� button.� It will ask you to choose a Chromatographic System.� Choose the only one, �Waters 440 GPC�, and click on OK.� This will take you into the Quick Set data acquisition page.

The first step is to load the instrument method in the lower right hand box. �There is only one default instrument method available.� It is called �Waters 440 Default�.� Then click on the Setup button, and wait until setup is done.� The status line at the bottom of the page will say �setting up�, and when it is finished it will say �idle�.

Next, click on the Monitor button to monitor the baseline readings of the two detectors.� Note that you can click and drag the vertical divider in the box in order to get more viewing area for the detector monitoring graphs.�� Re-zero the detectors as needed.� When the baselines have become stable, click the red button to stop the monitoring process.

To prepare to inject your samples, fill in the sample information.� Note that you can click and drag the vertical divider to give this table more viewing room.� Click on the function column to get a drop down menu that can be used to select the type of sample you want to run, such as �Inject Broad Samples�.� Fill in a data file name, set the run time to 45 minutes, and set the injection volume to 75 ul.�� Also select a Method set.� There is currently only one default method set, called �Waters 440 mthd set�.� Click on the right-hand injection icon to start the injection process.� The status line at the bottom of the box will say that it is now waiting for the sample to be injected.

E.� Sample Preparation

�� Sample solutions must be made using solvent that has come directly from the GPC solvent source for best results.� Solvent for this purpose should have been withdrawn from the Prime/Purge port on the front of the pump, before you increased the pump flow to 1.0 ml/min.

Please Note: Do not remove solvent from the Prime/Purge port on the pump when the system is at its operating pressure of 450 psi, and 1.0 ml/min.� This could damage the columns.

�The solvent you initially withdrew should be used to make a solution of the sample at a concentration of approximately 1mg/ml.� A tiny drop of toluene is then added to the solution using the 25ul syringe.� This syringe is labeled �Toluene Only�.�� The sample solution must then be drawn into a 3ml disposable syringe and filtered though a 0.45 micron PTFE filter (Fisher Part No. 09-911-2) into a clean dust-free container. This is an important step that removes particulate matter from the sample to prevent damage to the system. The solution is now ready for injection and analysis.�� The disposable syringe must not have any rubber parts on it. .� Recommended is a Norm-Ject 3 ml syringe, part # A3, available from Air-Tite Products, Inc.� (1-800-231-7762).�

Please Note:� It is extremely important that the solvent reservoir not get contaminated.� Please transfer the effluent tube from solvent reservoir to the waste container after a stable baseline has been established and before you inject your sample.� Be sure to close the reservoir stopcock to keep air out of the solvent.

�� After the software is set up for data acquisition, the detector signals are stable and the solvent effluent line is transferred to waste, you are ready to inject the sample.� Click on right-hand injector icon.� 75 microliters(ul) of sample solution are drawn into the 250ul Hamilton syringe which is fitted with a square tipped needle.� Draw up more than 75 microliters, and then hold the syringe upright and push out some of the solution to expel any air bubbles.� Holding a Kim Wipe around the needle will prevent making a mess while doing this.� The load/inject lever is moved to the load position.� The injection plug-locking lever is released and the plug removed.� The syringe is inserted slowly and fully into the injection port and the plunger depressed.� Note that a few drops of solvent are displaced out of the tube to the right of the injection port.� The syringe is removed, the plug is replaced and the plug lock lever is returned to the locking position.� The load/inject lever is moved to the inject position, which automatically starts the acquisition of data.� The sample has now been injected and the results can be monitored on the computer screen.

****Please Double-check that the solvent effluent tube is now inserted into the solvent waste bottle.****

F. Data Review

To review sample data that has been collected, minimize the Quick Set acquisition window and double click the �Browse Project� icon.� Select a project and click on OK.� Click on the Channels tab in order to view data channels that have been injected.� Channels that are labeled �SATIN� are data that have been collected from the UV detector.� Channels that are labeled �SATIN-2� are data that have been collected from the RI detector.� Highlight the channels that you wish to use (you can CNTRL-Click to select more than one, or use Shift-Click to highlight a range of� them).� Then click on the Review icon.� Alternatively, you can click on the Tools menu, and then Review.� Alternatively you can also right click one of the highlighted files to get a menu, and then click on Review.� This will take you into a Review window.� Note that you can also go directly�� into Review from the Quick Set data acquisition window by clicking on the Review icon, to look at files that you have just collected.

�� The first step to do in the Review window is to open a processing method.� Do this by clicking on File � Open � Method.�� Select the processing method called �UV_mm_dd_yy� for SATIN UV channels, or select the method called �RI_mm_dd_yy� for SATIN-2 RI channels, and click on Open.� Use the most current calibration as indicated by the month, day & year.�� Now you can process the data.� Be sure that you are looking at the Main Window under the Window menu.� Click on the Integrate button on the toolbar to integrate, (or alternatively you can click on the Process menu and then on Integrate).� Next click the Quantitate button on the toolbar to assign molecular weights and calculate molecular weight distributions.� If you want to look at the tabular results, click on Results under the Window menu.� You may need to click and drag the horizontal dividers to provide enough room to view each of the tables.� Clicking on the tab at the bottom of this window called �Distribution Plot� will show the distribution plot data.� If you are happy with the results, you should click on File � Save � Result.� If you want to look at other channels in the channel table, you can click on the next channel button on the toolbar.� You will have to click on Main Window under Window in order to see the next chromatogram.

If you cannot get molecular weight distribution numbers on your broad unknown peak after integrating and quantitating, the problem may be that the peak is out of the default range entered in the chromatogram slicing table.� To correct this, first make sure that your processing method is open.� If not, do a File � Open � Method, and choose the proper processing method.� Next, click on the Window menu, and then click on �Processing Method� in order to view the parameters associated with this processing method.� Click on the tab called "Slicing�.� In the table, enter the approximate retention time of your broad unknown peak.� Then enter a retention time window value, such that your broad unknown peaks fall within the retention time plus or minus the window value.� Then click on File � Save � Method.� It will ask you whether you want to clear out or copy the calibration curves associated with this method.� Click on �Copy Curves�.� Now your processing method should give you a molecular weight distribution on your broad unknown peak when you integrate and quantitate.

G. Printing Reports

To print a complete report from results that have been generated, go back to the project window.� Click on the Results tab to view the result files.� Highlight the files you wish to print, and then click on the Tools menu, and click on Preview.� Alternatively you can right click one of the highlighted files and click on Preview.� This will launch the report editor. It will first ask if you wish to use the default report method.� Click on OK.� It will then show you how the report will look.� If you want to use this format, just click on the print icon.� You can use the Next Report button on the toolbar to view the next report in your list of results.

�� If you do not like the default report format, then click on Close, and use the report publisher to customize your report format.� You can simply click and drag items available from the left-hand side into your report.� Items in the report can be moved and resized as desired.� If you wish, you can save your custom report format as a report method.� To do this click on File � Save and give the report method a name.

H. System Shutdown

To shutdown the system, close all of the windows and items on the taskbar except for the

main Millennium window.� Then click on logout.� After you have logged out of Millennium,� do a File � Exit� to quit the Millennium program.� This should take you back to the initial user log-on window.� Once you see that window, you know that you are completely logged off.

�� Put the waste effluent tube back into the solvent reservoir.� Slowly decrease the flow rate on the pump back down to 0.1 ml/min.