Setting Up and Running the HPLC at Sweet Briar College

HPLC System

 
Pumps: Three Waters 510 HPLC Pumps

Detector: Waters 996 Photodiode Array Detector

Software: Millennium Version 3.20 
 
 

Solvents

*Use freshly filtered solvents each day. 
 

      *Discard ultrapure water and replace with new ultrapure water. 
 

Filtering Solvents: Use 1000 mL flask 
 

1. Rinse flask with solvent to be filtered.
2. Put top on flask. Add filter (re-use them until dirty). Filters are expensive.
3. Vent trap before turning water off.
4. Rinse target flask with filtered solvent.
5. Then transfer solvent to target flask and cover with parafilm.
6. Dismantle filter.

Water:  Use ultrapure water 
 

1. Rinse flask with ultrapure water several times.
2. Then fill flask with ultrapure water.

Priming the Pumps and Setting up the Detector

Priming the Pumps

1. Unplug “black box” above the Pump Control Module (located to the right of Pump C) to switch from computer control.
2. Place all filters in methanol.
3. Set flow on all pumps to “0.0”.
4. Turn pumps on.
5. Turn reference valve on unit to the left of Pump A to “floppy” position (i.e., turn to the right).
6. Insert syringe into outlet valve on Pump A. Then unscrew the outlet valve. Pull out air bubbles in line. Close outlet valve. Empty syringe and redo until no air is in the line. Turn pump up to 0.4 flow rate. Do this for all pumps.
7. Keep flow rate at 0.4 on Pump A. Withdraw a syringe full of methanol. Remove syringe and invert it. Press in to remove air bubbles.
8. Keep pressure on syringe as you insert syringe into outlet valve of Pump A. Keep pressure on syringe as you open up outlet valve. Turn flow rate up to 9.9 mL flow rate and slowly with constant pressure inject at least 8 mL of methanol into Pump A. Watch outlet stream to see that pulsing has stopped. Leave at 9.9 mL.
9. Repeat Steps 9 and 10 for Pumps B and C.
10. Turn all pumps to 1.0 mL flow rate.

Setting Up Detector 
 

1. Prime pumps first with methanol as outlined above.
2. Then set the flow rate to 0.0 on all pumps. Turn reference valve on unit to the left of Pump A to left so that methanol will go through column. Then gradually increase the flow rate on Pump A to 2.0 mL. You should get a steady stream and have no air bubbles.
3. After » 15 minutes, turn on Waters 996. Wait a little and listen for “horses.” If status light is blinking, turn off and then back on immediately. Usually stops blinking. Leave frits from any unused pumps in methanol. If there are “leaks” in the column fittings, they must be eliminated before the Waters 996 detector will work properly.

Changing Pumps from Methanol to Desired Solvents 
 

1. It is conventional to put most polar solvent in Pump A and least polar in Pump C.
2. Always leave frits in solvents while pumps are running.
3. After all pumps are primed with methanol, methanol is going through the column, and the detector is ready, proceed as follows to change over to different solvents:

1. Set all pumps to 0.0 flow rate (but leave them “on.”) You can reduce the flow rate to 0.0 in one step.

2. Carefully pull out the frit of the pump you want to change. Shake off the frit. DOUBLE CHECK THAT YOU HAVE THE CORRECT ONE!! Stick the frit into the desired solvent. Put parafilm over the bottle top to hold down the frit.
3. Open up the outlet valve on the pump getting new solvent. Pull solvent through slowly (to avoid getting bubbles in pump). Pull three full syringes through.
4. Gradually increase the flow rate for the pump. The pump should still have a good (steady) flow rate (with no “beats”).

4. Repeat the step above to change over the solvent of other pumps.

5. If not ready to start, put all the frits in methanol and leave methanol flowing through the column at a 0.1 mL/min flow rate.

Creating Instrument Methods

For an HPLC experiment you will need three methods:

• Equilibrate Method. This method takes the column from its initial condition after priming with pure methanol to the initial solvent mixture specified for your run. It is created as described below. Often it is convenient to run this method over lunch to get the column ready for an afternoon run.
• Run Samples Method. This is the method you create to run your sample.
• Shut Down Method. See p. 5 ahead. This method takes the column from where you left off and leaves it equilibrated with methanol.

Directions for writing the first two methods are described below. 
 

1. Double click on the Millennium icon on the Desk Top to Login to Millennium.
2. Choose “Training” from the “Project” menu (if it is not already selected).
3. Double click on “Run Samples.” “HPLC at Sweet Briar” should be highlighted in the “Chromatographic Systems” window. Click on the OK button.
4. Select an Equilibrate Method from the “Instrument Method” tab similar to the one you are going to create. Then EDIT the method. Save your new method under a new name. When making up your method, follow these steps:
   1. Depress the PCM (Pump Control Module) button at the top of the screen (if it is not already depressed).
   2. From line to line in the table, change only one variable at a time.
   3. For your Equilibrate Method, you usually begin with 100% methanol going through the column at a slow rate from one pump (i.e., Pump C)
   4. Next increase the methanol flow rate to 1.0 mL/min. Can do this at clock time = 1 (min). Use default (6) for last entry on the right of table.
   5. Next choose longer clock time and set to change the solvent over towards your desired running mixture. Allow 30 to 60 minutes.
   6. Let final running mixture run through column for 20 minutes at 1 mL/min (or the equivalent).
   7. Add a final line to most methods with a very long time and a very slow flow rate. This protects against running the pumps dry.
   8. Depress the 996 PDA button at the top of the screen, and make any changes needed. In particular, be sure that the beginning and ending wavelengths are appropriate for your analysis.
   9. Save your Equilibrate Method under a new name.

5. Next create a Run Samples Method using a similar procedure to that described above. Begin the method with the solvent mixture with which your Equilibrate Method ends, proceed through whatever gradient you wish, and end by equilibrating the column with your initial solvent mixture so you are ready to begin another run. You create this method in a similar manner to that described above for the Equilibrate Method. However, you will also need to set up an “Event:”
   1. To set up an “Event,” click on the Event tab. Then click on the first line of table to set the default event. You will need an Event set for any method used to run samples.
   2. To establish an event, under “Event” click on “Event 1.”  Set “Function” to “Pulse.” The interval should automatically be set to 0.02.
   3. Save the method and close the window. This will bring you back to “Run Samples” mode.
   4. [New addition. May need revision or clarification] Click the “Develop Methods” button. Select your method name and click “Next.” Click “Next” again (no processing). Name your method. Click “Finish.”

Filling the Syringe

Sample Volume

The sample loop on our system holds 50 mL. For quantitative work it is best to completely fill the loop. To do this, use a 55 mL sample. 
 

Filling the Syringe

Syringe cannot have air bubbles! 
 

Clear with ultrapure water or filtered methanol. Fill it up just past where you want. Turn upside down. Pull plunger down. Tap if air bubbles. Push back till bubble of liquid comes out (not a sputter) 
 

Last of all, adjust volume down to desired injection volume. 
 

Equilibrating the Column and Running Samples

Follow the directions given below. Except for Step 12 (see below), your Equilibrate Method is run just like your Run Samples method.

• First run your Equilibrate Method (over lunch) to switch the pumps from running 100% methanol to running your initial solvent mixture, and to equilibrate the column. Once your Equilibrate Method has been run (after lunch), hit the “Stop Flow” button (lowest button on left at bottom of screen) and the “Abort” button (fifth button from left at top of screen) to stop the Equilibrate run.
• Next run your actual sample. Skip steps 1-5 and go to step 7 again to choose your Run Samples Method.

1. Have Waters 996 Photo Diode detector on. Both lights should be green and not blinking.
2. Leave pumps on, but set flow rate back to 0.0.
3. Plug in “black box” above control module (located to the right of Pump C) to switch to computer control. The green light on the Waters Pump Control Module (PCM) should go on.
4. Be sure reference valve on unit to the left of Pump A is turned to left so solvent flows through column.
5. Login to Millenium. (Note: If you are already logged into Millenium, Logout and Restart the computer; then Login to Millenium again.)
6. Double click on “Run Samples.”
7. Near the bottom of the “Run Samples” window, select the “ Single” tab in the “Single,” “Samples,” “Sample Set” line.
8. Click the “Develop Methods” box in the main window. Select the method which you want to run. Click “Next.” Click “Next” again (no processing). Then click “Finish.
9. Enter the “Sample Name”, “Volume” and “Run Time” in the main window.
10. Select the “Samples” tab in the “Single,” “Samples,” “Sample Set” line. In the first line of the window, fill in the sample name and injection amount; in the “Function” column select “Equilibrate”  (if running an Equilibrate Method) or “Inject Samples” (if running a Run Samples Method or a Shut Down method). Highlight the relevant row. Then select the “Single” tab again.
11. To run a sample, click “Prepare” (left hand inject-like-picture) under “Run Time,” and wait for screen to say that instrument is ready for injection.
12. Locate the Sample Loader/Injector (it is to the left of Pump A). Do one of the following:

• Equilibrate Method: Push in button on sample loader. Then immediately click on the “Inject” button that is located to the right of the “Prepare” button on the computer screen.

• Run Samples Method: Put injector in the (upper) “Load” position. Load sample in injector (or do that earlier). Push lever down to inject your sample, and simultaneously push in button on sample loader. Then immediately click on the “Inject” button which is located to the right of the “Prepare” button on the computer screen.

13. You can view data on the right of the screen during the run. Right click on a graph window to  “Customize Channels.” You can set number of channels to specific wavelengths (must reset for each run).

14. To end your run, hit the “Stop Flow” button (lowest button on left at bottom of screen) and the “Abort” button (fifth button from left at top of screen).

Reviewing Data

Data can be reviewed both during your run and after the run is complete. For detailed instructions, see Chapter 2, Viewing PDA Data, of the PDA Software Getting Starting Guide. It is recommended that you work through the tutorial presented in this chapter. Be sure to check out Section 2.5, Extracting a Chromatogram and Section 2.6, Extracting a Spectrum. 
 

The review process begins from the Millennium Login Window.

• From this window, right-click on the “Review Data” button. Then select “Review” from the context window and “Channels” from the cascade window.
• In the “Results Window”, highlight the row with sample name of interest.
• Click Review button above the “Channels” list.
• Pull out window hidden under contour plot.
• Click the “Extract Chromatogram Tool” which is the far left button in the row of buttons above the contour plot.
• Click the “Extract Spectrum Tool” which is the button second from the left in the row of buttons above the contour plot.
• You should now have time and wavelength indicator boxes below and to the right of the contour plot respectively. If you click in either of theses boxes and drag, the extracted spectrum for the time (wavelength) in the box will be displayed. You can also double click in the appropriate box to enter a time (wavelength).
• Click on the “Integrate” tool (sixth button from the left at the top of the window. This will integrate your peaks and display the retention times for each peak on the graph. To see a table of the peak areas and retention times, pull out the window hidden under this graph.

Shut Down Method

 1. Create and run a Shut Down Method which gradually transfers flow through the column from your final running mixture to pure methanol. (Note: If your run included a salt or buffer mixture, add a step to your method to run pure water through the column before the pure methanol.)

 2. Your Shut Down Method should let pure methanol flow through the column for 15 minutes at 1 mL/min (or the equivalent, i.e., a longer time at slower flow rate). It should include a final step with a very long time (500 min.) at a very slow flow rate (0.1 mL/min) of pure methanol.

 3. You can leave other pumps filled with their solvents.

 4. When Shut Down Method is complete, exit from Millenium. Set all pumps to a flow rate of 0.0 mL/min. Turn all pumps off.

 5. Unplug “black box” above PCM (pump control module located to the right of Pump C) to switch from computer control.

 6. Shake off frits and put them in their little bags. Cover the bottles with parafilm. 
 
 

Trouble Shooting

 1. Frits may be jammed. Remove and put in either ultrapure water or filtered methanol and sonicate for » 10 minutes.

 2. Must get rid of leak. Should detach with wrench and fit with new Teflon tape (small amount).

 3. Can use Kim-Wipes to help find leak).