Sharma, Manju 1-2

Quick Guide to Using the Agilent 1100 HPLC

Starting the HPLC:

1. Make sure all solvent bottles to be used are full
a. 8mM sulfuric acid (solvent bottle D) is used for acetate, fumarate,
maleate, and succinate analysis
b. Solvents should be vacuum filtered before being used
2. Turn on a the compartments on the HPLC
3. Open chemstation program - Instrument 2 Online
a. If the program is turned on before the instrument, the computer will
not recognize it.
4. Turn on system in the Chemstation program
5. Go to “Instrument”, “System On’
6. Open the purge valve on the HPLC
a. Black knob in the middle of the pump cabinet window
7. Adjust the pump flow to 5 mL/min
8. Click on the second arrow on the screen representing flow rate
9. Select “Set Up Pump”
10. Adjust solvent levels in the program
11. Click on the drawing of a bottle and fill out the volume for bottles in use
12. Allow the system to purge the solvent for about 5 minutes
a. This may help eliminate any air bubbles that may have been trapped in
the solvent lines while the instrument was inactive
13. Close valve
a. DO NOT tighten
14. Adjusts flow rate to 0.5mL/min
a. Pump pressure will be at about 70 bars
15. Let the system equilibrate for at least 20 minutes
a. The baseline should be a continuous straight line

Creating and Running a Sequence:

1. Under the “Sequence” menu, select “New Sequence”
2. Select “Sequence Table” in the same menu
3. Fill out the table with the names, methods, and injection/sample and sample
type info
4. For a long list of samples use the “Insert/Filldown wizard”
5. Go to “Sequence Parameters” in the “Sequence” menu
6. Assign a subdirectory and new name to your date
a. If you omit this step you will be overwriting older files
7. Save your sequence
a. Go to “File”, then “Save as”, then “Sequence” and give a new name to
your sequence
b. Usually the same name used in “sequence parameters”
8. Load your samples into the sampling try
a. Samples should be in a 2mL wide top crimp or screw-top vial with
septa
 Sharma, Manju 2-2

9. Make sure the tray is replaced correctly or else you will not be able to run
your sequence
10. Click on the “Start” button to begin analyzing your samples
11. You will be asked whether to save changes made to the method
12. Choose YES
13. Fill out the log book!

Creating/Editing a Method:
1. Load the method you would like to edit or select “New Method” in the
“Method” menu and follow the instructions
2. In the “Method” menu select “Edit Entire Method”
3. Adjust your chosen parameters and click “OK”
4. Repeat until you have no more options you wish to change
5. Save your method as a new file

Processing Data from Sequence:

1. Open Chemstation offline (Instrument 2 Offline)
2. If it shows the calibration table click ok
3. Go back to batch and click “output, batch report”
4. In the “Batch” menu select “Load Batch”
5. Choose the batch file name of interest and click “OK”
6. Select the data file(s) you would like to process
7. Click “start”
8. You data will be analyzed with the standard curve saved in the method
a. If you do not have a standard curve, create one
i. See short reference manual in HPLC gray folder sitting on the
computer, page 30-32

Notes:
1. Always refer to the manual if you’re not sure of something.
2. If the computer is acting weird and you did everything right, reboot the
computer.
3. Always fill out the log book when you use the HPLC.
4. Do not change a method that does not belong to you.
a. Always save your method changes to another file.