HPLC Manual 1.

0 10/29/01

General Notes:

1. Create a folder for your own files. From Windows, go to Main -> File Manager and
use the File-> Create new directory command to make a new folder. D:/ is a good choice
for user files.

2. When changing buffer composition and/or flow rate, change relatively slowly i.e. 0.3-
1 minute for a change. Although may be no noticeable damage from changing speeds too
fast, it seems that putting high pressure through the system over a short period of time
can’t be good for the system and may damage connections or other things. Speeding up
the pumps too fast is probably worse than slowing them down.

3. Make sure the waste bottle is empty so that no overflow occurs and likewise, make
sure that you have enough buffer to finish your experiment.

4. The Beckman HPLC has a four-way solvent selector so theoretically up to 8 different

solvents may be used. Only 4 lines are set up currently, however, it is better to physically
switch the inlet lines instead of using the solvent selector since there is some sort of bug
which causes the solvent selector to not work some of the time. The solvent may be
selected from direct control using the pull down menu to the right of a particular solvent.
Currently they are set at 1 but if you would like to use position 2, select 2 instead. If the
solvent selector light on the front of the pumps is lit then the selector is working
correctly, however if it is not lit then you may have to switch back and forth between
different selections to get the selector to work properly.

5. Guard columns should be used to protect the reverse phase columns from compounds
that will not desorb from the columns. These guard columns should be used for
analytical columns (4.6 x 100) and may or may not be used for the semi-prep columns.
Also, a precolumn filter should be placed upstream of the guard column. These are cheap
(~$10) relative to a guard cartridge and protect the column from particulate matter or
protein aggregates.

Turning on the Instrument:

1. Turn on computer and monitor

2. Turn on UV detector and Pumps (switches are located on the back of the instrument)

3. From DOS type “win” to load Windows 3.1

4. Load the HPLC software called Gold Noveau n the Chromatography folder
5. Click on Instrument 1
Preparation of the instrument for running:

Purpose of prep. is to remove bubbles from the lines of the pump and to prime the pumps
with buffer. This should be done if the HPLC has not been pumping or if switching
solvents.

1. Find the round silver valve, located to the right of Pump head A. Turn the valve
counter-clockwise (to your left) to open the flow to waste.

2. Find pump head B, located on top left section of the instrument. Find the three way
valve which should be labeled with “prime pump,” “operate,” and “prime pump.” The
valve should be set to “operate.” Turn the valve to “prime lines.” Using a 10mL
disposable syringe, pull about 5 mL of solvent into the syringe. Turn the valve to
“operate.” Remove all bubbles by flicking the syringe. Turn the valve to “prime pump.”
Push the solvent into the pump. Repeat 2-3 times. Remember to turn the three way
switch to “operate” when finished.

3. Repeate with Pump head A.

4. From the computer, open the direct control window. This can be done by clicking the
“control” button on the top of the instrument 1 window.

5. Turn the flow rate on

6. Start pumping with a target of 10 mL/min in 1 minute using 100% buffer B. When
washing the pumps, pressure should be relatively low (< 0.1 kpsi and should be around
0.037 kpsi), if not then the pump frit may be blocked because of crystallized salt (See
below).

7. Pump at 10 mL/min until all bubbles are removed.

8. Decrease flow rate to 3 mL/min in 0.5 min.

9. Change to 100% Buffer A in 0.5 min.

10. Increase flow to 10 mL/min in 0.7 min using 100% Buffer A.

11. Pump at 10 mL/min until all bubbles are removed.

12. Decrease flow rate to 0 mL/min

13. Turn the round silver valve (from step 1) clockwise (to the right) to connect the
pump to the column.
14. Start pumping at the target flow rate. For a Semi-prep hydrophobic column, flow
rate should be 4mL/min and pressure should be <1.5 kpsi with 100% buffer A and around
0.9 kpsi with 100% buffer B. For an analytical hydrophobic column, flow rate should be
1 mL/min and the pressure should be <1.1 kpsi for A and 0.8 kpsi for B.

Shutting down the instrument:

1. Equilibrate the column in storage buffer. If using a C8/C18 or hydrophobic column,

use 100% Buffer B. This column SHOULD be washed with 100% buffer B at the end of
the day to remove adsorbed material. If using an ion exchange column, equilibrate the
column with the low salt buffer, i.e. buffer A.

2. *IMPORTANT* If using high salt buffers (i.e. if doing ion exchange) wash the
pumps out with 100% ddH2O to remove salts from the pumps, otherwise the salt has a
tendency to clog the pump frit and increase pressure. To do this, remove the Buffer A
and Buffer B lines and place them in a container of ddH2O. Pump at the normal flow
rate for 10-15 minutes.

3. Set flow rate to 0 mL/min.

4. Quit Gold (computer program), then turn of the pumps and detector. If done in the
opposite order, error messages may result.
Changing the Column:

1. Prime the pumps and lines with the desired buffer.

2. Remove the column and guard column.

3. Pump at 1 mL/min to equilibrate the lines leading to the column with buffer for a few
minutes.

4. While pumping, connect the front end of the column

5. Allow the column to pump through to remove air, then connect the end of the column

Changing the Injector Loop:

1. Place the injector to the load position.

2. Using the 1/4 in wrench, unscrew the loop from the injector.

3. Place the new loop with the black mark facing up and secure
Injecting a sample:

NOTE: Samples should be filtered through a 0.45 or 0.2 um Spin-X filter before
injecting to keep particulate matter from clogging the column.

1. Click the preview button (this is to insure that the HPLC does not start running a
gradient – if this is not done, the computer thinks that you are injecting a sample and will
start running the last method in memory).

2. With the injector in the load position, wash the injector with ddH2O using the syringe
with the white cap.

3. Switch to the inject position and wash again with ddH2O.

4. Switch to the load position and wipe any spilled water with a kim wipe.

5. Press stop to stop the preview mode.

6. Click on single run to start. Enter necessary information and choose the method and
file name.

7. Hit start and the computer will prepare for a run.

8. Wash the injection syringe with water. Suck water into the syringe and expel in a
waste container. Wipe the tip of the syringe to remove any drops of water that may be
left. Repeat 5-10 times.

9. Take the appropriate volume of sample into the syringe and remove any air bubbles.

10. Check the computer to make sure that it is ready for the run. The bottom of the
screen should be flashing “waiting for trigger.”

11. Inject the sample, and quickly flip the injector to the inject position. Flipping is best
done quickly to insure good reproducibility/peak shape.
Making Buffers:

1. Filter the HPLC solvents using the 0.45 um filters and the glass filtering apparatus.
Use the trap in the hood with the centrifuges.

2. No degassing of solvents is necessary; the pump uses a high-pressure mixing chamber.

Cleaning the Column: (C18)

Not all steps are necessary to clean the column. Usually steps 1 and 2 are sufficient to
clean the column. Step 3 may be used fairly infrequently while 4 and 5 should be used
very rarely. Note: these recommendations partially taken from the Vydac Column Care
manual.

1. 100% Buffer be should at the very least be run through the column for 5-10 minutes
at the end of the day. It is usually good practice to wash the column after every run,
although this does lengthen the run time.

2. Run blank gradients (i.e. no injected sample) which seem to remove material better
than running 100% Buffer B through the column. Run 2-3 blank gradients going from 0-
100% Buffer B in 10-15 minutes.

3. Inject 500 uL of 1% SDS at the normal flow rate and then run a blank gradient as
above. Usually more than 1 blank gradient is necessary to properly remove all the SDS.

4. Use DCM or Chloroform to wash the column. However, these solvents are not
miscible with water so an intermediate solvent must be used. Pump 100% isopropanol
through the column at a low flow rate for 5-10 minutes (about 1-2 mL/min for the semi
prep column – just make sure the pressure is not too high) followed by 100% DCM for 5-
10 minutes followed by isopropanol again for 5-10 minutes. Acetone can be used instead
of isopropanol. Use good laboratory practice! Don’t put the pump lines in a solvent
bottle and return that bottle to common usage.

5. Use 20% 0.1 N Nitric Acid/80% isopropanol to wash the column overnight at 20% the
normal flow rate.

6. For high back pressure, reverse the column and pump at a low flow rate (50% of the
normal flow rate) followed by a higher flow rate (150-200% of the normal flow rate).
Again, make sure the pressure is not too high.

7. Check the guard column if pressure is high. The cartridge may need changing.
Using the Fraction Collector:

In theory, the fraction collector and computer should work together, however the relays
to the computer are not set up currently. In practice, it is easy to just collect time
windows by programming the collector manually. See the collector booklet for more
details. One may use program 1, which will collect time windows of 30 seconds, and edit
the start and stop times as well as the number of tubes collected. To run the program
press “Start,” and when injecting your sample press “proceed.”