Last updated 9-15-98 by Chih Long Liu HPLC Column Operating Guidelines

HPLC Column Operating Guidelines

HPLC Column Operating Guidelines Ver. 4.0; 9/14/1998

There are four components to the HPLC that require operation and need to be shut off at the end of the day:


The main unit is the pump. It is capable of accepting four different solutions in inlets A, B, C, and D. DO NOT USE C and D as those two inlets most likely are empty and will send air i nto the system (if the column is attached at this point, and if the pump is running, hit the "STOP" key and reverse the flow, or you may ruin the column!).

Remove line A from the 10% acetonitrile solution and insert it into the LSB. Do so similarly for line B and the HSB. Each line will have two termini; the white is the buffer inlet, and the blue line is a gas outlet available for sparging during a HPL C run (not necessary for cy dye purification). Cover the 10% acetonitrile bottles and the buffer bottles with parafilm.

At this point, you may attach the column (Dionex reverse phase anion exchange 9 ī 250 Ė 1.25 mL volume capacity; the pump is currently equipped with a 1 mL injection loop) onto the lines. Note the flow direction on the colum n. Have the pump start at a low flow rate (e.g. 0.2 mL/min), and let fluid drip out before screwing in the line to the columnís inlet (the line is the larger of the two beige screws). Wait until fluid begins to appear on the other end of the column befo re attaching the other line onto the columnís outlet. Monitor the pressure (indicator on upper right corner of pump LCD) for any fluctuations or unexpectedly low readings, which may indicate air or leaks in the system.

Equilibrate the column, following the general purge method in the protocol. This may have to be keyed in. Access the step-wise method by pressing the F2 key ("PUMP"), scroll down, and enter the appropriate time interval, flow rate, and perc entages for A and B. Take care that C and D remain at 0%, because the pumpís computer will sometimes fill in a non-zero percentage for C and D! All entries must be scrolled through before you can enter the next step. For gradients, enter 1.0 for "CURV" (linear gradient). After the final step, enter the next step as "HALT". Push "STRT" to run the method. You will notice that the method will remain at Step 0 until the injection port is set to "INJECT." B e sure to purge the injection port (set port to "INJECT" for 4 minutes into Step 1; donít forget to turn it back to "LOAD", as it is advisable not to load HSB into the injection port). Be sure to monitor t he pressure for any anomalies during this time.

Load the fraction collector with approximately 45 2.0mL free-standing centrifuge tubes. Set the run interval to 0.6 min/fraction. Set the collectorís arm to the first tube. Activate the chart recorder and be sure the settings are 0.5 cm/min and 10 mV. Also uncap the pen and set it down onto the paper.

Set the UV/Vis detector range to 0.500 (hit "PARA" then "2" and then the appropriate key with the preset value). If not already, zero the detector with "AUTO-ZERO" during the first step of the method. Set the wavelength to 260nm and response time to FAST.


The main runís method is stored under "Method 08." Access "DIR" and "RCL" Method 08. Prepare the sample for injection. Use the appropriate syringe (rinsed with ddH2O) and with the port in the "LOAD&q uot; position, inject the sample. Start the pump.

The following actions must be performed as close to simultaneous as possible (and in the following order):

You may now let the run proceed. Around 2-3 minutes into Step 1, return the injection port to "LOAD." The run should take 32 minutes. Occasionally monitor the pressure for fluctuations; LSB pressure is around 600-800 psi; HSB pressure 900- 1200 psi.

Around fractions 18-20 (cy3) or 21-22 (cy5), the cy dye should come off, and there should be a corresponding peak.

Make sure the pump stops at the end of the run (it might revert to Step 0, which is time indefinite!).

After a run, you may reload the collector, inject the sample, and initiate the run again. Be sure the HSB and LSB levels do not run too low (else air would enter the system).

Cleanup Procedures

If you finished your final run, purge the lines with 10% acetonitrile, and donít forget to purge the injection port. It is permissible to remove the column beforehand with LSB in it.

If you are going to use the HPLC again the subsequent day, you do not need to purge the system, but you may want to equilibrate the column before your first run. Purge after second day of use, regardless of whether you will continue using the HPLC.

Remove the column, unscrewing the outlet first (and capping it) before the inlet is unscrewed. Shut down power to ALL parts of the HPLC.

For Brown Lab only:

Locations of reagents

**A sizable lot (i.e. 167 nmol for cy 3 and 238.5 for cy 5) of sep-pack purified cy dyes is located in the cy dyes filtrate box, as of 9/14/98.**

This on-line version of the guidelines maintained by Chih Long Liu.
Last edited by Chih Long Liu on September 15, 1998 anno Domini.
Chih Long for comments, questions, or concerns.
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