I'm currently running a HiPrep 16/60 sephacryl S-100 column which has some compaction due to running at too high a flow rate. Since this occurred, I have reversed the flow direction to help ease compaction. After running a sample, fractions were collected using a Gradifrac system, which was programmed to collect fractions of 1.5mls and run the pump at a flow rate of 0.1ml/min. After running for 20 hours (which should have been one column volume and 80 x fractions of 1.5mls), I got 80 fractions, however they only contained 0.9mls each.
Is this likely to be an issue with the pump or the column? Any suggestions for how to fix it? There are no leaks from the column hardware and no air bubbles are visible. Solvents are degassed and filtered, protein sample was filtered but at a high concentration (50mg/ml).
hi,
First check the flow rate of your system by doing flow rate calibration, or may be some problem with auto collector volume collection, so that you may get low volume. If you get expected recovery from fraction collected after evoparation then there will be no problem with the low volume you have received.
Hi Taylor,
As Ashish mentioned, I would check that your pump is working properly and that you don't have a leak (the most likely reason for your under-performance).
I like to do a pump/leak test the following way: remove the column from the system and prime the full system to ensure no air is trapped anywhere. Now place the tubing that you normally put into the column into a e.g. 20 mL volumetric flask and set the flow rate to 1 mL/min. Stop the time it takes to fill the volumetric flask to the mark; this should be 20 min.
If filling the volumetric flask takes more than this (and 21 min is already on the long side since it is 5% longer than anticipated), it is an indication that you have either (1) a leak in the system or (2) still air in the system. To rule out number (2), prime the system again and repeat the experiment. If you get the same time again, perform a leak test on the system; this varies slightly between systems. But basically you place a blank nut after the injector and before the column, prime the system in isopropanol and then build up pressure in the system. If the pump has to work to maintain the pressure, you have a leak. But your software should tell you that. If the test fails with having the blank nut placed after the injector, place it before the injector and repeat. If it passes now, your leak is in the injector, if it fails again, the leak is somewhere in the pump. Often this means troubleshooting the pump heads which might be as simple as replacing washers but might include replacing certain valves.
Good luck!
Thanks for the suggestions Ashish and Sabine! I ended up testing the pump outflow pre-column and found that it was at about 65% of the set flow rate when measured using a measuring cylinder and timer, so it appears to be more of an issue with the pump than the column. Because I'm using the pump attached to a gradifrac system, I'm able to set a calibration factor for the pump to correct this and I've been able to bring the output up to the expected level using this calibration. As you suggested Sabine I suspect that the pump may need some parts replaced so I will have to get in touch with the manufacturers. Thanks again for your help :)
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