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). 

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