I am trying to isolate lipoprotein A1 by running liquid chromatography on Sephacryl S200 packed column, 5x150cm.
The buffer (moving phase) goes thru the top via gravity, and on the bottom i have a fraction collector with a drop counter. The system is gravity operated. I am having trouble setting the flow rate. When i open the column, the buffer elutes either too fast or too slow.
i would appreciate your feedack
Hi Ronny, for open gel filtration columns, you should not block the flow.
Have the column open and collecting fractions with buffer before you add the sample. Gel filtrations columns will slow down if the sample is viscous. If you have a continuous gravity buffer feed to the column, then just allow it to run.
Hello Steingrimur,
Thank you for your response. I allow a 75-100ml/hr flow rate and run about 3 to 4 liters of buffer before the addition of the sample. Once the sample is added, the SOP calls for a 25-30ml/hr flow rate, and that is why i partially close the valve on the bottom, to restrict the flow.
The easiest way to regulate the flow rate is with a peristaltic pump, either on the top or bottom of the column.
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