Hi, recently I used HiPrep™ 16/60 Sephacryl® S-500 HR column to purify the sample, loading the sample before, the filtered TEMB buffer balance the column and I set the AKTA system is 1.2CV buffer for balancing. But when this program operation end, the column bottom becomes dry about 10 cm distance and about 10 hours. I don't know what happens. The buffer just filtering not degas. When I found this column became dry, I used 20% methanol (filter and degas) to balance this column again about 4 CV. The column seems to rehydrate but when I used the MiniQ-H2O to wash this column, the bottom column became dry again. Could anyone give me some ideas on how to operate this column?
Are you pumping into the bottom end (ascending flow)? Only then will a column run dry from the bottom end. At any rate, you need to use enough buffer that no air enters the column. Once a column has air bubbles in it, it can't be restored to full theoretical plates by flushin it with methanol. Rather, the column should be emptied, the gel suspended in buffer, and then repacked. The booklet "Gel filtration theory and practice", available free from the Sigma-Aldrich site, has a detailed description of the procedure.
Hi Engelbert Buxbaum , I pumped into the bottom end. I thought the buffer is enough for balance (I prepared a 1L buffer) and just set 1.2 CV for balancing. Yesterday, I checked the AKTA whole system. The pump sometimes appeared to not work so that arise to the air into the column.
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