I am using the 5 mL Hi-Trap Q FF column to separate my protein/peptide sample. I got the correct responding of UV readings and conductivity before I connected the column to the FPLC unit, but as soon as I linked the column to the unit, the spectrum shows irregular peaks which did not make any sense. I tried several run with different flow rate etc. yet got the similar patterns. I also checked the leakage of the system and column and cleaned the pumps, tubings thoroughly and re-conditioned the column. However, I am still getting this pattern. If anyone encountered the same problem, please share your idea and experience. I thought I could be the problem of the column, but the same thing happened for my spare columns as well.
Many thanks.
It's a bit of a wild guess, but I've seen something similar that was caused by a small bubble oscillating in the detector, but it was on quite an old machine and I do not know if your detector could have the same issue. The solution was to flood the detector with buffer in reverse flow using a syringe attached to the outlet tube, while simultaneously tightening the inlet tube connector with the pump running. And then switch off the pump immediately after making the connection.
Can you apply a bit of back pressure on the detector? That could also help if bubbles are to blame.
If not the above, I suppose it could be electrical noise/faulty detector?
Degas the check valves of the pump in IPA. Otherwise, replace it with a new batch. Rarely plungers may be scratched due to typically used buffer salts in FPLC if proper washing procedures were not be applied but if I understood correctly the pump is starting malfunctioning when the backpressure exists. You must be observing the pump pressure fluctuation at the same time. This indicates the faulty check valves...
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