I used C18 semi-prep column to purify natural products. I've found the great gradient for my compound purification and ran 2 run for 50um of my sample and I have wash procedure after each sample run.
I switched off HPLC flow and went for dinner. When I went back, it took me an hour, which is abnormal, to balance the column to same pressure before and stable absorbance.
And the RT of my sample peak delayed about 20 min
What's the problem?
mobile phase: ACN + H2O
sample: 20ug/50uL dissolved in 10%CHCl3+90%MeOH (each run)
Which HPLC system? This helps us to evaluate the problem, knowing how the mobile phase is being mixed.
I'm guessing air in the pump head, try priming your pumps. If you are doing high-pressure mixing, the B solvent pump may not be drawing correctly. Depending on your solvent composition, that could change the mixture to more aqueous, causing higher back pressure (to the level you saw before dinner), but delaying elution of your compound.
Check your flow rate from each pump by removing the column and generating back pressure with some tubing with a small inner diameter to seat the check valves. See how long it takes to fill a small volumetric flask. If the flow is slow with pressure, and the pumps are primed check the pump seals.
We found that maybe my sample was stuck in the whole capillary, so pressure and RT fluctuated randomly. My sample seems not fully dissolved in ACN. We clean the whole capillary and column and I'll tried next run.
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