I am running HPLC with plasma samples. I have prepared sample by adding ACN and MeOH (40ul plasma + 60ul organic solvent) in the plasma to precipitate the proteins, and centrifuge it for 10 min at 13000 rpm. samples were filtered with 0.2uM membrane. I am seeing that back pressure is increasing with with every injection. Can anybody please help in this respect, why pressure is increasing continuously. I am using a guard column too.
Dear Vandana Srivastava,
It sounds like some HPLC maintenance is required-perhaps some of your sample has precipitated out of solution. I would start to eliminate the root cause of the pressure build up by the doing the following;
1) Purge-out as much of the mobile phase that you can from the instrument and prepare and use new mobile phase.
2) Clean or replace check values, and any tubing you think may be suspect.
3) Remove guard column and run a clean sample (preferably just solvent) on just the LC column. This enables you to ascertain if the guard column is the problem.
4) You should also inspect the injection port and try running a clean sample (preferably just solvent) to see if the root cause is the plasma samples you are injecting.
Dear Ma'am
This is probably due to insoluble matter in the sample or to a soluble substance in the sample solvent that precipitates in the mobile phase. That mobile phase may not be suitable for your analysis and sample preparation too.
What is the composition and final pH of your mobile phase? How long has it been on the system? Mobile phase with low organic and at near neutral pH are subject to imperceptible microbial growth that once introduced into the column could cause increases in back pressure over time. In cases like this always use fresh mobile phase and don't allow it to stand idle on the system during periods of non-use.
Hello Brent thanks for suggestions. It was 65-35 organic solvent (50:50 ACN:MeOH) and.01% formic acid. I cleaned the column and guard coulm with water, methanol and ACN. Pressure has gone down. I think sample preparation was an issue too, so I use ZIP tips. it is working better but still I see some increase 100 psi by the end of the day. I am using fresh solvent every day.
Try to run a standard or some calibration solution with and without the guard column, don't use plasma or your extract for this tests. See if there is a great difference into the system with and without guard column. The rationale is to check if you have a contaminated guard column, main column or both. If you have a big difference in pressure with and without the guard column then the guard column should be substituted with a new one. As much as you could clean any column could stand only so much contamination and use.
You also could try to backflush the column using the manufacture instruction (look like you're using a C18, am I right?)
Also check all the inline filters your system may have, it could be an idea to substitute all of those.
That's my suggestions to improve the analysis from the instrumentation side.
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