Chromatography Forum

Hi everyone,

I am new here, so, Hi everyone

I worked on a new (to me) instrument today, and its pressure was acting really weired. First, it was going up like crazy (nearly 400 bar) at 0.3 ml/min ( IPA, col 100 mm, 3mm, 2.5u). Second, the flow rate was not stable, it had a pattern of going from 0.3 ml/min to 0.27 ml/min, back to 3, then 0, and then 3 again, so it had its pressure dropping to nearly zero and back up.

I tried washing the (one month old) column, and then tried revesing it (anti-flow-wise) and washing at 0.1 ml/min with IPA for 30 min, tried replacing IPA to MeOH, to MeOH:water, ACN- same pressure pattern, unstable and dropping.

Will be glad to hear your ideas/ instructions what should be done.

BTW, I was warned by previous users that this instrument had had these problems before. They called a technician, who took the pump to their lab and washed it, and also (supposedly) thoroughly cleaned other parts of the system. But as we can tell the problem is still here...

Many thanks!

Pretty new, apparently. So we don't have to guess which unit and how equipped (e.g. degassser, quaternary pump, etc.), let us know.

Oftentimes failure to de-gas and failure to purge all channels (even unused ones) are the causes.

if you used buffter solution such as KH2PO4,may be it was crystas, you should remove tubing then ultrasonic them, wash seal

Thank you for your replies!
Pretty new apparently indeed, sorry for skipping these details.
Agilent 1100, degasser, quaternary pump.

I did purge all four channels, but will try again more intently, as well as using sonication instead of relaying on the degasser.
I have not used buffer solutions, but do not know if these have been used on that instrument. I will try to sonicate tubing and if all these do not work then I'll try the seal.

Thank you

That sounds like your checkvalves are dirty or broken. Use a volumetric flask to see if you get enough flow without the column. Make sure when the problem appears (with or without column). Sometimes it helps to let it run with IPA over night. Make sure you have a prefilter in any liquid you want to use in your pump.

Detach the tubing over the check valve to see if the flow constantly comes out and builds a drop. If it's pulsing, they don't work proper.


When changing liquids, do not draw the liquid with a syringe thrue the degaser, that will destroy his membranes.

If you have an ultrasonic bath, use that to pre degas your solvents.

Hold up. You had a PRESSURE of 400 bar at a FLOW RATE of 0.3 ml/min? Then is it your FLOW RATE that is "going from 0.3 ml/min to 0.27 ml/min, back to 3, then 0, and then 3 again".

I think you might have gotten a little confused with your flow rate and pressure.

Yes, sorry about that, changed it in the original message (at least it had its units...).

Adler, thank you so much for your specific instructions on how to check this. I will try doing as you suggested and see what happens, although if I find out my check valve is faulty, I will need some help in replacing it (I have seen it done before but never done it myself).

I am filtering my solvents, but will try to see what happens w/o a column. Will be back to report.

Many thanks again