Chromatography Forum

Hello
I was here a few weeks ago, because I had some problems with my HPLC. The problem was an increasing pressure during running a few samples in gradient mode (start: 80% H2O, 20% MeOH; MeOH up to 100% during the run). All of you mentioned a clogged column or clogged tubings. I cleaned and also changed everything! Nothing has helped.
Now I changed the pressure sensor too, but the problem still exists!

Do you have any new suggestions about solving the problem?

Thank you in advance!

Mikro

Get HPLC running at high pressure, then (working backward from column inlet) start loosening fittings one at a time until you notice a big pressure drop. That should help isolate. Our experience is that if the pressure blockage is not due to column or column inlet frit (or prefilter), then most likely it's due to a degraded, worn autosampler rotor (or manual sample rotor) clogging up the valve.

Have you checked the Frit at the head of the column?

If that gets blocked you may be able to cure the pressure problem by replacing this frit.

Column head frit blockage can be avoided by placing a disposable in-line filter immediately before the column.

As you have been using a buffer previously, I would thoroughly flush all channels of the system ( without column ) with warm water, and then methanol. Ensure that you also perform some large volume injections to flush the injector. Given that degassers have large internal volume, pay special attention to them.

Check any inline filters after the pump. Once the system is clean add the column and guard, and flush with high aqueous and solvent phases without buffers to ascertain how the pressures behave. At that stage I would perform the pressure checks suggested by CPG. I would not dismantle individual modules until I was sure the pressure was not due to precipitated gunk.

Assuming that your samples are not causing the pressure increase, my guess is that the buffer has precipitated previously. It's probably not completely soluble in high methanol when on the column. I'd want to ensure that the buffer components remained soluble, and that you are flushing the system with water/solvent, and not leaving buffer in it..

I would perform a couple of runs with just water and methanol to establish baseline pressures, then try the buffer.

Please keep having fun,

Bruce Hamilton

Hello
first of all, thank you for your suggestions. I probably forgot to tell you, that the problem is certainly not according to the application, because I observe the same increase in pressure just with a peek tubing as a resistence!

In the time you wrote this you could have done the systematic test suggested by CPG.