Chromatography Forum

I have a strange problem in my HPLC instrument. The pressure is increasing till reaching the maximum set of 3500 psi. I am using a low gradient mixing system with a degasses, C18 column with a guard column, autoinjector, 70% ACN/Water by mixing from two solvent reservoir.

I tried to investigate the problem since last week and here are the investigations I made:

1.Suspecting the solvents, I replaced them with new ones and replaced the frits, but high pressure still developed
2.I disconnected the column and ran the pump, high pressure still developed
3.I disconnected the autoinjector, pressure dropped about 1000 psi, but with continuous build up again
4.Replaced the inlet and outlet check valves in the pump, disconnected the mixer and the degasser, but no change in the pressure.
5.I ran the pump with only water, the pressure started to go DOWN! I tried ACN only, pressure back to build UP!
6.Tried a new ACN from a different supplier, No better results.
7.GAVE UP AND I AM CONSULTING THE EXPERTS IN CHROMFORUM! ==> Will be solved

Waiting for your thoughts and ready to have more fun!

From your description I’m getting the feeling that you’ve had some sort of buffer that precipitated – probably due to introduction of a high portion of organic solvent.
Suggestion: Pump a lot of water (no ACN or other organic solvents) in a couple of hours (flow rate; up to 3 – 4 mL/min, gradually increasing! – no column!) or until the pressure is down to 0 psi. That should remove the salt precipitations and you’re ready to go to your usual work.

Best REgards

I think Danko may have identified the problem, but you didn't mention a buffer, so it may have been a historical use?. I'll make a couple of comments. Did you replace the PTFE frit on the pump purge valve assembly?.

I would fill both mobile phase channels with degassed ( ultrasonicated or He sparged ) warm water, and use that ( set at 1:1 A:B ) to flush first the degasser ( for at least 30 minutes at maximum flow, as this module has the largest internal volume ) and pump ( at least 10mins ) , then the injector, column oven heat exchangers ( if used ) for at least 10 minutes , and leave the detector disconnected.

I'd closely watch the pressure, trying to keep flow rate as fast as possible, using pressures 100-200 Bar if necessary for the column upstream components. Ensure that all capillary lines between modules are also flushed.

Then I'd perform some large volume 100ul injections of warm water ( without a column and only 4 min run time ) to clear the sample loop/lines.

I'd then switch the solvent channel to 1:1 CH3CN:H2O, and then finally to CH3CN.

If that doesn't work, I'd filter the water and solvents through a 0.22um
filter, and see if the pressure rate rise changes.

Please keep having fun,

Bruce Hamilton

Thank you all.

This particular instrument has NOT been used with buffers at all.

I think I will try first to replace the frit on the pump purge valve assembly, before having more fun.

I replaced the frit on the pump purge valve assembly, and I found it really DIRTY. The pressure dropped down by 1000 psi to the NORMAL levels. I thought that the problem solved. However, when I connected the system again and ran it with 70% ACN/Water for about 4h without injecting anything, the pressure started to build up again but with lower rate. It increased by about 400 psi during the 4h.

Any comments?

I think I have to try the harder FUN suggested by Bruce.

If you didn't flush the system with warm water and then solvent, as suggested earlier, I would do that first, including cleaning the injector. Just to remove the possibility of residual gunk.

Then I would definitely filter both mobile phase components, and if that works, I'd just filter the water, and hopefully that should identify which component is the problem.

Ensure that your filter system is thoroughly clean before you start, because it's quite hard to maintain 0.2um cleanliness on occasionally-used items.

Please keep having fun,

Bruce Hamilton

The first thing I would do in a case like that is to clean that same frit again to see whether it got clogged again or whether something else is clogging.
Actually, a pressure problem can hardly be solved via a forum. One has to systematically look for the cause. Of course, some general suggestions can help to "sharpen the eye". For instance, the most difficult problems to locate in this lab where those connected with bad or squashed tubing cuts, or PEEK tubing whith swaging difficulties. Opening and reclosing such a connection usually relived the flow resistance, but only for a short time. This is why I go right back to check the same place after a recurring problem. (No idea why this happened a few times with PEEK tubing: After a resistance increase at the fitting and opening it, it was evident that the tubing end had slipped slightly closer to the ferrule. Can think of only one explanation, namely, that some PEEK very slightly changes volume even with solventa which are supposedly "inactive").