Chromatography Forum

I'm having problems of late where my system pressure will increase stepwise following each injection. If I stop injecting and let the system sit for 30-60 minutes I notice that the pressure begins to return to the original pressure. Since I have been having this problem I have changed to a new column, have changed out pumps (had back pressure), and have changed the mobile phase and autosampler filters incase of microbrial growth. This seemed to help for about 20 injections but I am starting to see the pressure increase again.
I run an isocratic method with premixed mobile phase (40% acetonitrile in 50 mM potassium phosphate buffer), use an autosampler, and have a UV detector. My samples are microdialysate samples which are fairly clean and have not been problematic in the past.
I've tried hooking up a manual injector and did not see this problem. I have also removed the column and tried just injecting into the guard column while using the autosampler and see this increase. I have also tried flushing the autosampler out using water and then methanol.

I'm at a loss of late. Does anyone have any suggestions!

Thanks in advance

Heidi Doyen
Grad Student

Heidi

You have done a thorough job of troubleshooting. One other possibility is that the seals in the auto-injector valve are shedding particles. To double check that it is not the mobile phase or the samples pump pure (filtered) water with about 10% methanol and inject filtered water.

Peter

If there is a stepwise increase of the problem with the injections and a slow disappearance when nothing is injected one can presume that the problem is connected to the injection. The most likely culprit would then be the samples. Microdialisis sounds biological, my experience would indicate that you can never assume that such samples will have constant characteristics forever. A super troubleshooting doesn´t help if done at the wrong place.

Have you tried to change the injection needle? if you are experiencing pressure problems, try disconnecting the various bits without stopping the flow of the mobile phase. If there is a blockage anywhere, you will see it because pressure will not drop if you disconnect that part. I hope this makes sense.

Work your way backwards from the detector.

Good Luck,

Salma

I'm in accord with HW on this one. The fact that the pressure increases step-wise with each injection points to either particulate matter (as suggested by Peter) or precipitation. The fact that the pressure gradually decreases after you stop injecting suggests a precipitate re-dissolving.

If you don't have any luck with the particles from injector source, consider looking at the interaction of your sample and mobile phase.

If you use a small test tube, you can gently add drops of sample to mobile phase, and vice versa. Look for any signs of haziness, and don't shake the tube. You should be looking at the drop interface as it slowly dissolves, trying to ascertain if any insoluble material forms.

Because the first part of the column can also interact with the injected sample as the injection solvent moves away, the simple visual check doesn't show all problems, but it's helped me in the past when pH effects have caused problems with diluted fermentation broth. Adjusting the sample diluent fixed the problem.

If the pressure increases with just the guard, it usually suggests either precipitation or immiscibility, with the former likely in your case. You have to try and find out what has changed recently, and thoughtful systematic efforts usually ensure success.

Good luck.

Bruce Hamilton