Can anyone please shed some light on my problem? I have a Nucleosil C18, 100A, 3u, 4.0x 80mm column on which I am running a gradient - phosphate buffer , pH 5.2 : Water from 99% buffer: 1% acetonitrile to 60% buffer : 40% acetonitrile. The column temp is 50C and flow rate is 1.5 mL / min. The starting pressure is ~2000psi. My problem is that after running this gradient several (<20) times the pressure skyrockets up past 5000psi and I can't even get 100% water or acetonitrile through the column at 0.1 mL / min. We filter both the buffer and acetonitrile through a 0.45um filter prior to running. Any insight? Thanks.

WHAT CONCENTRATION IS YOUR BUFFER? Sounds to me like a classic precipitation issue, each run is depositing more precipitated buffer on your column/frit. I'd change frit or backflush the column gently to flush out the precipitated buffer, and probably back off on buffer concentration or figure out how to do isocratically and pre-mix the aqueous buffer and ACN, and then filter the mixture before using.

NoName

The list below has the acid dissociation constants for phosphoric acid. Buffers work best around the pKa. Phosphoric acid falls at 2.16 and 7.21; 5.2 would have about 1:99 rather than the desireable 1:1 ratio.
phosphoric acid (1) H3PO4 6.92 E-3 2.16
phosphoric acid (2) (H2PO4)- 6.17 E-8 7.21
phosphoric acid (3) (HPO4)= 2.09 E-12 12.32

I have forgotten the 210nm absorbance but look at acetate, lactate, etc for a buffer and the clogging if it is a precipitate may go away too.
Jim

Thanks for the input. I tried backflushing but that didn't work; couldn't even move 0.1 ml/min through. I'm guessing this column is trashed. The buffer is 10 mM, so I don't understand why it would ppt out like that but perhaps you're right. As for changing method params (i.e. buffer conc or type) this is a QC method so I'm not at liberty to change those types of things. Thanks again.

NoName,
If the other collegue was correct the buffer should dissolve with even a slow flow, try just water and even at .01ml/min it will unclog.

Your information that it was a QC method throws a different light upon your problem. If the method ran before, development type answers will not help. How many columns can you distroy before your company will give up on the method? Can you validate a modification to your method? This procedure is approved in general and if your method is a new one, perhaps the best choice.
Jim

At 10 mM, I don't think the buffer is the problem. It's not a good buffer at pH 5.2, but this is not related to the pressure problem. Let us talk about the sample that you are injecting! What junk does it contain? Is it a plasma sample with maybe some proteins left (maybe not in QC)? Some high molecular weight agent in it that can get stuck on the column? Other sources of stuff that can clog columns could be the solvents, solvent lines etc. Anything new or unusual there?

Many columns also cannot handle 99% water for extended periods of time. There are very few columns that I would expose to those conditions.

Also, even validated test methods can be tweaked within limits without re-doing a whole new validation. There are published guidelines for both GC and HPLC parameter limits.

We inject samples that have a gelatine matrix and over time this will lead to high pressure. We have used a dilute enzyme to break up the gelatine and reduce the pressure. I don't know if this would help in your case. But I agree with Uwe; look at the sample matrix.

Yup, it sounds like the samples (maybe!). Filter the samples instead of the mobile phase constituents. Also, what type of filter material are you filtering the ACN through? For example, ACN will dissolve cellulose acetate, a very common filter material.

Does your column plug after 20 injections or 20 gradients? For example, if you performed 20 injections of water, does it plug? If so, it's precipitation or bugs growing in your 100% aqueous mobile phase A. If not, it's your sample.

My suggestions: don't filter ACN, filter your samples through a 0.2 micron spin-type filter and, if possible, to be sure, change your buffer from phosphate to acetate (lithium acetate if UV only, ammonium acetate if MS) for the two reasons given above: 1) phosphate is just a salt at pH 5.2 and 2) it will eliminate the chance of precipitation. Lithium based salts will keep the bugs from growing and is available in very pure grades.

That ought to do it.

I THINK YOUR COLUMN FRITE (AT THE END OF COLUMN BOTH SIDE ) ARE CHOKE-UP. BETTER YOU CHANGE THAT OR OPEN IT AND SONICATE THATE FRITE FOR HALF AN HOUR OR WASH IR IN MIXTURE OF WATER : NITIC ACID : 50 : 50 MIXTURE ON BY BOILING IT IN. HOPE THIS WILL SOLVE YOUR PROBLEM.
THANKS

i think the person mentioned this is a QC method, so whatever reason it is, the sample matrix, the column, i think this method needs to be revised and revalidated. and what happened during the initial validation of the method? these problem should show up at that time.