Chromatography Forum

Dear forummembers,

I am running an LC-MS/MS method using a gradient with water and acetonitrile (both with 2 mM ammoniumformate and 0.2% formic acid) on a PFP propyl column, 50*2.1 mm, 5 µm with guard column 10*2.1 mm, 5 µm.

I have a problem: after each run, the (equilibrated) pressure increases with 10-20 bar.

The problem occurs even when the sample is just water (which works fine on our other LC-MS/MS), so the sample is not the problem.

If I backflush the column when pressure is increased, the pressure returns to normal. If I start my analyses again, the pressure again rises. The guard column alone has always the same pressure, so the increased pressure is a problem of the column!! This is confirmed since backflushing the column with or without the guard column solves the problem.

Has anybody an idea how I can fix this, since it is not very convenient to backflush the column after every 5 runs or so. And I suppose this is not ideal for the column lifetime...

Thanks for your help!

Ruth

Hi there,
For assays that run a buffer mobile phase, such as the one you are using, we flush the column with DI water after each run finishes. This removes any salts that may be building up on the column. We notice a marked difference in backpressure and chromatography and a major decrease in column life if we do not flush a column after use.
Best wishes,
Laura

Your problem is an interesting one.

You did not say what type of samples you were injecting or how they were prepared.
SPE extract? Liq/liq? Protein precipitation? dilute and inject?

How long have you been using your guard column? Is it possible that the guard column is "saturated" with whatever and it is coming off and ending up on your analytical column?

That you can backflush and the pressure returns to normal suggests that something is building up on the inlet side of the column.

Another possibility is that your gradient also includes a flow rate change either by accident or by design, and it is not returning to the starting flow rate.

You are probably very well aware that running a gradient will cause a pressure change and unless the column equilibrates to the starting conditions the pressure may be quite different from the start of the run.

A final thought occurred to me... perhaps there is something on your column (some contaminant, protein or such) that your gradient causes to "thicken" or "swell" and back flushing with water helps eliminate this. Does backflusing with the highest organic percentage (I think you said acetonitrile/water) help or does it not?

One more thing, when you say "run" do you mean a batch of several samples or just one injection?

Alp

Hello there,

The pressure increases as soon as you start the analysis? If that's the case, it might be a problem with your MS switching/selection valve rather than the column.

Good luck