Chromatography Forum

Hi guys, need your advice.
In a C8 RP short separation there are 3 peaks A, B and C, A eluting first and C last in the gradient. When a new column is used, peaks B and C have the right retention time after a couple of injections but not A. A takes three to four days of repeated sample injections to finally get to the right retention time, it slowly decreases its retention time until the retention time is stable. Can anyone explain this to me? and are there any tricks to get this equilibrated faster? Thanks for any advice.

I've encountered this with end-capped columns at low pH (the short-chain end-capping hydrolyzes off), but you haven't provided enough details (e.g., what mobile phase) to say much.

i think matrix sample is dirty, if your sample have llipid or protein, it can micelle formation and attach C18 chain and make baseline very ugly. I think you need clean up your sample by SPE before filter.
Tom

The mobile phase is phosphate buffer pH 3.1 in 5% methanol and MP is ACN with 5% methanol. I run a gradient from 25% B to 60% B in 6 minutes and give 2 minutes for reequilibration. All of the analytes pKa are above 5 so at pH 3.1 should be ok. I just came to find out that the column takes 5 days of gradient runs with the occasional std injection to have peak A settle at the right time. The column is a C8 acquity BEH column. Any help or trick would be greatly appreciated

Maybe the interaction of your compound A should not be considered as being simple reversed phase mechanism. If there are noteworthy ion or polar interactions for your molecule possible, you may think more in this direction.

Without knowledge of your experiments and resulting chromatograms (and application), I can add only some general suggestions:
Implement a real washing step with > 90 % organic for some minutes
Check the influence of the re-equilibration time

Edit: Was is meant with "MP"?