Chromatography Forum

We are trying to validate a gradient method which uses a TFA buffer, A: 0.1% TFA and B: 0.1% TFA:ACN 20:80. We are using YMC C18 column. The diluent is 2% DMF in MeOH. The sample is difficult to dissolve.

The problem is that after a few injections a large hump is appearing before the main peak. It does not seem to increase or decrease with injection volumes, the increase/decrease appears to be random. There is no hump seen in the blank (both diluent and mobile phase). Any blank injection placed throughout the sample run is clean.

We have tried changing all the usual parameters, mp, column, system, sample prep but have not pinpointed the root cause.

Does anyone have any suggestions on where to go next?

Assuming that you got some reasonable peaks, initially, (which should mean that your injection volume is low enough so that your solvent incombatibility doesn´t show), and assuming that you only injected clean standard it could be that air is causing the hump.