I'm trying to analyse a protein of about 30,000 Da using the usual TFA acetonitrile gradients on a 300A C4 cloumn. The problem is I seem to get a unitary loss of protein as if it was 'sticking' somewhere in the system. It has been suggested that the addition of a carrier protein such as BSA might help. Has anyone else experienced this problem and I'd really appreciate any suggested solutions.
Regards
John

run a gradient with the sample, then follow it with a gradient without sample injection. You may find that the peak shows up again in the blank gradient. There have been theories published why this happens, but I do not remember.

Could your protein (standard?) be a mixture?
Again, we have had nothing but trouble with TFA. Maybe replacing it with a buffer, optimizing pH, will do? If not try in addition to replace ACN with an alcohol, maybe even propanol. If still no go: experiment with chaotropics, detergents. Maybe you also ruined your column with TFA, use a new one for further experiments.

Thanks for that. SDS I think is the next thing we will try. We have done some MALDI on the protein and it is a mixture. I wasn't aware of the problems you highlighted with TFA. We usually use it but for once off applications which I suppose doesn't give time for it to deteriorate columns.
John