I have been running a biological water extract C18 RP (Ultrasphere 250x4.6, 5um) with A: Water+0.1%TFA and B:ACN+0.1%TFA. My method is 0 to 8% B over 50 mins; then 8 to 100% B over 35 mins, then hold for 10 mins at 100% B. I have been interested in a group of peaks eluting late (about 80 mins) which have been broad (3-5 min peaks).

I recently change the system to B: THF+0.1%TFA as part of a method development process, then on return to the above program, same sample, saw what I call unbelievable chromatography - excellent peak shape and separation at the same 80 min region !!!!

My question is:

What is this "new" superior separation a result of ???? is it column regeneration ??

Thanks