Hi, I need to separate angiotensin II, III, IV and des-asp angiotensin I. I have tried 0.1% TFA in water (A), 0.08% TFA in 80% ACN (as well as 70% ACN)(B) with changing of flow rate, gradient and column (from 3.9 x 150mm to 2 x 150mm C18 column). However, all these don't seem to help. Angiotensin II, III and IV eluted very closely to each other while des-asp angiotensin eluted reasonably far from them.
Recently, I came across a paper which have separated these Angiotensin before. The author used 4mm TEAF, 30mM formic acid and 20% ACN. I will try to optimise on this system. Otherwise, I am thinking of adjusting pH of TFA using TEA. Anyone familiar with these systems?
Do you have any advice for my separation?
Would other methods like ion exchange help (I am worried that the sensitivity might not be good enough as the amount that I am going to quantitate might be quite low.)?