I'm trying to use reversed-phase HPLC to seperate and quantify an acidic
(A) peptide and a basic (B) peptide. The A peptide is only soluble > pH
6ish, but at this pH the B peptide sticks irretrievably to the column
(Vydac TP218, C18).
I presume it's getting stuck to silanol groups, as it is very basic,
definitely soluble, and elutes fine at pH2. I also suspect that although
changing my buffer system (Phosphate/MeCN) may help, it won't completely
negate the problem, meaning that I'm still not getting quantitative
results.
>From reading the archives, I suspect a base-deactivated column is what I
need. I can't find anything specifically labelled such in our catalogues -
are there other terms for the same thing, or is it fairly specialised?
Does it cost loads more?
Alternatively, is there a way to solve my silanol problem without buying a
new column? Or is there a completely different way to seperate these very
differently charged peptides (PI's about 3 & 10) quantitively?
Thanks!
Jenny Shipway
Centre for Biomolecular Drug Design and Drug Development
University of Sussex