RP-HPLC carryover problems - (May/23/2011 )

Hello All, I haven't posted here in a while but always found this place very helpful when needed! So I'm trying to develop a RP method for a vaccine and am having significant carryover problems (2% in blanks after sample). I cannot solve the problem for the life of me, I've tried various needle washes and injector programs, various columns (C3, C4, C8, Phenyl, etc.) and a variety of mobile phases (H2O/ACN, H2O/IPA) with a variety of different modifiers (TFA, FA, HCl, H3PO4) and I still can't get rid of the carryover.

I've never had this much trouble developing an RP method before and was wondering if anyone had any similar experiences or any tips. Currently I'm using a C3 column as I can at least get no carryover on smaller injections (4 ug) but we really need to inject 10 ug per client request. The carryover is definitely in the column as well and not in the autosampler as a blank run after the sample on a different column came up 100% clean!

Thanks in advanced!

What angstrom are you using on the columns, as that has a significant impact on how well the column cleans up between runs. I am guessing you are using something in the 100 angstrom range, which could lead to problems. I normally use a 300 angstrom resin, and it cleans up really well. I'd recommend trying a Phenomenex Jupiter C5 column, as the C5 columns tend to work better than the C4 columns. I haven't personally tried a C3 column, so I can't comment on that.