We want to synthesize citrate salt of a peptide and we used ammonium citrate to form a counterion to trifluoroacetate. citrate salt is formed perfectly but after lyophilization the weight gain was 4 times the actual peptide and we detected the excess crystalline citric acid with peptide. How to remove that citric acid from peptide ?
we tried HPLC, bicarbonate washings also but no results.
please help us
RPHPLC is great for exchanging to produce salts of volatile counterions, for example if you wanted to make the TFA salt. But eluting the peptide from the column in a citrate buffer will give you a mixture of peptide and buffer. So when you dry that down you'll still have peptide and buffer, in this case crystalline citric acid.
There are two things I would try. If your peptide is big enough you can dialyze it against dH2O. If it's too small to stay inside a dialysis membrane you can try RPHPLC with no modifier added to the mobile phase, i.e., just neat MeCN and H2O. If your peptide binds citrate well it's probably got plenty of basic AA. In that case it will perform poorly in RPHPLC with no modifier. Depending on how much end-capping is on your stationary phase you might observe unusually short RT and/or a long tail on the peak. But if your peptide is pure, you only need resolution from the solvent/salt front.
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