Perfluorinated acid alternatives to trifluoroacetic acid for reversed-phase high-performance liquid chromatography

Over the past decade trifluoroacetic acid (TFA) has become the ion-pairing agent (IPA) of choice for reversed-phase high-performance liquid chromatography (RP-HPLC) of peptides and proteins. Reagent grade TFA is highly pure, water soluble, transparent at 220 nm and readily volatile. A drawback of this universal appeal is that several alternative perfluorinated carboxylic acids tend to be overlooked when TFA does not work in a particular separation. Examples are given comparing TFA selectivity with those of pentafluoropropionic acid, heptafluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid and perfluoroheptanoic acid. We have found that increasing the IPA n-alkyl chain length can resolve sample components that otherwise co-elute in the void volume of TFA-based RP-HPLC. Examples are given for the resolution of an oligoglycine series and enhanced selectivity for a bovine hypothalamic extract.