Optimum concentration of trifluoroacetic acid for reversed-phase liquid chromatography of peptides revisited

Trifluoroacetic acid (TFA) remains the dominant mobile phase additive for reversed-phase high-performance liquid chromatography (RP-HPLC) of peptides after more than two decades since its introduction to this field. Generally, TFA has been employed in a concentration range of 0.05-0.1% (6.5-13 mM) for the majority of peptide separations. In order to revisit the question as to whether such a concentration range is optimum for separations of peptide mixtures containing peptides of varying net positive charge, the present study examined the effect of varying TFA concentration on RP-HPLC at 25 and 70 degrees C of three groups of synthetic 10-residue synthetic peptides containing either one (+1) or multiple (+3, +5) positively charged groups. The results show that the traditional range of TFA concentrations employed for peptide studies is not optimum for many, perhaps the majority, of peptide applications. For efficient resolution of peptide mixtures, particularly those containing peptides with multiple positive charges, our results show that 0.2-0.25% TFA in the mobile phase will achieve optimum resolution. In addition, the use of high temperature as a complement to such TFA concentration levels is also effective in maximizing peptide resolution.