Reversed-phase liquid chromatography of proteins with strong acids

Small organic acids are generally used as pairing agents at less than 1% concentration in reversed-phase chromatography (RPC) of proteins. When the protein is very hydrophobic and insoluble, as in the case of membrane proteins, up to 60% aq. formic acid has been used. This paper reports a study of the influence of acid concentration on both chromatographic retention and protein structure in RPC. Chromatographic retention increases in proportion to the concentration of organic acid in the mobile phase up to some intermediate concentration. Use of still higher concentrations of acid results in a sharp drop in chromatographic retention with a change in selectivity. The data indicate that the structure of proteins in strong acids are different from that in weak acids. This work examines the reason for this decrease in chromatographic retention at formic and trifluoroacetic acid concentrations above 30% (v/v). Spectroscopic studies show that protein conformation continuously changes with the addition of acid.