Abstract
Reversed-phase HPLC and UHPLC separations of peptide and protein samples most often use an acetonitrile/water gradient with an acidic mobile phase additive. Although many different acidic additives have been described in the literature and have been employed in the laboratory, two are heavily favored and chosen most often—trifluoroacetic acid (TFA) and formic acid (FA). The former is used most often for LC-UV analyses and the latter for LC-UV and LC–MS, because of TFA’s analyte signal suppression in LC–MS. In this seminar, I will discuss the use of these two acidic modifiers, as well as some alternatives, which have been selected for their favorable characteristics and performance in LC and LC–MS applications. In particular, I will describe the use of difluoroacetic acid (DFA), which we have found to have useful properties for LC separations of peptides and proteins, while affording less signal suppression than TFA. Depending on the application, wise selection of the correct acidic additive may improve results substantially. Finally, it will be demonstrated that having additional choices in mobile phase additives permits easy manipulation (adjustment) of retention, selectivity, and peak shape.