ABSTRACT
Of the major modes of high-performance liquid chromatography, reversed-phase chromatography (RPC) offers the widest scope for manipulation of mobile phase characteristics during peptide separations. This chapter demonstrates how the effects of anionic ionpairing reagents on peptide elution profiles in RPC may be manipulated in a highly predictable manner to achieve the desired separation. In addition, the effect of counterion concentration on peptide resolution and retention is discussed. Peptides are charged molecules at most pH values and the presence of different counterions will influence their chromatographic behavior. Differences in the polarities of peptides in a peptide mixture can be maximized through careful choice of ion-pairing reagent. All anionic counterions are potentially capable of ion-pairing with the positively charged basic residues of a peptide, thereby reducing its overall hydrophilicity and increasing peptide retention. However, they differ in their ability to interact with the reversed-phase sorbent, thus producing a useful basis for defining the nature of a particular counterion.
