[19] Analysis of synthetic peptides by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) has proved extremely versatile for the isolation/purification of peptides varying widely in their sources, quantity, and complexity. High-performance chromatographic techniques are suited to the purification of a single peptide from the kind of complex peptide mixtures encountered following solid-phase peptide synthesis, where impurities are closely related to the peptide of interest (deletion, terminated, or chemically modified peptides), perhaps missing only one amino acid residue, and, hence, may be difficult to separate. This chapter focuses on HPLC applications of particular interest to researchers utilizing solid-phase synthesis methods: (1) problem-solving approaches to difficult peptide separations, (2) HPLC monitoring of formation of desired product, (3) and HPLC as a diagnostic tool to detect unwanted side-chain modification. The use of reversed-phase chromatography (RP-HPLC), the most important mode of HPLC for synthetic peptidepurification, is focussed; in addition, the exciting potential of a novel mixed-mode HPLC technique, hydrophilic interaction/cation-exchange chromatography (HILIC/CEC), is demonstrated.