Strong cation-exchange high-performance liquid chromatography as a versatile tool for the characterization and purification of peptides

Synthetic peptides play a major role in all areas of biomedical research. The ease with which virtually any sequence of both natural and unnatural amino acids can be synthesized is a primary reason for their popularity. Often, the final product not only contains the target peptide sequence but contaminating species that differ in subtle ways such as minor deletions, incomplete deprotected side chains, fragmentation at unexpected residues, and residue adducts as a consequence of improper resin cleavage. The investigator must then purify the desired target molecule from the crude mixture in order to conduct meaningful experiments. A widely practiced and highly successful method of analysis and purification utilizes reversed-phase high-performance liquid chromatography (RP-HPLC). An alternative Chromatographic procedure uses strong-cation exchange (SCX)-HPLC. Here the primary mode of separation is the interaction of the peptidesʹ positive charges with the negatively charged functional moiety of the Chromatographic support. At a mobile phase of pH 3, peptides are expected to possess a net positive charge as a result of protonation of carboxyl groups and C-terminus, leaving arginine, lysine, histidine and the N-terminus to contribute to the net positive charge. Thus, as a first approximation separation will be a monotonic function of differences in net positive charge. Considerable success has been realized with a sulfoethyl aspartamide SCX-HPLC support for the analysis and purification of well over 500 distinct synthetic and proteolytically derived peptide fragments. This short practical review will present examples of these separations with emphasis on synthetic peptides. For completeness, a section on SCX-HPLC of proteolytically derived peptides will also be presented.