Separation of peptides by strong cation-exchange high-performance liquid chromatography

Abstract

The effects of pH and gradient conditions on the separation of a series of ten peptides (9–36 residues) and carboxamidomethylated troponin I (CM-TnI, 178 residues) on a new commercially available strong cation-exchange silica based 300-Å column (Synchropak S300) were examined. The elution times of the peptides were linear with respect to their net charge at pH 3.0 and pH 6.5. The basic protein CM-TnI (p I ≈ 9.5) and peptides with net charges from + 2 to + 10 were separated with linear AB salt gradients varying from 5 m M to 10 m M B per min (A  5 m M KH 2PO 4 buffer, pH 6.5 or 3.0; B  5 m M KH 2PO 4 buffer, pH 6.5 or 3.0, containing 1 M KCl). All peptides and CM-TnI were eluted with KCl concentrations below ca. 0.6 M. The advantage of performing cation-exchange chromatography over anion-exchange chromatography was demonstrated for the separation of peptides which, while acidic or weakly basic at neutral pH, through protonation of the acidic functions results in positively charged peptides at pH 3.0.