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

Chromatographic separations of a series of highly basic peptides on commercially available 300-Å pore size CM 300 weak cation-exchange columns have been compared at various loads, pHs and ionic strengths of the eluent. On analytical columns (250 x 4.1 mm I.D.), mixtures of basic peptides containing 7 - 9 nmole of each component were separated with a 50 mM KH₂PO₄-KC1 gradient (pH 4.5) and under isocratic conditions (pH 4.5 and 6.5). The isocratic conditions demonstrated the effects of pH and ionic strength on retention time and resolving power on the CM 300 column. The load capacity of a CM 300 preparative column (250 x 10 mm I.D.), studied under gradient conditions (50 mM KH₂PO₄, 0.2 - 0.4 M KC1, pH 4.5 and 6.5), revealed that its capacity is much greater at pH 6.5. Loads up to 10-20 mg (6.6-13.3 μmol) could be applied before peaks in the crude peptide sample tested were seen to fuse.