The Effects of Pore Diameter and Ligand Chain Length on Fast Liquid Chromatography of Proteins and Peptides

Abstract

Fast flow separations of proteins and peptides were successfully carried out on microbore columns containing reversed phase supports of three pore diameters. Increasing flowrates from 0.25 to 3 ml/min (1.2 mm/s–14.4 mm/s) did not show any adverse effects on the separations when SynChropak RPP-1000 and RPP-4000, which have 1000Å and 4000Å pore diameters, were used. At 3 ml/min, the 1000Å and 4000Å pore diameters yielded narrower peaks for proteins than the 300Å support, whereas the latter exhibited better resolution for peptides. Analyses in less than five minutes were achieved.

A series of ligand chains (C-4, C-8 and C-18) showed few differences in retention or resolution for either protein or peptide standards. Longterm stability of the 300Å and 4000Å supports was in excess of 25,000 column volumes when run at 3 ml/min with 0.1% trifluoroacetic acid.