Effects of chain length and carbon load on the performance of alkyl-bonded silicas for protein separations

The retention and recovery of proteins in reversed-phase liquid chromatography with gradient elution using aqueous-organic mobile phases were found to be similar from large pore (30 nm) silica bonded to maximal extents with either propylor octyldimethylsilanes. Proteins were less retained and less completely recovered from silica partially bonded with octyldimethylsilane when used with an acetonitrile-containing mobile phase but not with 1-propanol as modifier. The elution position of proteins under gradient conditions was shown to be independent of flow-rate for fixed gradient slope, but to move later in the gradient with increases in gradient slope. The effects of gradient slope and flow-rate on resolution, separation time, and sensitivity are illustrated.