SELECTIVITY OPTIMIZATION OF REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC PEPTIDE AND PROTEIN SEPARATIONS BY VARYING BONDED-PHASE FUNCTIONALITY

Several chemical bonded-phase modified silicas were prepared using sterically protected monofunctional silane reagents which varied widely in structure and polarity. Since some of these bonded-phase packing materials are highly polar (hydrophilic), resistance to acid-catalyzed bonded-phase loss by hydrolysis was examined, and observed to remain high even for the highly polar Diol bonded-phase functionality. Modification of the surface of 300 Angstrom pore size, fully hydroxylated and base-deactivated silica microspheres with these sterically protected silanes yielded HPLC column packing materials for examination of separation selectivities in reversed-phase separations of peptide and protein mixtures. Distinct separation selectivities were apparent for each bonded-phase functionality. Selectivity differences ranged from limited band spacing changes for steric-protected C-18 and C-8 bonded-phases, to reversal of elution order for the more polar C-3 and CN bonded phases. The use of column-based selectivity differences between sequential reversed-phase separation steps is used for the two-step HPLC isolation of a recombinant human amyloid precursor polypeptide fragment from a crude bacterial extract.