Comparison of Alkyl-Bonded Alumina Stationary Phases for Peptide Separations by High Performance Liquid Chromatography

Abstract

The separations of synthetic octapeptide mixtures and tryptic digests of larger proteins were compared on columns packed with several alkyl-bonded alumina-based stationary phases of differing chain lengths, particle pore diameters and particle shapes. Employing both standard and optimized mobile phase gradient conditions, it was found that (1) columns of different lengths (15 cm and 25 cm) packed with the cctadecyl-bonded alumina phase gave similar peptide separations: (2) narrow pore (diameter = 11 nm) alumina-based phases gave equal or superior peptide separations compared to those obtained on corresponding wide pore (diameter = 21 nm) phases; (3) octadecyl (C-18) bonded alumina phases gave superior peptide separations compared to those obtained on corresponding octyl (C-8) phases; and (4) alumina-based phases consisting of fused microplatelet particles gave superior separations compared those obtained on corresponding phases consisting of spherical particles. Peptide chromatographic recoveries were also found to be similar on all alkyl bonded alumina-based phases tested. Differences in these effects with those observed for peptide separations on silica-based phases are attributed to the enhanced solute mass transfer properties of the fused microplatelet alumina particles.