HPLC retention behavior on hydride-based stationary phases
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Updated: Jul 15, 2026

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
Joseph J Pesek1, Maria T Matyska, Susan Larrabee
1Deparment of Chemistry, San Jose State University, San Jose, CA 95112, USA. pesek@sjsu.edu
Abstract:
Two stationary phases attached to a silica hydride surface, cholesterol and bidentate C18, are investigated with a number of pharmaceutically related compounds in order to illustrate the various retention mechanisms that are possible for these bonded materials. The test solutes range from hydrophilic to hydrophobic based on log P (octanol/water partition coefficient) and pKa values. The mobile phases consist of acidified (formic and perchloric acid) water/methanol or water/ACN mixtures. Of particular interest are the high organic content mobile phase compositions where the retention would increase if the bonded material was operating in the aqueous normal phase (ANP) mode. Plots of retention factor (k) versus mobile phase composition are used to elucidate the retention mechanism. A number of examples are presented where solutes are retained based on RP, ANP, or dual retention mechanisms. The silica hydride-based stationary phases can also retain compounds in the organic normal phase.
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