Comparison and evaluation of HIC columns of different hydrophobicity

Abstract

Retention and selectivity in hydrophobic interaction chromatography (HIC) depend both on the type of stationary phase and on the mobile phase. In the last few years various high performance packing materials and columns have been introduced for HIC resulting in a range of different retentions and selectivity. We have investigated the effect of the stationary phase on the retention of various proteins. The retention of some solutes of different hydrophobicities were measured on three commercial HIC columns (TSK-Phenyl, Synchropack-Propyl, CAA-HIC) under isocratic conditions using water-methanol mixtures as eluent. The log kw values determined according to the literature were devalues determined according to the literature were dependent on the type and structure of the stationary phase and indicated a much less hydrophobic character for these columns than that obtained for reversed phase columns. Gradient separations were then carried out on a standard protein mixture using ammonium sulfate and sodium citrate to change the gradient time. In order to compare the effect of the stationary phase and the two salts investigated apparent capacity factors (kg) were determined and plotted against the gradient time obtained for the three columns in the two eluent system. It was shown that the type of stationary phase had a significant effect on the retention of proteins. In addition, the effect of the mobile phase composition, i.e. salt type, was considerably different on the various stationary phases. In order to exploit the potential of HIC to modulate selectivity for the separation of proteins, the combined effect of the stationary phase and the type of salt should be taken into account.