Characterization of hydrophobic interaction and hydrophobic interaction chromatography media by multivariate analysis

Abstract

Mapping of the selectivity of different hydrophobic interaction chromatography (HIC) media was performed using principal component analysis. The elution positions of proteins in a descending salt gradient were determined for a selection of commercially available and experimetal gels. Data regarding the proteins obtained from the literature, such as hyrophobicity indices, polar/non-polar volume ratio and charge, were compared with the elution positions. Principal component analysis revealed that the media could be divided into several groups. The map also showed that the different hydrophobicity indices could be related to the different retention patterns of the media groups. Some media were selecting mainly according to protein hydrophobicity as expressed by the fraction of hydrophobic amino acids, and their mechanism of interaction was insensitive to protein charge. On other media the charge on the protein, or rather the lack of charge, was the most important factor. The results suggest that HIC in many cases works by a combination of two different mechanisms, thus explaining the irregular behaviour which is so often experienced when using HIC media. Principal component analysis and other multivariate techniques were found to be valuable tools in the process of understanding and optimizing hydrophobic or salt-promoted interaction chromatography.