Optimal operation conditions for protein separation in hydrophobic interaction chromatography

Protein retention in hydrophobic interaction chromatography is determined by protein physicochemical properties and by system characteristics. In this paper we present an attempt to determine the optimal operation conditions that would allow the separation of binary protein mixtures. The statistically significant system variables were determined, and then empirical models were obtained which explained more than 92% of variability in dimensionless retention time based on salt properties, ionic strength of the initial eluent and substitution degree of the resin. These variables were optimized in order to achieve the maximum retention time difference between two proteins in a mixture. The optimum operation conditions as predicted by the models were tested experimentally, showing a good agreement with predicted separation. We concluded that it would be possible to determine the system conditions that allow the maximum separation of two proteins based on the main system properties. The methodology proposed here presents potential to be applied to partially characterized systems, however, it could be improved if protein's properties were included explicitly in the models.