Biomolecular adsorption phenomena in hydrophobic interaction chromatography

Hydrophobic Interaction Chromatography (HIC) separates biomolecules on the basis of surface hydrophobicity. Support, solute and mobile phase interactions were studied using chromatography and densimetric techniques. Salt chromatography studies showed that the kosmotropic or chaotropic nature of the salt determined its interaction with a mildly hydrophobic stationary phase. Densimetric techniques were used to probe the salt-support interaction phenomena further by measuring preferential interaction parameters of salts with sorbents. The preferential interaction parameter of a salt with a component denotes the net salt inclusion or exclusion in the immediate environment of a component. Polar kosmotropic salts were excluded from supports on the order of the lyotropic series, and the interaction parameter was concentration dependent. HIC adsorption phenomena was related to mobile phase interaction parameters using the thermodynamic theory of linked functions. Retention studies of lysozyme and myoglobin show a linear relation between the anionic lyotropic number and the protein\u27s capacity factor. A model was developed to predict a solute\u27s retention behavior in various mobile phases, given a set of conditions defining properties of the stationary phase and the protein