Additionally, the model was applied only to isotherm data, and the model derivation results in a predicted log–log relationship between the chromatographic retention factor and salt concentration that is generally inconsistent with experimental observation. However, the model was applied to only a single protein species and ligand type, so that the relationship between model parameters and the properties of the protein and ligand are not clear. The model improved upon previous efforts by explicitly accounting for ligand density and protein loading effects.
Further, the model includes a logarithmic dependence on salt concentration that diverges in the limit of no salt in the mobile phase, and the model cannot be extended to describe the effects of protein loading.Ī different approach to modeling salt effects in protein HIC was taken by Chen and Sun, who describe a “desolvation” of hydrophobic patches on the protein surface and hydrophobic resin ligands by salt ions, followed by adsorption of the dehydrated protein to the dehydrated ligands. However, the derivation of the governing equation results in an integrating constant that is independent of salt concentration and is not related to any physical system attributes, but is required to describe the behavior of adsorption systems.
In this model, the effect of salt concentration in the mobile phase is related to the number of interfacial water molecules and salt ions that are displaced to the bulk solution upon protein adsorption. The preferential interaction theory has been successfully applied to the description of salt effects in HIC retention. Fausnaugh and Regnier studied the HIC adsorption of several proteins in the presence of different types of salts and found that the solvophobic theory alone could not adequately explain retention differences. The solvophobic theory, based on the association and solvation of the participating species, describes retention in terms of the molal surface tension increment of the salt. There have been considerable efforts toward theoretical understanding of the mechanism of protein retention on hydrophobic chromatography surfaces. Design efforts would be greatly aided by a model that enables the reduction of the design space based on a fundamental understanding of the thermodynamics of HIC retention. Process design and optimization is usually achieved through exhaustive experimentation, and substantial work has been conducted to develop high-throughput experimental methods to facilitate the screening of a large process design space. Hydrophobic interaction chromatography (HIC) continues to be an important technique used in the purification of biological macromolecules however, the relationships between many process variables and the observed chromatographic behavior are poorly understood.
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