Hydrophobic interaction chromatography (HIC) is a commonly used chromatography technique for purifying proteins. It utilizes salting-out salts to facilitate the binding of native proteins to weakly hydrophobic ligands. There have been three proposed mechanisms for the promoting effects of salting-out salts, which include the dehydration of proteins by salts, cavity theory, and salt exclusion. To evaluate the above three mechanisms, an HIC study was conducted on Phenyl Sepharose using four different additives. These additives included a salting-out salt (NH4)2SO4, sodium phosphate that increases the surface tension of water, a salting-in salt MgCl2, and an amphiphilic protein-precipitant polyethylene glycol (PEG). Results indicated that the first two salts resulted in protein binding, while MgCl2 and PEG led to flow-through. These findings were then used to interpret the three proposed mechanisms, which showed that MgCl2 and PEG deviated from the dehydration mechanism, and MgCl2 also deviated from the cavity theory. The observed effects of these additives on HIC were reasonably well explained for the first time by their interactions with proteins.
Keywords: Cavity theory; Dehydration mechanism; Hydrophobic interaction chromatography.
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