Binary and ternary salt gradients in hydrophobic-interaction chromatography of proteins

Hydrophobic-interaction chromatography of mixtures of acidic and basic proteins having a wide range of molecular weights and hydrophobic character was carried out by using binary and ternary salt gradients. Chaotropic and antichaotropic salts as well as organic salts were incorporated in the eluents. The stationary phase consisted of macroporous silica with surface-bound polyether moieties. At constant eluent surface tension, gradient elution with two or three aqueous salt solutions was found to be superior to single-salt gradients in modulating hydrophobic-interaction chromatography retention and selectivity. The effect was attributed to the competitive salt-specific binding to the protein molecule and/or the stationary phase surface. Chaotropic/antichaotropic salt gradient systems exhibited vastly different selectivities upon changing the nature and concentrations of salts in the eluents. In general, the retention of basic proteins increased while that of acidic proteins either decreased or remained unchanged with the use of chaotropic salts. At the same surface tension of the eluent, KSCN and KC1O4 yielded different selectivities. The addition of organic salts, such as tetrabutylammonium bromide was found to be suitable for the separation of proteins having a wide range of isoelectric points.