Hydrophobic interaction chromatography : The synthesis and the use of some alkyl and aryl derivatives of agarose

Abstract

Aliphatic and aromatic alcohols in the form of glycidyl ethers have been coupled to agarose gels. These neutral agarose derivatives, which thus contain hydrophobic substituents, have been used as adsorbents in hydrophobic interaction chromatography. The coupling yield and the degree of substitution have been determined for one aliphatic and one aromatic model substance. Different fractionation problems require different degrees of hydrophobicity of the substituents. To “tailor make” gels, the hydrophobicity can be varied in small steps by the use of aliphatic alcohols of different chain length. The agarose derivatives described have been used for the purification of proteins, demonstrated with a plasma fractionation, viruses (STNV) and even whole cells (baker's yeast). Under suitable experimental conditions, the interactions can be very mild (enzyme activities have been recovered in a 50–100% yield). Enzyme reactors with a high capacity can be prepared in a simple manner by applying the enzyme solution at any pH on to a suitable hydrophobically interacting bed. As the enzymes are not covalently linked to the bed, they can easily be recovered in the free form. Contrary to ion-exchange chromatography, the adsorption in hydrophobic interaction chromatography decreases with a decrease in ionic strength and temperature.