Hydrophobic Interaction Chromatography

impression was that here was a novel method applicable not only to hydrophobic or lipophilic but also to hydrophilic, possibly to all proteins. The name 'hydrophobic chromatography' coined by Shaltiel therefore soon came to widespread use. Only a few months after Shaltiel's first paper, B.H.J. Hofstee published a series of papers leading to similar results.

As stated above, all of these hydrophobic gels were synthesized by the simple CNBr method. Some criticism however arose that positive charges, introduced by a side reaction into the matrix by the CNBr procedure, were influencing the chromatographic results on hydrocarbon-coated agaroses. A rational approach and solution to this problem proved difficult since the chemical mechanism of the CNBr coupling reaction was conclusively clarified only some time later by M. Wilchek in 1981. Wilchek found that the number of charges introduced into the matrix dependend on the pH of the washing solution and the length of the washing procedure after CNBr activation of the agarose, since intermediate cyanate esters were selectively hydrolysed in alkali in contrast to the imidocarbonates which were hydrolysed in acid. Thus pure charged isourea gels, pure uncharged imidocarbonate/carbamate gels or mixed ionic-hy-drophobic gels can be obtained by the CNBr procedure. In a later paper, Shaltiel conclusively showed that under his conditions the influence of charges in his hydrocarbon-coated agaroses had been small. In addition it was shown by various other groups that salts also effectively quenched the charges introduced by the CNBr method.

Fully uncharged hydrophobic gels were therefore synthesized in 1973 by Porath's group who reacted benzyl chloride with agarose at high temperatures. The synthesis of a graded homologous series of hydrocarbon-coated agaroses was however not possible by this method. In addition Porath demonstrated the inverse salt behaviour of proteins adsorbed on such gels. In contrast to ion exchangers, proteins were applied to these gels at high salt concentrations and eluted by decreasing the ionic strength (negative salt gradients). Interestingly the protein cytochrome c was adsorbed when 1-3 M NaCl was included in the buffer, a salt which in itself had very little salting-out potential. A similar salt behaviour of protein binding on hydrophobic gels synthesized by the CNBr procedure was reported later by Hjerten who demonstrated that Shaltiel-type gels showed similar properties as the Porath-type gels. Hjerten also suggested the term 'hydrophobic interaction chromatography' which is now popularly accepted. In 1974 Hjerten described a novel preparation of uncharged hydrophobic gels of broad potential by coupling alkyl and aryl groups via the glycidyl ether method.

In retrospect, although there is no doubt that fully uncharged hydrophobic gels are, by virtue of displaying a single (pure) type of noncovalent interaction, superior to the CNBr-prepared gels, it appears that all groups involved in the development of hydrophobic (interaction) chromatography observed the binding and fractionation of proteins by predominantly hydrophobic interactions. Both terms, 'hydrophobic chromatography' and 'hydro-phobic interaction chromatography' can be used synonymously, the shorter term 'hydrophobic chromatography' being no more a misnomer than 'affinity chromatography'.

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