Role of Physical Forces in Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is a new non-biospecific liquid chromatography method for the separation of proteins, and other biological macromolecules; the mobile phases are aqueous and the adsorbents are agarose beads coated with ionogenic, or non-ionogenic, hydrocarbonaceous ligands. A non-traditional interpretation is given here for the mechanisms of retention and elution in HIC in terms of the several physical forces between the protein and the adsorbent, chiefly the van der Waals attraction (comprising dispersion, orientation and induction) and the electrostatic double layer interaction. From a qualitative analysis of the hydrogen-bond and the structural features of water, it is shown here that the role of the alkyl ligands on the adsorbent, the lyotropic salt effects and the effect of additives to the mobile phase such as ethylene glycol can all be unifyingly represented in the Hamaker coefficient of the van der Waals attraction between protein and adsorbent in water. An increase in the number and length of alkyl ligands, and the addition of structure-making (“salting-out”) salts at high ionic strengths increase the latter attraction while the addition of structure-breaking “salting-in” salts or organic solvents such as ethylene glycol decreases the attraction. The potentials corresponding to the different physical forces add up to the total interaction potential. The shapes of the total interaction potential relevant to HIC are identified. Coefficients of adsorption and desorption are shown to be related to this potential and the high sensitivity of the latter to its parameters such as the Hamaker coefficient, is illustrated. Retention, elution and the natures of the different fractions into which a protein mixture may be separated by HIC are visualized in terms of the interaction potential.

Numerous experimental reports in HIC are classified, tabulated, and in certain cases, discussed in detail. Experimental evidence is presented for the application of ionic strength manipulations, in the low ionic strength range (electrostatic effects), in the high ionic strength range (lyotropic salt effects) as well as for the combined use of low and high ionic strength effects, retention onto adsorbents with no ligands as well as onto adsorbents with alkyl ligands of various chain lengths and number densities, temperature effects, and the effects of heterogeneities of proteins and adsorbents. Applications and variants of HIC are cited. In total, the paper summarizes various types of HIC experimental facts and explains most of them in a simple, unifying fashion.