Hydrophobic Interaction Chromatography
Published Online: 15 FEB 2013
Copyright © 2001 John Wiley & Sons, Ltd. All rights reserved.
How to Cite
Jennissen, H. P. 2013. Hydrophobic Interaction Chromatography. eLS. .
- Published Online: 15 FEB 2013
Hydrophobic interaction chromatography (HIC) involves the separation of protein molecules in their native and biologically active state owing to a differential interaction of these molecules with hydrophobic sites on the surface of a solid support. In the separation process, hydrophobic patches on the protein surface interact with hydrophobic molecules (e.g. alkyl residues) immobilised on the hydrophilic solid phase surface (e.g. agarose). In the paper, a historical overview is followed by a consideration of the biochemical and biophysical mechanisms involved in HIC and a discussion of the critical hydrophobicity method and other approaches to hydrophobic interaction chromatographic methods. A detailed protocol for the individual synthesis of gel libraries for specific needs has been published. The treatise is concluded with a discussion on the predictability of chromatographic results and an outlook into the future.
Positive and negative cooperativity manifests itself by binding isotherms of proteins not governed by the Langmuir equation, that is, a single binding constant but depends on site–site interactions leading to multiple binding constants.
Adsorption–desorption hysteresis manifests itself by an adsorption isotherm, which is not retraced by the desorption isotherm, due to conformational changes on the surface, dissipation of entropy and an increase in affinity.
Metastability of adsorption states occurs during adsorption–desorption hysteresis and indicates energetically that a local energy minimum exists (i.e. nonequilibrium state) in distinction to the global energy minimum, that is, true equilibrium.
Critical hydrophobicity is the threshold hydrophobicity under low salt concentrations (e.g. 50 mM Tris, 150 mM NaCl and pH 7.0) at which the adsorption of a protein can first be measured.
A quantised hydrophobicity gradient is a hydrophobicity gradient separated into a library of distinct gels of varying hydrophobicity for determining the critical hydrophobicity.
A homologous gel library consists of gels carrying covalently immobilised hydrocarbons (e.g. alkyl residues) comprising a homologous series.
The lattice-site binding function is a protein adsorption isotherm (often showing positive cooperativity) as a function of the surface concentration of immobilised hydrocarbons (e.g. alkyl residues).
The bulk ligand binding function is a protein adsorption isotherm on immobilised hydrocarbons (often showing negative cooperativity) as a function of the bulk protein concentration.
Predicting chromatographic behaviour from sorption isotherms is a major aim in understanding HIC.
Predicting chromatographic behaviour from protein structure is the ultimate aim in understanding and utilising HIC.
The critical hydrophobic patch area hypothesis is a method of predicting the chromatographic behaviour of proteins in HIC from protein structure, utilising the area of hydrophobic patches on the surface of proteins.
- agarose/Sepharose 4B;
- adsorption hysteresis;
- critical hydrophobicity;
- hofmeister series;
- multivalent interactions;