Hydrophobic-Interaction Chromatography
 
Hydrophobic (literally meaning “water hating”) describes nonpolar compounds that have little affinity for polar molecules, such as water, and self-associate in a polar environment because of the hydrophobic effect . Hydrophobic interactions are energetically favorable because of an increase in entropy (DS > 0) that originates when the solvent water molecules leave the more ordered structure around the nonassociated nonpolar compounds for the more unstructured, bulk polar environment. If matrices in a chromatography column provide hydrophobic surfaces to interact with analytes and one can separate mixtures of molecules with hydrophobic moieties, we are dealing with hydrophobic-interaction chromatography.
The principle of hydrophobic-interaction chromatography was originally known (1) by the name “salting-out chromatography,” but it acquired its current name only after the synthesis of specific stationary phases in the early 1970s (2-4). In hydrophobic-interaction chromatography, retention is promoted by high concentrations of an appropriate salt, and elution is achieved with low salt concentrations. The matrices in the column are very hydrophilic and have only a very mild hydrophobicity. The increase in concentration of a salting-out salt drives the analytes out of the mobile phase and onto the stationary phase, much like the precipitation of highly water-soluble proteins or hydrophilic polymers that occurs under conditions of high ionic strength.
Although many view hydrophobic-interaction chromatography as an extension of reversed-phase chromatography to an aqueous mobile phase without organic modifiers, there are a number of basic differences between these two separation techniques. In hydrophobic-interaction chromatography, the matrix is hydrophilic and is substituted with short-chain groups, such as phenyl, butyl, or octyl (see Ref. 5 for a list of different matrices), and the mobile phase is usually an aqueous salt solution. In reversed-phase chromatography, the matrix is an octyl (C8) or octadecyl (C18)-derived silica, and the mobile phase is usually a mixture of water and a less polar organic modifier (eg, methanol or acetonitrile). These two techniques exploit the different sources of the hydrophobicity of proteins. Hydrophobic-interaction chromatography depends on surface hydrophobic groups, which arise from the protein's tertiary and quaternary structures, and is carried out under conditions that maintain the integrity of the protein conformation. Reversed-phase chromatography is carried out on the unfolded protein, in which nearly all the hydrophobic groups are exposed to the matrix, and depends on the protein's primary structure. Proteins frequently denature under conditions of reversed-phase chromatography because organic solvents are needed for elution and/or because of the strength of the interaction with the stationary phase, whereas the mild elution conditions and mild hydrophobic interaction of hydrophobic-interaction chromatography matrices allow the elution and recovery of native proteins.
 The most interesting aspect of hydrophobic-interaction chromatography is the frequent increase of retention with increasing temperature because this chromatographic method is an entropy-driven process. This is the opposite of any other type of retention chromatography. It should also be pointed out that the proteins are loaded onto the hydrophobic-interaction chromatography column using a buffer with a high salt concentration as the starting mobile phase and are then eluted by a gradient with decreasing ionic strength. This is the opposite of the procedure employed in ion-exchange chromatography .
 Hydrophobic-interaction chromatography is extremely useful for purifying proteins and peptides. The principle of this chromatographic method is orthogonal to those of ion-exchange and size-exclusion chromatography methods. Therefore it is used effectively in schemes that combine all three chromatographic methods to separate proteins from complex mixtures. However, the solubilities of proteins in high-salt buffers are limited by the salting-out effect. This constrains the applications of hydrophobic-interaction chromatography for preparative purposes.
 
References
1. A. Tiselius (1948) Ark. Kemi. Mineral. Geol. B26, 1. 
2. S. Shaltiel and Z. Er-el (1973) Proc. Natl. Acad. Sci. USA 70, 778–781. 
3. B. H. J. Hofstee (1973) Anal. Biochem. 52, 430-448. 
4. S. Hjertén, J. Rosengren, and S. Pahlman (1974) J. Chromatogr. 101, 281–288. 
5. K.-O. Eriksson (1989) in Protein Purification: Principles, High Resolution Methods, and Applications (J.-C. Janson, and L. Rydén, eds.), VCH, New York, pp. 207–226. 
6. R. M. Kennedy (1990) in Guide to Protein Purification (M. P. Deutscher, ed), Methods in Enzymology 182, Academic Press, New York, pp. 339–343. 
7. U. D. Neue (1997) HPLC Columns: Theory, Technology, and Practice, Wiley-VCH, New York.