Hydrophobic interaction chromatography operating principle
Separation of molecules by hydrophobic interaction chromatography (HIC) relies on differences in hydrophobic groups on the surface of solute molecules. In turn, these hydrophobic groups tend to bind to hydrophobic groups on the surface of an insoluble matrix.
The legend below will identify the salient features of the diagram above. The reversible interaction between the solute and the matrix is increased at high ionic strength. Hence, typically, protein solutes are introduced onto the column at high concentrations of ammonium sulphate, and eluted by running a decreasing gradient of this salt.
The hydrophobic ligands on the matrix can be chosen with differing binding strength: a strongly hydrophobic matrix will have phenyl or octyl groups, while weakly hydrophobic matrices may bear ether or isopropyl groups.
Applications of hydrophobic interaction chromatography
Hydrophobic interaction chromatography is best used as an intermediate purification technique. For example, after precipitation with ammonium sulphate, a mixture of proteins may be brought back in solution by decreasing the concentration of ammonium sulphate to about 1.5M, and applied to the column. Washing with this solution will remove unbound material. Gradually decreasing the concentration of the eluting solution will then elute the different components.
- Protein Purification (Amersham Handbook) (see page 77)