Hydrophobic Interaction Chromatography


Ammonium Sulfate Precipitation

Ammonium sulfate precipitation was one of the earliest forms of protein purification. By increasing the concentration of ammonium sulfate in steps, differential precipitation of the protein mixture would occur. Centrifugation of the precipitate resulted in “cuts” of protein populations, significantly enriching the purity over the starting material. The precipitate would normally resolubilize once the salt was removed, either by filtration or by dialysis.

Hydrophobic Interaction Chromatography

The same principles used in ammonium sulfate precipitation can be used as a paradigm to describe hydrophobic interaction chromatography (HIC). HIC is the most recent addition to the different modes of chromatography.

Most proteins, and to a much lesser extent hydrophilic molecules (e.g. DNA and carbohydrates), have hydrophobic areas or patches on their surface. Solvation of these patches is energetically unfavorable and results in the formation of hydrophobic cavities in the aqueous mobile phase. The promotion of the hydrophobic effect (by addition of lyotropic salts) drives the adsorption of hydrophobic areas on a protein to the hydrophobic areas on the solid support. This is thermodynamically favorable in that it reduces the number and volume of individual hydrophobic cavities. Reducing hydrophobic interaction by decreasing the concentration of lyotropic salts results in desorption from the solid support.

HIC is unique in that proteins bind at high salt concentration and elute at low salt concentration. This is manifested in a reverse salt gradient which is an immediate indication that HIC is being employed.

HIC, a milder form of RPC

HIC is sometimes referred to as a milder form of RPC. However, HIC utilizes gentler binding and elution conditions which typically retain the biological activity of the target protein. Also, the use of lyotropic salts in HIC is preferred at large scale over the use of organic solvents.

Find out more

TSKgel columns for HIC analysis »
Toyopearl and TSKgel resins for purification by HIC »

For an overview refer to our chromatography mode poster General Principles of Chromatography, send us an e-mail to receive a printed copy.