The aim of hydrophobic interaction chromatography (HIC) is to separate proteins based on their differences in surface hydrophobicity. As high salt enhances the hydrophobic effect, proteins are bound to the column under high salt conditions and eluted in a gradient to low salt. Organic solvents can also be used for the elution, but as most proteins are destabilized by organic solvents, this is not commonly utilized (except for peptides). Often an ammonium sulfate gradient is used for elution (for example a gradient from 2 M (NH₄)₂SO₄ to low salt).

The characteristics of the HIC medium depend on both the matrix material and the ligand. The choice of matrix will be determined by the degree of resolution that is required, the binding capacity and the flow rate that will be used. The nature of the ligand (hydrophobicity) and the degree of ligand substitution on the matrix are important for the selectivity of the HIC medium. Commonly used matrices include for example SOURCE 15 material, Sepharose High Performance, Sepharose 6 Fast Flow and Sepharose 4 Fast Flow. Popular ligands are for example Phenyl, Butyl-S, Butyl, Octyl, Ether and Isopropyl. As the most suitable HIC medium is hard to determine a priori, often some screening is required to find the most optimal HIC medium and conditions.

In reverse phase chromatography (RPC) more hydrophobic surfaces are used than in traditional hydrophobic interaction chromatography, which leads to stronger interactions. As non-polar organic solvents (e.g. isopropanol, acetonitrile) are required for elution, RPC is not commonly used for proteins, but it is a popular technique to separate peptides. Often ion-pairing reagents are utilized to enhance the hydrophobic interaction (TFA can be used to pair with amino groups for example).