Hydrophobic interaction chromatography

What is hydrophobic interaction chromatography?

Hydrophobic interaction chromatography (HIC) separates proteins according to differences in their surface hydrophobicity by utilizing a reversible interaction between the proteins and the hydrophobic surface of a HIC resin.

The interaction between hydrophobic proteins and a HIC resin is influenced significantly by the presence of certain salts in the running buffer. A high salt concentration enhances the interaction while lowering the salt concentration weakens the interaction.

How does hydrophobic interaction chromatography work?

Proteins with different degrees of surface hydrophobicity can be separated using hydrophobic interaction chromatography. The proteins interact with the hydrophobic surface of the HIC resin.

As the ionic strength of the buffer is reduced, the interaction is reversed and the protein with the lowest degree of hydrophobicity is eluted first. The most hydrophobic protein elutes last, requiring a greater reduction in salt concentration to reverse the interaction.

Hydrophobic interaction chromatography

When should I use hydrophobic interaction chromatography?

Hydrophobic interaction chromatography can be used for capture, intermediate purification, or polishing steps. As samples should be in a high salt concentration to promote hydrophobic interaction, HIC is well-suited for capture steps after sample cleanup by ammonium sulfate precipitation or for intermediate steps directly after an ion exchange separation.

In both situations, the sample is already in a high salt solution and, apart from the addition of more salt, no further preparation is required. As a HIC separation will concentrate the protein of interest into a reduced volume, fractions can also be transferred directly to size exclusion chromatography.

How does reversed phase chromatography, RPC, differ from HIC?

Reversed phase chromatography also separates proteins and peptides on the basis of hydrophobicity. Compared with a HIC resin, the surface of an RPC resin is usually more hydrophobic, leading to stronger interactions that, for successful elution, must be reversed using non-polar, organic solvents such as acetonitrile or methanol.

As many proteins are denatured by organic solvents, RPC is not generally recommended for protein purification. Instead, RPC is well-suited for applications such as peptide mapping or purity checking.

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