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What is Hydrophobic interaction chromatography?

Hydrophobic interaction chromatography (HIC) is a preparative and analytical purification technique that helps isolate biomolecules such as proteins and monoclonal antibodies (mAbs) based on surface hydrophobicity. HIC uses a reversible interaction between the target molecules and hydrophobic ligands coupled to an HIC resin.

HIC vs reversed phase chromatography

Reversed phase chromatography (RPC) also isolates proteins and peptides based on their hydrophobicity. However, RPC resins are often more hydrophobic than HIC resins. Their hydrophobic properties create powerful interactions that require organic and nonpolar solvents like acetonitrile for efficient elution. Because most proteins are denatured by organic solvents, HIC is well-suited for protein separations and purification. Common applications for RPC are purity checking and mapping of peptides.

HIC chromatography columns FAQs

Here are answers to some frequently asked questions about HIC chromatography columns.

What is a HIC column?

HIC columns are prepacked with HIC resin, used to separate biomolecules from a mixture based on differences in hydrophobicity of the ligand and target molecule. The most common ligands found in HIC resins include butyl, octyl, and phenyl.

Why does high salt promote hydrophobic interactions?

High salt concentration enhances hydrophobic interactions because salt reduces the solvation of sample molecules to expose their hydrophobic regions. The amount of salt required for optimal binding is inversely proportional to the hydrophobicity of target molecules.

How does HIC column chromatography separate proteins?

HIC chromatography columns separate protein or other molecules based on their surface hydrophobicity. In this technique, mixtures containing proteins with hydrophobic and hydrophilic regions are applied to columns in high salt buffer conditions. This allows the adsorption of the target molecule to the HIC chromatography column or resin.

How are HIC columns used?

Use HIC chromatography columns in protein purification for analytical and preparative applications. HIC processes can be used for capture, intermediate, and polishing chromatography. HIC chromatography columns also help remove impurities such as product-related contaminants.

Which HIC chromatography column is right for my application?

Use our Purify app to find the right HIC column or resin for your needs.