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Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a separation method that isolate biomolecules from complex mixtures using reversible hydrophobic interactions. This separation technique is often the polishing step in a protein purification strategy. It exploits the hydrophobic regions on protein surfaces and interaction of target proteins with ligands on hydrophobic resins. Running buffer properties influence the reversible interaction between proteins and hydrophobic ligands.

Principle of HIC

HIC is useful when separating proteins with varying degrees of surface hydrophobicity. The principle for adsorption of proteins to HIC media complements ion exchange and size exclusion chromatography. In hydrophobic interaction chromatography, hydrophobic groups like butyl, octyl, or phenyl are attached to the stationary column. Sample molecules with hydrophobic amino acid side chain regions are applied to HIC columns in high salt buffers. These proteins interact with and bind to the hydrophobic groups in the column.

The salt in buffers minimizes the solvation of samples. Decreased solvation exposes the hydrophobic regions for speedy adsorption by media. Molecules with high hydrophobicity require less salt to ensure binding. Use a decreasing salt gradient to elute your samples from the column.

Factors to consider during HIC

General considerations to make during hydrophobic interaction chromatography include:

  • Ligand: The type of immobilized ligand in your hydrophobic interaction chromatography resins affects protein adsorption.
  • Degree of substitution: An increased substitution of the immobilized ligand increases protein binding capacity.
  • Matrix: Commonly used supports are hydrophilic carbohydrates, such as cross-linked agarose and synthetic copolymers.
  • Salt concentration: Adding structured salts to equilibration buffers and samples enhances protein-ligand interactions during HIC.
  • Temperature: A change in temperature affects the hydrophobic interaction. Temperature also affects the solubility and structure of proteins. Fluctuations in temperature influence how proteins interact with hydrophobic resins.

Hydrophobic resins FAQs

Here are answers to some frequently asked questions about hydrophobic resins.

What is a HIC resin?
Hydrophobic resins or HIC resins are the stationary phase of liquid chromatography. HIC resins are composed of a porous matrix, such as agarose beads coupled to a ligand. Hydrophobic resins are available in bulk or in prepacked columns for the capture and purification or polishing of biomolecules like antibodies or plasmids.

What are HIC beads?
HIC beads form the resins used in HIC. HIC beads have varying particle diameters and concentrations. Agarose beads are a common matrix for hydrophobic resins. Polystyrene/divinylbenzene is also used. The matrix structure allows movement of the mobile phase over the resin during chromatography.

What are the different hydrophobic ligands?
Common ligands used during HIC include phenyl, butyl, and octyl groups. Cytiva offers a range of hydrophobic resins that use these ligands.

How do I know which HIC resin to choose?
Select hydrophobic resins based on the hydrophobicity of your target biomolecules. We offer screening and selection kits to help you choose an appropriate resin for high flow, capacity, and resolution. You can also use our Purify web app to help choose the right resin or prepacked column for your needs.