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The Basics of Hydrophobic Interaction Chromatography

Posted by The Protein Man on Dec 24, 2014 2:11:00 PM
The Protein Man

Basics of Hydrophobic Hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state.  HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding. So, how does HIC work? What are the principles behind this technique? To understand the mechanism behind this type of chromatography, here are some things you definitely need to know.

How Does HIC Work?

In a nutshell, HIC (also known as 'salting out') separates protein molecules using the properties of hydrophobicity. In this method, proteins containing both hydrophilic and hydrophobic regions are applied to an HIC column under high salt buffer conditions.

The salt in the buffer (usually ammonium sulfate) reduces the solvation of sample solutes and exposes the hydrophobic regions along the surface of the protein molecule. This facilitates the adsorption of these hydrophobic regions to the hydrophobic areas on the solid support and precipitates (crystallizes) proteins out of the solution.

Keep in mind that when performing this type of chromatography, the addition of lyotrophic salts enhances the hydrophobic effect and reduces the number and volume of individual hydrophobic cavities while decreasing the salt concentration will result in desorption from the solid support. As such, sample elution can be facilitated through decreasing salt gradient. Mild organic modifiers or detergents may also be added to the elution buffer to aid in the elution process.

Using the principles of HIC, you can bind proteins from aqueous solutions in varying degrees depending on the structure of your protein of interest, salt concentration, pH, temperature and organic solvents used.

Hydrophobic Interaction Chromatography: Uncovering the Underlying Theories

There are three major theories that may help explain the mechanism behind HIC – (1) the salting out theory, (2) the thermodynamic theory and (3) the surface tension or Van der Waals forces theory.

Salting Out Theory

According to the salting out theory, the hydrophobic amino acids form protected hydrophobic areas upon folding in aqueous solution while the hydrophilic amino acids allow proteins to bond with the hydrogen molecules in the water. As a result, the probability that the protein will dissolve in water will increase if enough hydrophilic areas are present in the surface of the protein molecules.

However, upon the addition of anti-chaotrophic salts (such as ammonium sulfate and sodium sulfate) in the solution, some of the water molecules will interact with the salt ions instead of the charged part of the protein. When the protein-protein interactions in the solution become stronger than the solvent-solute interactions, the protein molecules react freely with one another - allowing them to aggregate and eventually precipitate out of the solution. Hence, the addition of salt in the solution reduces the solubility of different proteins to varying extents.

Thermodynamics Theory

This theory states that the interaction between hydrophobic molecules is an entropy-driven process, based on the second law of Thermodynamics. The hydrophobic interaction between two or more non-polar molecules in a polar solvent solution is a spontaneous process governed by a change in entropy. However, such interactions can be altered by controlling the temperature or by modifying the solvent polarity.

As such, when a non-polar molecule comes into contact with a polar solvent such as water, there will be an increase in the degree of order of the solvent molecules surrounding the hydrophobic molecule. As long as enthalpy does not increase significantly, this will produce a decrease in entropy and provide an overall positive change in the Gibbs energy.  As such, the dissolution of a non-polar molecule in a polar solvent will not occur spontaneously since it is not thermodynamically favorable.

However, when you put two or more non-polar molecules in a polar environment, the hydrophobic surfaces of the macromolecules will be hidden from the polar surrounding and the hydrophobic molecules will aggregate spontaneously. The highly structured solvent molecules surrounding the exposed surface of the hydrophobic molecules will be displaced towards the bulk of the solvent consisting of less structured molecules.  As a consequence, entropy increases while the Gibbs energy decreases. Under such cases, hydrophobic interaction becomes a thermodynamically favorable process.

Surface Tension, Van der Waals Theory

This theory suggests that the hydrophobic interaction in HIC is dependent on van der Waals forces between proteins and immobilized ligands since these forces increase as the ordered structure of water increases in the presence of salting out salts.

Image Source : University of Exeter

Topics: Protein Purification

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