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Sunday, April 27, 2014

Hydrophobic Interaction Chromatography - Theory and Principle


Hydrophobic Interaction chromatography, is a powerful technique for the separation and purification of biomolecules. Hydrophobic Interaction Chromatography is widely used for the separation and purification of proteins in their native state, as well as for isolating, protein complexes and studying protein folding and unfolding.




HIC was initially termed as salting out. Hydrophobic Interaction chromatography,utilizes the reversible interaction of protein and the hydrophobic ligand for the separation of protein mixtures.

Hydrophobic Interaction Chromatography (HIC) Mechanism

Proteins separates out based on the increasing order of hydrophobicity.

Proteins containing hydrophobic and hydrophilic regions are applied to an HIC column, in a high-salt buffer.

The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that, become exposed are adsorbed by the media.

The more hydrophobic the molecule, the less salt is needed to promote binding.

Usually a decreasing salt gradient, is used to elute samples from the column, in the order of increasing hydrophobicity.

Sample elution, may also be assisted by the addition of, mild organic modifiers or detergents to the elution buffer.

Hydrophobic Interaction Chromatography Theory

There are three major theories, explaining the mechanism of hydrophobic interaction chromatography. They are
  • Salting Out and Hydrophobic Interaction Theory,
  • Thermodynamic Theory, and
  • Surface Tension, van der Waals Forces Theory.

Salting Out and Hydrophobic Interaction Theory

The elution or precipitation strength, of an ion is described by the Hofmeister series. Small, highly charged ions are strong precipitators, whereas organic acids and bases, have a more stabilizing effect, in the presence of proteins in solution. The term chaotropic, refers to the ability of the ion to produce chaos in the water structure.

salting in and salting out

Anti chaotropic salts, such as ammonium sulphate and sodium sulphate, expose hydrophobic patches on proteins by removing the highly structured water layer, which usually covers these patches in solution. As a result hydrophobic residues on a protein molecule can interact with the hydrophobic ligands of the matrix. Salts can also reduce the solubility of proteins by shielding charged groups which normally keep proteins apart in solution. When the electrostatic charge on protein molecules are shielded, the molecules can easily interact, form aggregates and eventually precipitate. The solubility, of different proteins is reduced to different extents by salt addition.

hofmeister series salts HIC


Chaotropic agents disrupt the intermolecular forces between water molecules, allowing proteins and other macromolecules to dissolve more easily. Chaotropic agents interfere with stabilizing intramolecular interactions mediated by non covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects.

Thermodynamics Theory of Hydrophobic Interaction Chromatography

Thermodynamic theory of HIC, directly relates to Gibb’s Free Energy equation.

The phenomenon of increased interaction, in the presence of salting out ions, is explained by a higher gain in entropy when water is transferred from the surface of a nonpolar molecule to the bulk of water.

hydrophobic interaction chromatography

In hydrophobic interaction, the entropy change is of greatest importance . If hydrophobic molecules such as aliphatic carbon chains are immersed in water, the monolayer of water molecules in contact with a carbon chain, will be in higher order than those in the bulk of the water. If two or more hydrophobic structures come together, the surface that has to be covered by, ordered water molecules decreases, some water molecules join the less ordered bulk water, and the entire system gains in entropy and thus, decreases its free energy. This state will be favourable, for energetic reasons and thus promoted.


Hydrophobic Interaction chroamtography thermodynamics


Surface Tension, van der Waals Forces Theory.

A third theory suggests that, van der Waals forces are responsible, for the hydrophobic interaction in HIC.

This is supported by the fact that, these forces should be increased as the order of water increases, in the presence of salt.

Hydrophobic Interaction Chromatography Media

In HIC, media is described based on:
  • Ligand, 
  • Ligand Density, 
  • Available Capacity.
Available Capacity is the, actual amount of protein that can bind to the media. If flow rate is included in the defined conditions then it is called as Dynamic Binding Capacity.

Dynamic Binding Capacity, is based on these factors
  • Salt Concentration, 
  • Flow Rate,
  • Temperature and 
  • pH to a lesser extent.
The Characteristics, of good Hydrophobic Interaction Chromatography Media /  Matrix are,
  • High Binding Capacity, 
  • Physical Stability, 
  • Chemical Stability and
  • The Inert Matrix.
Hydrophobic Interaction Chromatography Ligands

Ligands Used in, Hydrophobic Interaction Chromatography meida has Alkyl or Aryl Groups.

Phenyl 650 HIC matrix
Phenyl 650

butyl S 650 hydrophobic interaction matrix
Butyl S 650







Alkyl shows, pure hydrophobic character, while Aryl shows mixed behaviour.


Ligand attachment, to the matrix in HIC is done through glycidyl ether.

Binding and Elution in HIC

Binding is done at high salt concentration: 1 to 2 Molar Ammonium Sulfate or 3 Molar sodium chloride

Elution is performed, by reducing the salt concentration.

Elution of proteins from the HIC, will be in the increasing order of hydrophobicity.

References
  • Protein Purification Technical Resources, GE Amersham.
  • Protein Purification Technical Resources, Biorad.
  • Protein Purification Technical Resources, TOSOH Biosciences.
  • Hydrophobic Interaction Chromatography:Fundamentals and Applications in Biomedical Engineering, Andrea Mahn.
  • Hydrophobic Interaction Chromatography, Encyclopedia of Bioprocess Technology.
  • Calibration and Optimization of Hydrophobic Interaction Chromatography , Alex Olsson.