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Wednesday, September 14, 2011

Hydrophobic Interaction Chromatography Principle



Salting Out:
There are hydrophobic amino acids and hydrophilic amino acids in protein molecules. After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules. If enough of the protein surface is hydrophilic, the protein can be dissolved in water.
When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein. As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other. This process is known as salting out.
HIC Mechanism
Separation Based on Hydrophobic Interaction
The hydrophobic ligands on HIC media can interact with the hydrophobic surfaces of proteins. In pure
water any hydrophobic effect is too weak to cause interaction between ligand and proteins or between
the proteins themselves. However, certain salts enhance hydrophobic interactions, and adding such salts brings about binding (adsorption) to HIC media. For selective elution (desorption), the salt concentration is lowered gradually and the sample components elute in order of hydrophobicity.
The final result of a HIC separation is based therefore on interplay between the prevalence and
distribution of surface-exposed hydrophobic amino acid residues, the hydrophobicity of the medium
the nature and composition of the sample, and the type and concentration of salt used in the buffers.

HIC media are composed of ligands containing alkyl or aryl groups coupled to an inert matrix of
spherical particles. The matrix is porous, in order to provide a high internal surface area, while the ligand plays a significant role in the final hydrophobicity of the medium.

Sodium, potassium or ammonium sulfates produce relatively high precipitation. It is these salts
that effectively promote hydrophobic interaction and have a stabilizing influence on protein
structure. In practice sodium, potassium or ammonium sulfates effectively promote ligand-
protein interactions in HIC and have a stabilizing influence on protein structure. Hence the most
commonly used salts are (NH4)2SO4, Na2SO4, NaCl, KCl and CH3COONH4.
If the protein of interest does not bind under high salt conditions, use a more hydrophobic
medium. If the protein of interest binds so strongly that non-polar additives are required for
elution, decrease the salt concentration in the start buffer or use a less hydrophobic medium.