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Friday, November 25, 2011

Principle behind the use of Salts in Hydrophobic Interaction Chromatography

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

Salting in & Salting out of Protiens 

Use of Ammonium sulphate (Anti-Chaotropic agent) for binding in HIC
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 (In case of hydrophobic interaction chromatography these protein molecules interact with the hydophobic ligands of the matrix). The solubility of different proteins is reduced to different extents by salt addition.

use of Acetic acid (chaotropic agent) for Elution in HIC
A denaturating agent is a chaotropic agent, but chaotropic agents aren't necessarily denaturating agents. 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.