Hydrophobic interaction chromatography

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In the hydrophobe interaction chromatography (HIC) is a bioanalytical separation method of proteins in which their native form these - and hence its biological activity - maintained.

Separation principle

The separation is based on interactions of non-polar surface regions of a protein with a hydrophobic stationary phase. As in the case of salt precipitation, these interactions arise only through an increased salt concentration in the solution. An increased salt concentration leads to an increase in the surface tension, which leads to a partial removal of the hydrate shell and thus to exposure of hydrophobic areas of the protein. These hydrophobic surface areas of an analyte molecule now in turn interact with the hydrophobic residues of the stationary phase. It should be noted that the greater the hydrophobicity of a protein, the lower the salt concentration required for binding to the stationary phase. To elute the proteins, the chromatography is therefore carried out with a decreasing concentration gradient. This is probably the most important difference to reverse phase chromatography.

Stationary phase

Polymers whose surface has been modified with phenyl and alkyl radicals are usually used as the stationary phase at HIC . The selectivity and capacity of the HIC depend on the density and the hydrophobicity of the groups used. Which stationary phase is used for what must, however, usually be determined empirically, since, for example, with phenyl groups, not only hydrophobic but also aromatic effects occur, and thus the interactions can change.

Mobile phase

The salt most frequently used at HIC is ammonium sulphate, whereby the salt is generally selected with the help of the Hofmeister series , which classifies ions in terms of their precipitating effect. Ions (such as: ammonium cations ) that initiate hydrophobic interactions are called antichaotropic or kosmotropic, those that prevent interactions (such as: thiocyanate anions ) are called chaotropic .

Influence of temperature and pH

As the temperature drops, the hydrophobic interaction is weakened. No exact, generally valid statements can be formulated about the influence of the pH value ; rather, its influence potential has to be determined empirically for the respective experiment. As a rule, however, an HIC around pH 7 is carried out, since here most of the proteins are in the uncharged state and thus the hydrophobic interactions are greatest.

literature

  • Friedrich Lottspeich, Joachim W. Engels: Bioanalytics. 2nd Edition. Spektrum Akademischer Verlag, Munich 2006, ISBN 3-8274-1760-0 .