User talk:Jeremy Rajaballey/sandbox

Hydrophobic Interaction Chromatography/Protein Purification

Hydrophobic interactions between proteins and the chromatographic matrix can be exploited to purify proteins. In hydrophobic interaction chromatography the matrix material is lightly substituted with hydrophobic groups. These groups can range from methyl, ethyl, propyl, octyl, or phenyl groups.[textbook source] At high salt concentrations, non-polar sidechains on the surface on proteins "interact" with the hydrophobic groups; that is, both types of groups are excluded by the polar solvent (hydrophobic effects are augmented by increased ionic strength). Thus, the sample is applied to the column in a buffer which is highly polar. The eluant is typically an aqueous buffer with decreasing salt concentrations, increasing concentrations of detergent (which disrupts hydrophobic interactions), or changes in pH.

In general, Hydrophobic Interaction Chromatography (HIC) is advantageous if the sample is sensitive to pH change or harsh solvents typically used in other types of chromatography but not high salt concentrations. Commonly, it is the amount of salt in the buffer which is varied. In 2012, Müller and Franzreb described the effects of temperature on HIC using Bovine Serum Albumin (BSA) with four different types of hydrophobic resin. The study altered temperature as to effect the binding affinity of BSA onto the matrix. It was concluded that cycling temperature from 50 degrees to 10 degrees would not be adequate to effectively wash all BSA from the matrix but could be very effective if the column would only be used a few times. [source] Using temperature to effect change allows labs to cut costs on buying salt and saving money.

If high salt concentrations along with temperature fluctuations want to be avoided you can use a more hydrophobic to compete with your sample to elute it. [source] This so-called salt independent method of HIC showed a direct isolation of Human Immunoglobulin G (IgG) from serum with satisfactory yield and used Beta-cyclodextrin as a competitor to displace IgG from the matrix [source]. This largely opens up the possibility of using HIC with samples which are salt sensitive as we know high salt concentrations precipitate proteins.