Editing Chromatography (section)

===Hydrophobic interaction chromatography===

Hydrophobic Interaction Chromatography (HIC) is a purification and analytical technique that separates analytes, such as proteins, based on [[Hydrophobic effect|hydrophobic interactions]] between that analyte and the chromatographic matrix. It can provide a non-denaturing orthogonal approach to reversed phase separation, preserving native structures and potentially protein activity.  In hydrophobic interaction chromatography, the matrix material is lightly substituted with hydrophobic groups. These groups can range from methyl, ethyl, propyl, butyl, octyl, or phenyl groups.<ref>{{cite book | vauthors = Ninfa AJ, Ballou DP, Benore M | title = Fundamental Laboratory Approaches for Biochemistry and Biotechnology | location = Hoboken, NJ | publisher = John Wiley | date = 2010 }}</ref> 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, which drives an association of hydrophobic patches on the analyte with the stationary phase. The eluent is typically an aqueous buffer with decreasing salt concentrations, increasing concentrations of detergent (which disrupts hydrophobic interactions), or changes in pH. Of critical importance is the type of salt used, with more [[kosmotropic]] salts as defined by the [[Hofmeister series]] providing the most water structuring around the molecule and resulting hydrophobic pressure. Ammonium sulfate is frequently used for this purpose. The addition of organic solvents or other less polar constituents may assist in improving resolution.

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 40 to 10 degrees Celsius 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.<ref>{{cite journal | vauthors = Müller TK, Franzreb M | title = Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions | journal = Journal of Chromatography A | volume = 1260 | pages = 88–96 | date = October 2012 | pmid = 22954746 | doi = 10.1016/j.chroma.2012.08.052 }}</ref> Using temperature to effect change allows labs to cut costs on buying salt and saves money.

If high salt concentrations along with temperature fluctuations want to be avoided one can use a more hydrophobic to compete with one's sample to elute it. This so-called salt independent method of HIC showed a direct isolation of Human Immunoglobulin G (IgG) from serum with satisfactory yield and used β-cyclodextrin as a competitor to displace IgG from the matrix.<ref>{{cite journal | vauthors = Ren J, Yao P, Chen J, Jia L | title = Salt-independent hydrophobic displacement chromatography for antibody purification using cyclodextrin as supermolecular displacer | journal = Journal of Chromatography A | volume = 1369 | pages = 98–104 | date = November 2014 | pmid = 25441076 | doi = 10.1016/j.chroma.2014.10.009 }}</ref> This largely opens up the possibility of using HIC with samples which are salt sensitive as we know high salt concentrations precipitate proteins.