Hydrophobic chromatography or Hydrophobic Interaction Chromatography (HIC) is a separation method that relies on the hydrophobicity of the adsorbent to bind proteins which are then selectively eluted from the column.
Hofstee (1973) examined the binding of various proteins to the matrix agarose (Sepharose 4B) which is commonly used in preparative chromatography. This matrix was substituted with n-alkylamines of 3 different chain lengths, C1, C4 and C8. The negatively charged proteins in a pH8 and 0.05 ionic strength buffer had a strong binding affinity for adsorbents that had the longer carbon-length chains. The positively charged proteins had a weaker affinity.
It was reasoned that binding was based on a mix of hydrophobic and electrostatic forces. The electrostatic forces were due to the substitution process which introduced positive charges that attracted the negatively charged proteins.
Examples include the fractionation of a lipase from a Chromobacterium species that was conducted with polypropylene glycol – Sepharose gel. The amount of lipase retained on the gel depended on the salt used and ionic strength. The adsorption was stronger as the ionic strength of buffer increased. The pH did not have much of an influence.
A buffer of 20% (w/v) ammonium sulphate in phosphate buffer was the ideal solution for encouraging binding of the lipase protein to the adsorbent. Decreasing the ionic strength of the eluting buffer caused desorption. This paper also looked at binding of the lipase to Sepharose CL-6B modified by covalent immobilization using 1,4-butanediol diglycidyl ether, polyethylene glycol and polypropylene glycol.
References
Hofstee, B. H. J. (1973). Protein binding by agarose carrying hydrophobic groups in conjunction with charges. Biochemical and Biophysical Research Communications, 50(3), pp. 751-757.
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