What are the steps involved in hydrophobic interaction chromatography (HIC)?

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Column Setup: Alkyl or aryl ligands are attached to a porous matrix, forming HIC media, which is then packed into a column. 
Buffer Preparation: A moderately high salt buffer, typically containing 1–2M ammonium sulfate or 3M sodium chloride, is used to fill the pores and spaces between particles in the matrix. This buffer enhances the interaction between the protein sample and the medium while minimizing interactions with less hydrophobic proteins. 
Protein Binding and Washing: The protein sample is applied to the column, and non-bound proteins are washed away.
Gradient Elution: The salt concentration is gradually decreased to begin eluting proteins. Manipulating salt gradients allows for the differential elution of proteins, with those having the lowest hydrophobicity eluting first. 
Final Wash: A salt-free buffer is used to wash the column and remove tightly-bound proteins. Additives in the buffer, such as water-miscible alcohols, detergents, and chaotropic salt solutions can further cause the desorption of bound proteins. 
Harsh Conditions (if needed): In rare cases, harsher conditions like 0.5–1.0M sodium hydroxide, 70% ethanol, or 30% isopropanol may be required to remove all bound proteins.

What are the steps involved in hydrophobic interaction chromatography (HIC)?

Posted May 8, 2024