Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography is exploiting the special chemical property of protein that there are extensive hydrophobic patches on the protein surface, in addition to expected hydrophilic regions. The hydrophobic patches are the part on the protein surface that would bind to the hydrophobic ligands on the adsorbent, which are in favour of hydrophobic interaction in the presence of high salt concentration. Hence, the type and concentration of salt play an important role in the efficiency of the HIC. The influence of the salt on the hydrophobic interaction follows the Hofmeister series. Such non-ionic interaction is enhanced by the salting-out principle. In salting-out, the main cause of aggregation is the strengthening of hydrophobic interaction between proteins. The presence of high concentration of salt causes the formation of water cavity. The formation of water cavity with a salt that gives a high surface tension needs a bigger input of energy than the salt that gives low surface tension. It is the salt that gives the highest surface tension that provides greatest hydrophobic interaction.

 

Similar to other chromatography, HIC also follow the following steps:

1. Equilibration
   
2. Sample application
   
3. Washing
   
4. Elution
   

Sample Application

The sample has to be in a salt in order to enhance hydrophobic interaction. Most commonly used salt are ammonium sulphate or sodium sulphate, but sometimes chloride salts are used.

Elution

The process of elution can be done by:

1. Changing the concentration of salt
   
2. Changing the pH of the buffer
   
3. Changing the polarity of the solvent
   

Elution process can be carried out by decreasing the concentration of salt, either a downward gradient or by stepwise lowering. As resolution is not high, gradients may not achieve any better result than stepwise process. Changing the pH of the buffer for the process of elution might not work as it is impossible to predict how the strength of the hydrophobic interaction between a protein and HIC adsorbent will effect upon change in pH. Besides that, solvent that can lower the polarity of the column such as ethanol and ethylene glycol can be used for elution of strongly-bound protein. Such solvent can be added after the salt has been removed from the column.