Operation of the technique-Several parameters needs to be consider to perform hydrophobic interaction chromatography.

1. Equlibration-HIC column material packed in a column and equilibrate with a buffer containing 0.5-1.5M ammonium sulphate (mobile phase). The salt must be below the concentration where it has salting-out effect.  

2. Sample Preparation- The sample is prepared in the mobile phase and it should be free of suspended particle to avoid clogging of the column. The most recommended method to apply the sample is to inject the sample with a syringe.

3. Elution- There are many ways to elute a analyte from the hydrophobic interaction column. (1) decreasing salt concentration, (2) changing the polarity of the mobile phase such as alchol, (3) By a detergent to displace the bound protein. 

4. Column Regeneration- After the elution of analyte, HIC column require a regeneration step to use next time. column is washed with 6M urea or guanidine hydrochloride to remove all non-specifically bound protein. The column is then equiliberated with mobile phase to regenerate the column. The column can be store at 4⁰C in the presence of 20% alchol containing 0.05% sodium azide.