Chromatography Forum

Hi

Is there a difference in retention mechanism between Hydrophobic Interaction Chromatography and Reversed phase chromatography??

The selectivity is the same; both focus on the hydrophobic residues and ignore the hydrophilic ones. Results for HIC and RPC of peptides are quite similar, except that the peaks are appreciably sharper in RPC because the mobile phases are much less viscous. The big difference comes when a peptide or protein is large enough to have significant secondary or tertiary structure. That is largely lost in RPC but preserved in HIC. At that point the selectivity of HIC becomes superior because it is focusing only on the relatively small number of hydrophobic residues that are on the surface of the protein's tertiary structure. RPC sees all of the hydrophobic residues in the protein's core, so its selectivity is then on the basis of small differences between large numbers. I might note that many proteins don't elute from RPC columns at all, or else elute in peaks 20 minutes wide. HIC is more compatible with proteins in general.