In this technique, one uses hydrophobic interactions between the sample and the ligand on the chromatographic support to obtains separation.
For proteins mobile phase additives increase hydrophobicity by forming ion pairs that strongly adsorb to the stationary phase.
Adsorption is so strong that a gradient of increasing concentration of organic solvent such as acetonitrile or 2-propanol, is required for elution.
Because of the high ligand density of reversed phase chromatography (RPC) media and the drastic elution conditions required, the enzymatic and immunologic activity of proteins in generally not maintained after RPC separation. It is used for separating small molecules and peptides.
RPC has a high peak capacity and is particularly effective for separating small molecules, peptides, nucleotides and restriction fragments.