Reversed Phase Chromatography (RPC)
RPC separates proteins and peptides on the basis of hydrophobicity. RPC has become increasingly important for high resolution separation and analysis of proteins, peptides, and nucleic acids.
The method requires the use of organic solvents and is widely used for purity check analyses when activity and tertiary structure are not a focus. Because many proteins are denatured by organic solvents, the method is not generally recommended for preparative protein purification.
Proteins tend to denature and bind strongly to RPC medium, and can be difficult to elute. Elution is usually performed by an increase in organic solvent concentration. Acetonitrile, methanol, ethanol, and propanol are most commonly used. Use RPC for the final purification step of proteins that are stable in the organic solvents used.