What is reversed phase chromatography?
Reversed phase chromatography (RPC) is a separation technique that separates molecules according to hydrophobicity. Unlike in hydrophobic interaction chromatography (HIC) the RPC medium is highly substituted with hydrocarbon chains making it very hydrophobic. Proteins, peptides and oligonucleotides adsorb to the hydrocarbon chains even in pure water. In HIC, hydrophobic interaction has to be promoted by salts to force the sample molecules to adsorb while RPC requires organic solvents to desorb them. Elution uses gradients of increasing concentrations an organic solvent like acetonitrile in water. RPC provides very high resolution and is used to separate molecules with only minor structural differences such as peptides produced by enzymatic digestion or failures after peptide synthesis. In fact, RPC is the preferred separation technique for peptides. Unlike peptides, proteins contain a substantial degree of tertiary structure important for its biological function. The strong adsorption
