What is Reverse-phase chromatography?

Reverse-Phase chromatography is a technique which implies to the use of hydrophobic stationary phase in order to separate the non-polar molecules. It is opposite of the HPLC where polar molecule will be retarded in the column. In this, the most polar molecules will elute first, and the non-polar molecules will be eluted at the end. The separation of the molecules is thus based on the hydrophobicity of the molecules.

The most non-polar will elute at the end. The matrix used in this chromatography is either silica or polystyrene. However, silica is chemically unstable at higher pH and also dissolved at pH 7.2 if kept for longer time. Thus, the matrix used in the Reverse-phase chromatography is polystyrene. The role of the matrix is to bound the stationary phase with itself.

The stationary phases used in the Reverse-phase chromatography are the alkyl group. The most commonly used stationary group is the octadecyl group, i.e., C-18. It has 18 carbon long chain. The other alkyl groups are butyl and octyl. The increase in the length of the alkyl group increases the hydrophobicity and thus can increase the resolution for separation. High shape selectivity for hydrophobic, long-chain or structurally related isomers such as carotenoids and steroids can be accomplished using C-30 column.