Reversed-phase chromatography

Reversed-phase chromatography (also called RPC, reverse-phase chromatography, or hydrophobic chromatography) includes any chromatographic method that uses a hydrophobic stationary phase. RPC refers to liquid (rather than gas) chromatography.

The term "reversed-phase" has a historical background. In the 1970s, most liquid chromatography was performed using a solid support stationary phase (also called a "column") containing unmodified silica or alumina resins. This method is now called "normal phase chromatography". In normal phase chromatography, the stationary phase is hydrophilic and therefore has a strong affinity for hydrophilic molecules in the mobile phase. Thus, the hydrophilic molecules in the mobile phase tend to bind (or "adsorb") to the column, while the hydrophobic molecules pass through the column and are eluted first. In normal phase chromatography, hydrophilic molecules can be eluted from the column by increasing the polarity of the solution in the mobile phase.

The introduction of a technique using alkyl chains covalently bonded to the solid support created a hydrophobic stationary phase, which has a stronger affinity for hydrophobic compounds. The use of a hydrophobic stationary phase can be considered the opposite, or "reverse", of normal phase chromatography – hence the term "reversed-phase chromatography". Reversed-phase chromatography employs a polar (aqueous) mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the column and are eluted first. Hydrophobic molecules can be eluted from the column by decreasing the polarity of the mobile phase using an organic (non-polar) solvent, which reduces hydrophobic interactions. The more hydrophobic the molecule, the more strongly it will bind to the stationary phase, and the higher the concentration of organic solvent that will be required to elute the molecule.

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