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. 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. 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 type of technique is now referred to as normal-phase chromatography. Since the stationary phase is hydrophilic in this technique, molecules with hydrophilic properties contained within the mobile phase will have a high affinity for the stationary phase, and therefore will adsorb to the column packing. Hydrophobic molecules experience less of an affinity for the column packing, and will pass through to be eluted and detected first. Elution of the hydrophilic molecules adsorbed to the column packing requires the use of more hydrophilic or more polar solvents in the mobile phase to shift the distribution of the particles in the stationary phase towards that of the mobile phase. Reversed-phase chromatography is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. The use of a hydrophobic stationary phase is essentially the reverse of normal phase chromatography, since the polarity of the mobile and stationary phases have been inverted – 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. Many of the mathematical and experimental considerations used in other chromatographic methods also apply to RPC (for example, the separation resolution is dependent on the length of the column). It can be used for the separation of a wide variety of molecules. It is not typically used for separation of proteins, because the organic solvents used in RPC can denature many proteins. For this reason, normal phase chromatography is more commonly used for separation of proteins.