Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Reversed-phase chromatography

Reversed-phase chromatography (RPC) includes any chromatographic method that uses a non-polar stationary phase. The name "reversed phase" has a historical background. In the 1970s most liquid chromatography was done on non-modified silica or alumina with a hydrophilic surface chemistry and a stronger affinity for polar compounds - hence it was considered "normal". The introduction of alkyl chains bonded covalently to the support surface reversed the elution order . Now polar compounds are eluted first while non-polar compounds are retained - hence "reversed phase". All of the mathematical and experimental considerations used in other chromatographic methods apply (i.e. separation resolution proportional to the column length). Today, reversed-phase column chromatography accounts for the vast majority of analysis performed in liquid chromatography.

Stationary Phases

Silica Based Stationary Phases

Any inert non-polar substance that achieves sufficient packing can be used for reversed-phase chromatography. The most popular column is a octadecyl carbon chain (C18) bonded silica (USP classification L1) with 297 columns commercially available This is followed by C8 bonded silica (L7 - 166 columns), pure silica (L3 - 88 columns), cyano bonded silica (L10 - 73 columns) and phenyl bonded silica (L11 - 72 columns). Note that C18, C8 and phenyl are dedicated reversed phase packings while cyano columns can be used in a reversed phase mode depending on analyte and mobile phase conditions. It should be noted at this point that not all C18 columns have identical retention properties. Surface functionalization of silica can be performed in a monomeric or a polymeric reaction with different short-chain organosilanes used in a second step to cover remaining silanol groups (end-capping). While the overall retention mechanism remains the same subtle differences in the surface chemistries of different stationary phases will lead to changes in selectivity.

Mobile Phase Considerations

Mixtures of water or aqueous buffers and organic solvents are used to elute analytes from a reversed phase column. The solvents have to be miscible with water and the most common organic solvents used are acetonitrile, methanol or tetrahydrofuran (THF). Other solvents can be used such as ethanol, 2-propanol (iso-propyl alcohol). Elution can be performed isocratic (the water-solvent composition does not change during the separation process) or by using a gradient (the water-solvent composition does change during the separation process). The pH of the mobile phase can have an important role on the retention of an analyte and can change the selectivity of certain analytes. Charged analytes can be separated on a reversed phase column by the use of ion-pairing (also called ion-interaction). This technique is known as reversed phase ion-pairing chromatography.