In RPLC the mobile phase is more polar than the stationary phase. RPLC is the dominant mode of LC, thanks to its enormous flexibility in separating samples from different origins. RPLC is also highly selective efficient and potentially fast.
The mobile phase usually contains water as a usually a major component. Retention is typically controlled by the addition of organic solvents (“modifiers”), such as acetonitrile or methanol. In addition, the mobile phase may contain a variety of additives to improve the separation or enhance the efficiency.
SEE MODULE 3.5
Chemically bonded phases #
The most common stationary phases used in RPLC are chemically bonded phases (CBPs) based on silica. Non-polar ligands, such as octadecyl (C18) chains, change the polar surface into a quite non-polar stationary phase. The weakest point of C18 stationary phases is their limited stability in basic or highly acidic solvents.
Gradient elution #
RPLC stationary phases provide a high surface areas and rapid mass transfer. This allows high chromatographic efficiencies (plate counts). Moreover, rapid equilibration makes RPLC highly compatible with gradient elution, i.e. separations in which the mobile-phase composition is purposefully varied during the run.
By gradually increasing the elution strength of the mobile phase (i.e. the concentration of organic modifier) increasingly large and apolar molecules can be eluted. Gradient-elution RPLC allows separation of complex mixtures containing very diverse analytes.
