Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds.[1][2][3] The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.[4]
The factors affecting the retention and separation of solutes in the reversed phase chromatographic system are as follows:
a. The chemical nature of the stationary phase, i.e., the ligands bonded on its surface, as well as their bonding density, namely the extent of their coverage.
b. The composition of the mobile phase. Type of the bulk solvents whose mixtures affect the polarity of the mobile phase, hence the name modifier for a solvent added to affect the polarity of the mobile phase.
C. Additives, such as buffers, affect the pH of the mobile phase, which affect the ionization state of the solutes and their polarity.
In order to retain the organic components in mixtures, the stationary phases, packed within columns, consist of a hydrophobic substrates, bonded to the surface of porous silica-gel particles in various geometries (spheric, irregular), at different diameters (sub-2, 3, 5, 7, 10 um), with varying pore diameters (60, 100, 150, 300, A). The particle's surface is covered by chemically bonded hydrocarbons, such as C3, C4, C8, C18 and more. The longer the hydrocarbon associated with the stationary phase, the longer the sample components will be retained. Some stationary phases are also made of hydrophobic polymeric particles, or hybridized silica-organic groups particles, for method in which mobile phases at extreme pH are used. Most current methods of separation of biomedical materials use C-18 columns, sometimes called by trade names, such as ODS (octadecylsilane) or RP-18.
The mobile phases are mixtures of water and polar organic solvents, the vast majority of which are methanol and acetonitrile. These mixtures usually contain various additives such as buffers (acetate, phosphate, citrate), surfactants (alkyl amines or alkyl sulfonates) and special additives (EDTA). The goal of using supplements of one kind or another is to increase efficiency, selectivity, and control solute retention.