Reversed Phase Chromatography (RPLC or RPC) is the most efficient of all biomolecule separation techniques. It has been the technique of choice for the analysis of small molar mass compounds in both the pharmaceutical and chemical industries, as well as in biomedical research, since the late 1970s. More recently, RPC has become the accepted tool for the separation of peptides, proteins and other biopolymers, making it largely responsible for the widespread popularity of HPLC as a chromatographic technique.
The opposite of normal phase chromatography, RPC requires a non-polar stationary phase and a mobile phase that consists of a mixture of water and polar-solvent mobile phase. The so-called “hydrophobic effect” is the major driving force for retention in RPC. The hydrophobic effect is related to the non-polar surface area of the solute molecule, which varies as a function of mobile phase composition, while the strength of the hydrophobic bond is proportional to the decrease in molecular surface area when the solute associates with the carbon-based stationary phase. Mobile phase additives, such as trifluoroacetic acid, increase protein hydrophobicity by forming ion pairs that strongly adsorb to the stationary phase. Typically, the mobile phase consists of a mixture of water (buffer) and acetonitrile, methanol or, less common, THF, or 2-propanol. The biological molecules are eluted from the chromatographic support by a change in the polarity of the mobile phase.
Silica particles are most commonly used as the support, which then is derivatized with octadecylsilane (ODS). Polymer-based supports have been introduced as an alternative to silica-based reversed phase columns, particularly for analyzing basic compounds in their neutral state at high pH.
RPC columns can be applied to the analysis of a wide variety of compounds, ranging from neutral polar and nonpolar solutes to acidic, basic, and amphoteric compounds. RPC is also an efficient technique for the analysis of derivatized amino acids, peptides and proteins, although protein structure is not always maintained due to the high concentration of organic solvent required for their elution.