rplC

Reversed-phase liquid chromatography (RPLC) is a mode of high-performance liquid chromatography where the stationary phase is nonpolar and the mobile phase is relatively polar. The most common stationary phase consists of silica particles bearing hydrophobic alkyl chains, with C18 (octadecyl) bonded phases being the standard. Other options include C8, phenyl, and cyano coatings. Separation occurs primarily through hydrophobic interactions: analytes with greater nonpolar character interact more strongly with the stationary phase and therefore have longer retention times than polar compounds that interact less.

Mobile phases typically combine water or aqueous buffers with organic modifiers such as acetonitrile or methanol. Adjusting the solvent strength, pH, and temperature modulates retention. Isocratic runs use a fixed mobile-phase composition, while gradient elution increases the organic content during the run to elute strongly retained species.

RPLC is widely used because of its broad applicability, high resolving power, and compatibility with detectors such as UV–visible, diode-array, fluorescence, and mass spectrometry. It is commonly employed in pharmaceutical analysis, environmental monitoring, clinical and bioanalytical work, and the analysis of small molecules, peptides, and proteins under suitable conditions. Modern systems include high-pressure UHPLC platforms with sub-2-micron particles for higher efficiency and faster separations.

Limitations include poor retention for very polar analytes on common C18 phases unless special conditions are used, potential column degradation with harsh solvents, and method development that can require optimization of many variables. The technique has a long history dating to the 1960s–1980s with the commercial introduction of bonded silica stationary phases, and it remains a staple of analytical chemistry.