Reversephase

Reverse phase chromatography, commonly referred to as reversed-phase liquid chromatography (RPLC), is a mode of liquid chromatography in which the stationary phase is nonpolar and the mobile phase is relatively polar. This arrangement is the reverse of older normal-phase chromatography and is the most widely used mode in analytical HPLC.

Typical stationary phases are alkylsilane bonded silica, with C18 chains (octadecyl) being the most common, followed by C8, C4, and other chemistries. The mobile phase usually consists of water or buffered aqueous solutions mixed with polar organic modifiers such as acetonitrile or methanol. Gradient elution, gradually increasing the organic fraction, is common for separating compounds over a broad polarity range; isocratic conditions are used for simpler separations.

Separation is governed by hydrophobic interactions: more nonpolar analytes interact more strongly with the nonpolar stationary phase and are retained longer. RP-LC is compatible with mass spectrometry, UV/Vis detection, and refractive index detectors, enabling analysis across pharmaceuticals, environmental samples, foods, and metabolites. It is also used in proteomics for peptide separation prior to MS analysis.

Advantages include high efficiency, broad applicability, robustness, and easy interfacing with MS. Disadvantages include poor retention of very polar compounds without special modifiers or ion-pairing reagents, potential long run times for highly hydrophobic solutes, and substantial solvent consumption. Method development often involves selecting column chemistry, optimizing the mobile phase composition, and choosing gradient profiles to achieve the desired separation.