Reversed-phase high performance liquid chromatography is a separation technique in which analytes are resolved according to their hydrophobicity using a non-polar stationary phase and a polar mobile phase. The stationary phase typically consists of silica particles bonded with long alkyl chains such as octadecyl gro…
Reversed-phase high performance liquid chromatography is a separation technique in which analytes are resolved according to their hydrophobicity using a non-polar stationary phase and a polar mobile phase. The stationary phase typically consists of silica particles bonded with long alkyl chains such as octadecyl groups, while the mobile phase is an aqueous mixture containing organic modifiers like methanol or acetonitrile. Compounds partition between the two phases according to their relative affinity, so more hydrophobic species are retained longer and elute later; selectivity is tuned by adjusting mobile-phase composition, pH and gradient profile. Because most organic molecules of pharmaceutical and biochemical interest are amenable to this mode, reversed-phase chromatography is the most widely applied form of liquid chromatography. It is used to quantify active ingredients in drug formulations, to assess compound stability and purity, to separate small molecules and biomolecules, and to characterise novel monolithic and bonded column materials that improve resolution and throughput. Coupling to ultraviolet detection enables routine assay of analytes such as statins and other drugs, while hyphenation to mass spectrometry extends the approach to complex mixtures in pharmaceutical, biotechnological, food and environmental analysis. Its versatility, reproducibility and quantitative accuracy make reversed-phase high performance liquid chromatography a foundational tool of analytical chemistry.
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