High-Performance Liquid Chromatography

Separation quality in this technique scales with how small the stationary phase particles can be made, and the pressure requirement follows directly from that: packing beds with particles of a few micrometres forces mobile phase pressures of hundreds of bar. Reversed-phase operation dominates practice, pairing a non-polar bonded stationary phase, commonly octadecylsilane, with a polar aqueous mobile phase, so that compounds elute in order of decreasing polarity. Isocratic runs hold the mobile phase composition fixed, while gradient elution varies it during the run to bring strongly retained species off the column in reasonable time. Detection is most often by ultraviolet absorbance, though mass spectrometry, fluorescence and refractive index detectors serve where the analyte demands. The method suits non-volatile and thermally labile compounds that gas chromatography cannot handle.