Highperformance liquid chromatography

Application of GC as a separation technique is limited to volatile molecules. However, HPLC can be used for separation of both polar and nonpolar molecules. Usually, derivatization is not necessary for HPLC analysis. HPLC is based on the principle of liquid-liquid chromatography where both mobile phase and stationary phase are liquid. Liquid chromatography using column was first described in 1941. In normal liquid chromatography, the stationary phase is polar and the mobile phase (eluting solvent) is nonpolar. In reverse phase chromatography, the stationary phase is nonpolar and the mobile phase is polar. Several detectors can be used for monitoring elution of peaks from HPLC column including ultraviolet detector, fluorescence detector, conductivity detector, and refractive index detector. Ultraviolet detection is commonly used in clinical laboratories although other detection techniques such as fluorescence technique and electrochemical detection technique are also used.

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