In high-performance liquid chromatography (HPLC), a liquid solvent containing a mixture of molecules to be identified is passed through a column densely packed with a small-diameter insoluble bead-like resin. In column chromatography, the smaller and more tightly packed the resin beads, the greater the resolution of the separation technique. In HPLC, the resin is so tightly packed that the liquid must be pumped through the column at high pressure. Therefore HPLC uses precise high-pressure pumps with metal plumbing and columns rather than glass and plastics used in gravity chromatography. Resin beads are coated with charged groups to separate compounds by ion exchange or with hydrophobic groups to retard hydrophobic nonpolar molecules. In hydrophobic chromatography, tightly associated nonpolar compounds are eluted from the hydrophobic beads in aqueous solvents containing various percentage of an organic reagent. The higher the percentage of organic solvent in the effluent, the faster the nonpolar component is eluted from the hydrophobic resin. This latter type of chromatography over nonpolar resin beads is called reverse-phase HPLC. HPLC separations have extremely high resolution and reproducibility.

Application of HPLC

In recent times HPLC has emerged as a method of choice for analytical purposes. The biggest advantage that it has over other techniques is the speed of analysis which is many times more than other techniques except, perhaps, for GLC. The sample requirement is also very low for this technique and as less as a few femtograms of the sample will be analyzed satisfactorily. On top of all this the detectors that are employed in HPLC are non-destructive in nature and thus the separated components can be recovered for further study.

HPLC has been successfully applied to the separation of proteins, nucleic acids, polysaccharides, plant pigments, amino acids, pesticides, medicines and their metabolites, animal and plant hormones and complex lipids.