The separations are greatly improved, however, through the use of high-resolution columns and the column retentions times are much reduced. The narrow and relatively long columns are packed with a noncompressible matrix of fine glass or plastic beads coated with a thin layer of the stationary phase. Alternatively, matrix may consist of silica whose available hydroxyl groups can be derivatives with many of the commonly used functional groups of ion exchange chromatography, RPC, HIC, or affinity chromatography.
The mobile phase is one of the solvent systems including gradient elutions with binary or even ternary mixtures. In the case of HPLC, however, the mobile phase is forced through the tightly packed column at pressures of up to 5000 psi leading to greatly reduced analysis times. The elutants are detected as they leave the column by such methods as UV absorption, refractive index, or fluorescence measurements.
The advantages of HPLC are
• Its high resolution, which permits the routine purification of mixtures that have defined separation by other techniques.
• Its speed, which permits most separation to be accomplished in significantly less than one hour.
• Its high sensitivity, which, in favorable cases, permits the quantitative estimation of less than picomole quantities of materials.
• Its capacity for automation.
Thus, few biochemistry laboratories now function without access to an HPLC system. HPLC is also often utilized in the clinical analyses of body fluids because it can rapidly, routinely, and automatically yield reliable quantitative estimates of nanogram quantities of biological materials such as vitamins, lipids and medicine metabolites.