High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography

Although gas chromatography is widely used, it is limited to samples that are ther- mally stable and easily volatilized. Nonvolatile samples, such as peptides and carbo- hydrates, can be analyzed by GC, but only after they have been made more volatile by a suitable chemical derivatization. For this reason, the various techniques in- cluded within the general scope of liquid chromatography are among the most commonly used separation techniques. Although simple column chromatography, first introduced by Tswett, is still used in large-scale preparative work, the focus of this section is on high-performance liquid chromatography (HPLC).

In HPLC, a liquid sample, or a solid sample dissolved in a suitable solvent, is carried through a chromatographic column by a liquid mobile phase. Separation is determined by solute/stationary-phase interactions, including liquid–solid adsorp- tion, liquid–liquid partitioning, ion exchange and size exclusion, and by solute/mobile-phase interactions. In each case, however, the basic instrumentation is essentially the same. A schematic diagram of a typical HPLC instrument is shown in Figure 12.26. The remainder of this section deals exclusively with HPLC separations based on liquid–liquid partitioning.