18] Two-dimensional liquid chromatography—capillary electrophoresis techniques for analysis of proteins and peptides

Abstract

Publisher Summary The probability of resolving all the components in a sample decreases, as the number of components in that sample increases. Mathematical models, set forth by Giddings, predict that, as sample complexity increases, complete separation of component peaks can be achieved only with high-efficiency separations. Even the most efficient one-dimensional (1D) techniques have insufficient peak capacity for the separation of highly complex biological samples. Two-dimensional (2D) separations have peak capacities approximately equal to the product of the peak capacities of the component 1D separations and are, therefore, preferable for these samples. This chapter discusses three approaches to 2D coupled-column designs. Each approach is comprehensive as defined and each uses a form of liquid chromatography, as the first dimension, with capillary zone electrophoresis, as the second. Liquid chromatography and capillary zone electrophoresis (CZE) are complementary techniques, because their solvent systems are compatible, but their separation mechanisms are different. The interface between the two systems is also simple: either an automated valve or a fluid tee. Detection is by ultraviolet (UV) absorption or laser-induced fluorescence (LIF). The first approach couples microcolumn size-exclusion chromatography (SEC), with CZE, for the separation of proteins. The second approach combines reversed-phase liquid chromatography (RPLC) and CZE for the separation of tryptic peptides. The last approach couples RPLC with fast-CZE, a variation of CZE, enabling fast 2D separations.