Concepts and practice of multidimensional high-performance liquid chromatography

High-performance liquid chromatography (HPLC) is a long established method used for the separation, identification, and determination of chemical components in complex mixtures [1]. This analytical tool is employed for the qualitative identification and quantitative determination of separated species [1, 2] and finds widespread application in almost all areas of science. Many different modes of liquid chromatography exist that depend on the type of liquid and the type of stationary phase. For example, the technique referred to as normal phase liquid chromatography (NPLC) employs a polar stationary phase and a nonpolar mobile phase. The reverse of this process, referred to as reversed-phase liquid chromatography (RPLC), utilizes a nonpolar stationary phase and a polar mobile phase. In both these techniques, the stationary phase is usually a chemically modified solid support with either a polar or nonpolar ligand, depending on the desired mode of separation. Ion exchange liquid chromatography is a technique for separating ionic species where the solid support is chemically modified using an ionic ligand. Separation of charged species depends on the pH and electrolytic strength of the mobile phase. Ion exclusion and ion-paired chromatographies are two other techniques employed for the separation of ionic substances. Size-exclusion chromatography (SEC) separates species according to their molecular size. Molecules traverse a porous stationary phase in a mobile-phase environment that is unfavorable for enthalpic interactions. The molecules are consequently separated according to their entropic exclusion from within the porous network. This technique is also referred to as gel permeation chromatography (GPC). Affinity chromatography is a technique with widespread use in the biosciences. In this technique, the stationary phase is modified with a solute receptor targeted for the retention and hence isolation of a specific solute. Chiral chromatography is employed for the separation of enantiomers. The stationary phase is modified with chiral ligands that preferentially retain one enantiomer; the technique is spatially selective. Chiral mobile phases can also be employed to exploit the "handedness" of the target molecules. These illustrations of the different modes of separation are by no means comprehensive but they do show the vast number of liquid chromatographic processes at the disposal of the chromatographer. In fact, too many different stationary phases exist to list here but a scan through any supplies catalogue will Concepts and Practice of High-Performance Liquid Chromatography 179 illustrate the vast choices available to the chromatographer. Even then, there are many specialty stationary phases that are not commercially available. As a consequence, by appropriate selection of the most suitable techniques, there is virtually no sample - regardless of the complexity - that cannot be reduced to a simpler form, allowing for analysis of the sample constituents. In this chapter, we are not concerned with mechanisms of retntion; rather, our interest lies in selectivity. The aim of this chapter is therefore to investigate processes in which selectivity can be exploited in order to gain separation. One way to increase selectivity and also increase the peak capacity of a liquid chromatographic system is to incorporate more than one separation dimension - that is, utilize the process referred to as multidimensional liquid chromatography. Here, multiple separating columns are coupled together in an automated system in such a manner as to improve the separation potential, allowing complex samples to be resolved into simpler systems. Before we begin our discussion on multidimensional chromatography, we must first discuss single-dimensional systems and understand factors that control selectivity.