An Introduction to Capillary Electrochromatography

KEITH D. BARTLE, MARIA G. CIKALO AND MARK M. ROBSON

1 What is Capillary Electrochromatography?

Capillary electrochromatography (CEC) is a recently developed variant of high-performance liquid chromatography (HPLC) in which the flow of mobile phase is driven through the column by an electric field, a phenomenon known as electroosmosis, rather than by applied pressure. This electroosmotic flow (EOF) is generated by applying a large voltage across the column; positive ions of the added electrolyte accumulate in the electrical double layer of particles of column packing, move towards the cathode and drag the liquid mobile phase with them. As in capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC), small diameter (typically 50-100 µm) columns with favourable surface area-to-volume ratio are employed to minimise thermal gradients from ohmic heating, which can have an adverse effect on band widths. CEC differs crucially from CE and MEKC, however, in that the separating principle is partition between the liquid and solid phases (Table 1.1).

Avoiding the use of pressure results in a number of important advantages for CEC over conventional HPLC. Firstly, the pressure-driven flow rate through a packed bed depends directly on the square of the particle diameter and inversely on column length; for practical pressures, generally used particle diameters are seldom less than 3 µm, with column lengths restricted to approximately 25 cm. By contrast the electrically driven flow rate is independent of particle diameter and column length so that, in principle, smaller particles and longer columns can be used. If follows that considerably higher efficiencies can be generated in CEC than in HPLC. A second consequence of employing electrodrive is that the plug-like flow -velocity profile in EOF reduces dispersion of the band of solute as it passes through the column, further increasing column efficiency. The combined effect of reduced particle diameter, increased column length and plug flow leads to CEC efficiencies of typically 200 000 plates per metre, and substantially improved resolution.

Voltages up to 30 kV are applied to generate the electric field usually for solutions of 1–50 mM buffers in aqueous reversed-phase mobile phases, although non-aqueous CEC has also been carried out. The dependence of EOF rate on solvent dielectric constant has been confirmed, but the electrical potential (the zeta potential) of the boundary between the fixed and diffuse layers (the double layer; see pages 43–5 for further discussion) of positive ions at the stationary phase wall (Figure 1.1) is less well understood. The conclusion of an early theoretical study which suggested that flat EOF profiles in a capillary of diameter d would result if d were considerably greater than the double layer thickness, δ, has been confirmed by experiment; for channels between particles, however, the influence of δ is less clear. Current indications are that it should be possible to use monodisperse particles with diameters down to 0.5 µm. Pore sizes of commonly used HPLC particles are too small to give rise to EOF, but larger pore packings show promise. Although CEC has been demonstrated for stationary phases bonded to the walls of open tubes, and in sol–gel derived phases, most work has been carried out on columns packed with HPLC stationary phases; a new generation of packings custom-synthesized for CEC is, however, now beginning to make an impact.

2 History of CEC

Strain first reported the use of the EOF in chromatography; he recognized the difference between electrophoresis and electrochromatography on the one hand, and partitioning of analytes between a mobile and a stationary phase on the other. Electrodrive (electrophoretic mobility and electroosmosis) was used to move the analytes through a separation medium, so that the importance of the EOF was recognized in electrochromatography. Early work in electrical chromatography was either in relatively large diameter columns (>1 mm) or in thin layers, which were used to analyse neutral, basic and acidic molecules by electromigration through a paper matrix.

The separation of polysaccharides using electrodrive through a colloidal membrane is probably the first reported use of EOF to drive a mobile phase through a stationary phase. For thin layers, Kowalczyk quantified the EOF velocity while Hybarger et al. proposed an annular bed system for preparative separations. Cylindrical columns packed with Sephadex were used by a number of groups for protein separation. Gel columns were employed in the separation of high-molecular-weight (>500 kDa) compounds in experiments in which counteracting electrophoretic and hydrodynamic forces were used.

The originators of CEC were, however, the Pretorius group, who reported that if the EOF were used to drive the mobile phase flow, as opposed to the hydrodynamic flow in conventional liquid chromatography, the plate height was reduced. They also pointed out the absence of pressure drop across the column if the EOF were used. Significant progress in CEC began in the 1980s; Jorgenson and Lukacs demonstrated the use of electroosmosis in capillaries and showed the possibilities for low reduced plate heights. Tsuda then showed that CEC was possible in coated open tubular columns and recognized the factors that control the EOF as well as the importance of practical effects, such as bubble formation, in packed columns.

The recent resurgence of CEC dates from the detailed theoretical analysis of Knox and Grant, published in 1987, followed by practical demonstrations by the same group in 1991. Both slurry-packed and draw-packed capillaries were used in a detailed study of factors affecting the EOF. Particle sizes down to 1.5 µm were used, and reduced plate heights near unity were demonstrated in 30–200 µm i.d. columns up to 1 m long. The important observation was made that columns driven electrically show higher efficiencies than the same column with pressure drive (Figure 1.2).

The potential of CEC in the analysis of mixtures relevant to the pharmaceutical industry was realized by Smith and Evans in 1994. The capabilities of CEC were demonstrated in high-resolution chromatograms of drug compounds (e.g. Figure 1.3) on a reversed-phase C18 stationary phase. The same group than went on to show how especially...