Analytical Chemistry

Table 4.22 Principal modes of HPCE

Mode Separation mechanism Separation medium Typical sample types

capillary zone electrophoresis

(CZE) or free-solution

capillary electrophoresis

(FSCE)

differences in electrophoretic

mobilities

electrolyte solution relatively small charged

species

micellar electrokinetic

capillary chromatography

(MEKC or MECC)

differences in hydrophobic

interactions with micelles

(partitioning)

micellar electrolyte relatively small neutral and

charged species

capillary gel electrophoresis

(CGE)

molecular sieving and

differences in electrophoretic

mobilities

polyacrylamide or agarose gel biopolymers

capillary isoelectric focusing

(CIEF)

differences in isoelectric

points

electrolyte/ampholyte pH

gradient

proteins

buffers are listed in Table 4.23. Each one should be used only over a pH range of pKa ± 1 (p. 47),

polybasic acids such as phosphoric and citric being particularly versatile because each has three pKa

values. Buffers having large ions or which are zwitterionic, such as Tris, borate, histidine and CAPS,
can be used at high concentrations because they generate relatively low currents, but the high UV
absorbance of some is a disadvantage.

Additional substances (buffer additives) are often added to the buffer solution to alter selectivity and/or
to improve efficiency, and the wall of the capillary may be treated to reduce adsorptive interactions with
solute species. Organic solvents, surfactants, urea and chiral selectors are among the many additives that
have been recommended (Table 4.24). Many alter or even reverse the EOF by affecting the surface
charge on the capillary wall, whilst some help to solubilize hydrophobic solutes, form ion-pairs, or
minimize solute adsorption on the capillary wall. Chiral selectors enable racemic mixtures to be
separated by differential interactions with the two enantiomers which affects their electrophoretic
mobilities. Deactivation of the capillary wall to improve efficiency by minimizing interactions with
solute species can be achieved by permanent chemical modification such as silylation or the adsorption
of a polymeric coating. Alternatively, dynamic deactivation by buffer additives has the advantage of the
modification being constantly renewed during electrophoresis so providing a more stable surface. Both
approaches can eliminate or even reverse the EOF.