Figure 4.58
Separation by CIEF.points after which they are passed sequentially through the detector by applying pressure at one end of
the capillary or by adding a salt to one of the buffer reservoirs. Minimizing the EOF and adsorption
effects by coating the walls of the capillary with a polymer is necessary to avoid disturbance of the
separating species until focusing is complete. Larger samples can be analysed than in other modes of
HPCE, but this is limited as some proteins may precipitate at their isoelectric points.
Figure 4.59 illustrates some typical separations employing different modes of HPCE.
Capillary Electrochromatography
CEC is a relatively new technique and is hybrid of capillary electrophoresis and high-performance
liquid chromatography, combining elements of both. A fused silica CE column is packed with an HPLC
stationary phase, usually a bonded-phase silica, and filled with a buffer solution (> pH 4). Under the
influence of an applied voltage, an electro-osmotic flow (EOF) of buffer towards the cathodic end of the
capillary is generated. Unlike CE however, the electrical double-layer formed is predominantly at the
surface of the individual particles of packing rather than the capillary wall. The flow of mobile phase
has a flat profile, as in CE, but unlike in HPLC there is no pressure drop because the driving force is
generated throughout the length of the column. Even higher efficiencies are observed than in CZE
because the column packing limits molecular diffusion in the mobile phase (p. 89). Very small particles
of stationary phase, currently 1.5– 3 μm nominal diameter, can be used and columns of 25–50 cm in
length are typical. Internal diameters are generally 50– 100 μm, but narrower columns are advantageous
because the EOF is faster thus speeding up the separations. Column packings can be porous, non-
porous, spherical or irregular in shape and of controlled pore size if required. In some cases, mixed-
mode separations can be achieved by using both non-polar and polar or ionic-bonded phases in the
same column. Separations are based on both electrophoretic migration for charged analytes and
chromatographic sorption for neutral species, hence the name capillary electrochromatography.