● capillary gel electrophoresis, CGE, is similar to classical gel electrophoresis,
the capillary being filled with a polyacrylamide or agarose gel that super-
imposes size exclusion selectivity onto the electrophoretic migration of ionic
solutes. The larger the solute species the slower the rate of migration through
the gel. Solute peaks are narrow because band spreading by diffusion in the
running buffer is hindered by the gel structure. The main applications of
CGE are in separating polymer mixtures, protein fractions and DNA
sequencing.
● capillary isoelectric focusing, CIEF, is similar to classical isoelectric focusing,
a pH gradientbeing first formed in the capillary using carrier ampholytes
having pIvalues spanning the required pH range, typically 3 to 10. Sample
solutes migrate and are focusedin positions along the capillary where their
isoelectric point, pI, is equal to the pH. Solute zones are self-sharpening
because diffusion away from the focal points causes the solutes to aquire a
charge which results in them migrating back towards their isoelectric point.
After the separation is complete, pressure is applied to the anodic end of the
capillary to move all the solutes sequentially through the detector cell.Capillary electrochromatography, CEC, is a relatively new technique, and is a
hybrid of capillary electrophoresis and high-performance liquid chromatog-
raphy, combining elements of both. Particular features of CECare:● the capillary is packed with an HPLCstationary phase, usually a bonded-
phase silica, and filled with a running buffer (>pH 4);
● as in CE, the applied potential generates a strong EOFwith a flat flow profile,
but the electrical double-layer formed is predominantly at the surface of the
individual particles of packing rather than the capillary wall;
● unlike in HPLC, there is no pressure drop because the driving force is gener-
ated throughout the length of the column;
● even higher efficiencies are observed than in CZE because the column
packing limits solute diffusion in the mobile phase. Very small particles of
stationary phase, currently 1.5 to 3 mm nominal diameter, can be used and
columns of 25 to 50 cm in length are typical. Internal diameters are generally
50 to 100 mm, but narrower columns are advantageous because the EOFis
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 separa-
tions can be achieved by using both non-polar and polar or ionic bonded
phases in the same column.CEC separations are based on both electrophoretic migration for charged
analytes and chromatographic sorption for neutral species, hence providing an
additional source of selectivity over and above differences in electrophoretic
mobility. The composition of the mobile phase can have dramatic effects on both
the EOFand the selectivity of the separation. CEChas a number of other advan-
tages over both CE and HPLC. Compared to HPLC, solvent consumption is
greatly reduced, which facilitates coupling CECto mass spectrometry (Section
F). Furthermore, there is no need to use a micelle-forming surfactant additive as
in MEKCwhen separating neutral solutes. This is also an advantage when
coupling the technique to mass spectrometry because solvents containing
high concentrations of surfactants such as SDS can cause difficulties with some
ionization sources.186 Section D – Separation techniques