acids and peptides. A stable pH gradientis first created along the gel by
polymerizing it with a mixture of polyamino-polycarboxylic acids having a
range of pKavalues. Applying a potential causes them to migrate to positions
in the gel where they become stationary on account of electrical neutrality by
forming neutral molecules or zwitterions. The pH values at these positions
define their isoelectric points, pI. Electrophoresis of samples results in
ampholytic components migrating to their respective isoelectric points in the
pH gradient, after which they can be located with a suitable dye.
● Immunoelectrophoresisdepends on specific antigen–antibody reactions for
the detection of separated proteins. Low-voltage electrophoresis of samples
in an agarose gel is followed by the introduction of antiserathat diffuse
throughout the gel forming visible precipitates with the separated antigens.Capillary electrophoresisseparations can be performed in one of four principal
modes, each depending on a different separation mechanism. They are
primarily used in the pharmaceutical, clinical and biomedical fields for the
analysis of mixtures of amino acids, peptides, proteins and other macro-
molecules, and drugs and their metabolites in body fluids. Analyses down to
nanogram (10-^9 g) or picogram (10-^12 g) levels are often quicker, giving better
resolution than corresponding HPLCprocedures (Fig. 2c).● Capillary zone electrophoresis, CZE, is the simplest and currently the most
widely used mode of CE. The capillary is filled with a running buffer of the
appropriate pH and ionic strength (Topic D8, Table 1a), and all solutes are
carried towards the cathodic end of the capillary by a strong EOF. Cationic
and anionic solutes are separated, but neutral species, which migrate
together at the same velocity as the EOF, are not separated from one another.
Cationicsolutes migrate fasterthan the EOFbecause their overall mobilities
are enhanced by their attraction to the cathode, whereas anionicsolutes
migrate slowerthan the EOF because they are attracted towards the anode.
Solutes reach the detector in order of decreasing total mobility(mTot=msolute+
mEOF), as shown diagrammatically in Figure 1a, the individual solute mobili-
ties being determined by their size and charge, that is:
(i) cationicspecies first in increasingorder of size;
(ii) neutralspecies next, but not separated;
(iii)anionicspecies last in decreasingorder of size.
Buffer additives, such as urea, surfactants and organic solvents are some-
times used to alter the selectivity of a separation through controlling the
EOF, solute mobilities, solubilities and other effects (Topic D8, Table 1b).
An example of a CZEelectropherogram, the separation of some artificial
sweetners and preservatives, is shown in Figure 1b.
● Micellar electrokinetic chromatography, MEKCor MECC, is a more versa-
tile mode than CZE because both neutral and ionic solutes can be separated.
A surfactantis added to the running buffer, forming aggregates of mole-
cules, or micelles, having a hydrophobic center and a positively or nega-
tively-charged outer surface (Fig. 2a). The micelles act as a chromatographic
pseudo-stationary phase, into which neutral solutes can partition, their
distribution ratiosdepending on their degree of hydrophobicity. Cationic
micelles migrate towards the cathode faster than the EOFand anionic
micellesmore slowly. Neutral solutes migrate at rates intermediate between
the velocity of the EOFand that of the micelles. By analogy with chromatog-
raphy, they are eluted with characteristicretention times, tR, that depend onD9 – Electrophoresis and electrochromatography: modes, procedures and applications 183