Capillary gel electrophoresis (CGE) is a variation where selectivity based on molecular size and shape
is introduced by filling the capillary tube with a polymeric material, usually a cross-linked
polyacrylamide or agarose gel. A molecular sieving mechanism is therefore superimposed onto the
basic electrophoretic process as is done in traditional gel slab or column separations (Figure 4.57). The
larger the solute species, the slower its rate of migration, hence elution is in order of increasing relative
molecular mass. A polymer-gel filled capillary provides additional advantages by minimizing band-
spreading through solute-diffusion, preventing solute adsorption onto the capillary wall and eliminating
electro-osmosis. The latter results in the maximum resolution in the shortest possible distance. More
recently, linear polymers capable of forming entangled polymer networks inside the capillary have been
used. Very careful control of conditions both in gel formation and during electrophoresis is necessary to
achieve acceptable reproducibility. The formation of bubbles or voids in the filled capillary can
interrupt current flow, whilst excessive temperature fluctuations can degrade performance by causing
movement of the polymer filling.
Figure 4.57
Size-separation in CGE.The main applications of CGE are in separating protein fractions and oligonucleotides, and for DNA
sequencing. Cyclodextrins have been incorporated into some polymers to introduce chiral selectivity.
Capillary isoelectric focusing (CIEF) separates amphoteric substances such as peptides and proteins on
the basis of differences in their isoelectric points (pI values) defined as the pH at which the molecule is
uncharged. A pH gradient is formed along the capillary using a mixture of carrier ampholytes having pI
values spanning the desired pH range, typically 3 to 10. This is achieved by filling the capillary with a
solution of the sample and ampholytes and applying a potential field (Figure 4.58). All charged species
migrate along the capillary, cations and anions in opposite directions, until they become 'focused' at a
point where they are uncharged, i.e. pI = pH. The zone occupied by each sample species is self-
sharpening because diffusion away from the focal point causes them to acquire a charge which results
in a reversal in the direction of movement. The current flow virtually ceases when all species have
become 'focused' at their respective isoelectric