Instant Notes: Analytical Chemistry

Capillary electrochromatography(CEC) is an even newer technique than CE

and uses capillaries packed with 5mm or smaller particles of a stationary phase

similar to those used in HPLC(Topics D6 and D7). Solutes are separated by

a combination of electrophoretic migration and chromatographic sorption

processes (Topic D2) giving the technique additional versatility in varying the

selectivity. Efficiencies are particularly high because the packing minimizes

band spreading by solute diffusion in the buffer solution.

Running buffer The function of the running buffer is to provide an electrically conducting
medium and pH stability. The latter is essential in ensuring that solutes have a
constant mobility throughout the separation. Typical buffers covering a wide
range of pH values are listed in Table 2(a). Concentrations of 0.05–0.5 M provide
an optimum ionic strength that allows rapid migration of solutes without the
generation of excessive heat or losses by evaporation. Buffer additives in the
form of surfactants, complexing agents and organic solvents are sometimes
added to control migration rates and selectivity. Some examples are given in
Table 2(b).

Various solid media are employed to support the running buffer in traditional

electrophoresis. Polymeric gels, such as polyacrylamide, agarose, starch and

cross-linked dextrans (Sephadex) are the most common, although paper and

cellulose acetate have also been used. The gels are saturated with the running

buffer, and have a restricted range of pore sizes that can be controlled during

polymerization. This facilitates the separation of solutes by a size exclusion

mechanism (Topic D2) in addition to their differential electrophoretic migration.

Polyacrylamide gelsare the most versatile, offering superior resolution and

dissipating heat efficiently. Agarose gels are particularly effective for the

separation of mixtures of large biomolecules such as DNA proteins, as the

pore sizes are larger than those in polyacrylamide.

Electro-osmosis When a potential gradient is applied across a running buffer, hydrated buffer
cations tend to be drawn towards the cathode producing a bulk flow of
liquid known as the electro-osmotic flow(EOF). Although the effect is not
pronounced in classical electrophoresis, in capillary electrophoresis it is

Supporting
medium

D8 – Electrophoresis and electrochromatography 177

Electrolyte

buffer

Anode

Capillary

inlet

Reservoir

Electrolyte

buffer

Cathode

Capillary

outlet

Data

acquisition

Reservoir

Hight voltage

power supply

Detector

Fig. 2. Schematic diagram of a high performance capillary electrophoresis system.