Electrophoresis is a separation technique based on the movement of charged
molecules in an electric field. Dissimilar molecules move at different rates
and the components of a mixture will be separated when an electric field is
applied. It is a widely used technique, particularly for the analysis of
complex mixtures or for the verification of purity (homogeneity) of isolated
While electrophoresis is mostly used for the separation of charged
macromolecules, techniques are available for high-resolution separations, e.g.
capillary electrophoresis, of small molecules such as amino acids, anions and
The electrophoretic mobility of a charged molecule depends on:
- Net charge - negatively charged molecules (anions) migrate towards the
anode (+), while positively charged molecules (cations) migrate towards
the cathode (-); highly charged molecules move faster towards the
electrode of opposite charge than those with lesser charge.
- Size - frictional resistance exerted on molecules moving in a solution
means that smaller molecules migrate faster than large molecules.
- Shape - the effect of friction also means that the shape of the molecule
will affect mobility, e.g. globular proteins compared with fibrous proteins,
linear surfactants compared with micellular surfactants.
- Electrical field strength - mobility increases with increasing field strength
(voltage), but there are practical limitations to using high voltages,
especially due to heating effects.
The combined influence of net charge and size means that mobility is
determined by the charge-to-density or the charge-to-mass ratio, according to
|⇒ Equation [33.1]
is the net charge on the molecule, r
is the molecular radius and E
the field strength.
Most types of electrophoresis using supporting media are simple to carry
out and the apparatus can be easily constructed, although inexpensive
equipment is commercially available. High-resolution techniques such as twodimensional
electrophoresis and capillary electrophoresis require more
sophisticated equipment, both for separation and analysis (see later).
Simple electrophoretic separations can be performed either vertically (Fig.
33.1) or horizontally (Fig. 33.2). The electrodes are normally made of
platinum wire, each in its own buffer compartment. In vertical electrophoresis,
the buffer solution forms the electrical contact between the
electrodes and the supporting medium in which the sample separation takes
place. In horizontal electrophoresis electrical contact can be made by buffersoaked
paper 'wicks' dipping in the buffer reservoir and laid upon the
supporting medium. The buffer reservoir normally contains a divider acting
as a barrier to diffusion (but not to electrical current), so that localized pH
changes which occur in the region of the electrodes (as a result of electrolysis, are not transmitted to the supporting medium or the sample.
Individual samples are spotted onto a solid supporting medium containing buffer or are applied to 'wells' formed in the supporting medium. The power
pack used for most types of electrophoresis should be capable of delivering
approximately 500V and approximately 100mA.
|Fig. 33.1 Apparatus for vertical slab electrophoresis (components move downwards from wells,
through the gel matrix).
|Fig. 33.2 Agarose gel electrophoresis.