196 Charged interfacesIf the protein solution consists of a number of electrophoretically
different fractions, the sharp peak corresponding to the initially
formed boundary will broaden and may eventually split up into a
number of separate peaks each moving at a characteristic speed. The
above precautions against boundary disturbances enable a high
degree of resolution to be obtained, thus facilitating the identification,
characterisation and estimation of the components in such mixtures.
For example, the first purpose to which the Tiselius technique was
applied^184 was to demonstrate that the component of blood serum
once known simply as globulin actually consists of a mixture of
several proteins (Figure 7.9).
The moving boundary method is usually complicated by small
differences between the ascending and descending boundaries in the
two arms of the U-tube. These boundary anomalies result from
differences in the conductivity (and, therefore, the potential gradient) at
each boundary. They can be minimised by working with low protein
concentrations.Zone electrophoresis.94Zone electrophoresis involves the use of a relatively inert and
homogeneous solid or gel framework to support the solution under
investigation and minimise convectional disturbances. In addition to
being experimentally much simpler than moving boundary electro-
phoresis, it offers the advantages of giving, in principle, complete
separation of all electrophoretically different components and of
requiring much smaller samples; however, migration through the
stabilising medium is generally a complex process and zone electro-
phoresis is unsuited for the determination of electrophoretic mobilities.
Zone electrophoresis is used mainly as an analytical technique and,
to a lesser extent, for small-scale preparative separations. The main
applications are in the biochemical and clinical fields, particularly in
the study of protein mixtures. Like chromatography, zone electro-
phoresis is mainly a practical subject, and the most important
advances have involved improvements in experimental technique and
the introduction and development of a range of suitable supporting
media. Much of the earlier work involved the use of filter paper as the
supporting medium; however, in recent years filter paper has been
somewhat superseded by other materials, such as cellulose acetate,
starch gel and polyacrylamide gel, which permit sharper separations.