relative to the position reached by a neutral molecule. Propanone, urea or glucose is sometimes added to
a sample for this purpose. Electro-osmotic flow (EOF) is particularly important in high performance
capillary electrophoresis, vide infra.
Supporting Medium
The function of the supporting medium is to provide an inert porous structure for the electrolyte
solution. Filter paper, cellulose acetate and various gels are used for this purpose. Although paper is
cheap and convenient to use, its fibrous structure and the presence of ionic groups result in poor
resolution of migrating species due to tailing. Polymerized cellulose acetate is available in sheets and
has a small uniform pore size. This fact coupled with the absence of ion-exchange or adsorption sites
results in separations showing better resolution than those on paper. However, electro-osmosis is
pronounced and the maximum sample loading is only about a quarter that of paper. Agar and
polyacrylamide gels have found the most widespread use. Both are used in the form of thin flat beds or
in columns. Agar is particularly useful for column work because its high mechanical strength enables it
to be readily extruded for visualizing or further analysis after the separation process is complete.
Polyacrylamide gels are used in gel filtration procedures where varying the degree of cross-linking
produces materials fractionating over different molecular size ranges. In electrophoresis separations,
fractionation according to size is therefore superimposed on the migration process. This results in
enhanced degrees of separation for complex mixtures of macromolecules.
Detection of Separated Components
For separations on paper, after a preliminary drying, spraying or dipping with any of the chromogenic
reagents used in paper and thin-layer chromatography serves to visualize the individual components.
Cellulose acetate has the added advantage that it can be made water-clear by treatment with an oil or
with a mixture of acetic acid and ethanol. This facilitates subsequent measurements of spot densities.
Proteins and other macromolecules are visualized by treating the electropherogram with a stain or dye
that becomes adsorbed onto the solute molecules but which is easily washed off the solute-free areas.
Gel electropherograms cannot be dried before spraying or dipping, and a precipitant is incorporated into
the visualizing reagent to prevent materials dissolving during treatment.
Applications of Traditional Zone Electrophoresis
This is considered to be largely a qualitative technique. Difficulties that arise in obtaining reproducible
quantitative data are similar to those encountered in thin-layer chromatography. In addition, adsorption
characteristics of dyes on macromolecules are so variable that only semiquantitative comparisons can
be made. These are, however, still very useful