Figure 4.52
Electropherogram of blood serum on cellulose
acetate. (Buffer: 0.50 M barbitone, pH = 8.6)Effect of Temperature, pH and Ionic Strength
As mobility and rate of diffusion both increase with increasing temperature, close control is necessary
to ensure that valid comparisons can be made and that component bands are sharply resolved. The net
charge and hence the mobility of many species, especially organic compounds, is pH dependent. To
ensure that the migration characteristics of the components of a mixture remain constant during a
separation, the electrolyte solution employed is usually a buffer. In an unbuffered medium, pH changes
could lead to gross changes in the ionic concentrations of partially dissociated compounds such as weak
acids and bases. Ampholytes, e.g. amino-acids, and proteins, might even reverse their direction of
migration as a result of a change in pH
Large reservoirs of buffer solution are maintained in the electrode compartments to minimize the effect
of electrolysis which is to produce hydrogen ions at the anode and hydroxyl ions at the cathode. The
choice of buffer is critical in its effect on the degree of separation of a mixture, and is dictated largely
by the values of the various dissociation constants.
The total ionic strength should be kept fairly low (0.01–0.1 M) as mobilities decrease with increasing
salt concentration. Furthermore, at high electrolyte concentrations, more current flows through the
solution causing increasing difficulty in the dissipation of the evolved heat. Macromolecules are
frequently insoluble in solutions containing a high level of salts.
Electro-osmosis
During the migration of cations and anions towards their respective electrodes, each ion tends to carry
solvated water along with it. As cations are usually more solvated than anions, a net flow of water
towards the cathode occurs during the separation process. This effect, known as electro-osmosis, results
in a movement of neutral species which would normally be expected to remain at the point of
application of the sample. If required, a correction can be applied to the distances migrated by ionic
species by measuring them