Electrophoresis

Electrophoresis is the term given to the migration of charged particles under the influence of a direct electric current. It is a single-phase system and depends upon the relative mobilities of ions under identical electrical conditions.

>■ Ionization of amino acids - see Section 10.1.3.

>■ The electrochemical potential is the theoretics charge developed b> a molecule at a specific PH.

► The zeta potential is the actual charge carried by a molecule as a result of interactions with other ions In the solution.

> The ionic strength is a measure of either the total anionic or cationic charge of a solution.

Moving-boundary electrophoretic techniques, originally demonstrated by Tiselius in 1937, employ a U-tube with the sample occupying the lower part of the U and the two limbs being carefully filled with a buffered electrolyte so as to maintain sharp boundaries with the sample. Electrodes are immersed in the electrolyte and direct current passed between them. The rate of migration of the sample in the electric field is measured by observing the movement of the boundary as a function of time. For colourless samples, differences in refractive index may be used to detect the boundary. Such moving-boundary techniques are used mainly in either studies of the physical characteristics of molecules or bulk preparative processes.

Zonal techniques are the most frequently used form of electrophoresis and involve the application of a sample as a small zone to a relatively large area of inert supporting medium which enables the subsequent detection of the separated sample zones. A wide range of supporting media have been developed either to eliminate difficulties caused by some media (e.g. the adsorptive effects of paper) or to offer additional features (e.g. the molecular sieving effects of polyacrylamide gel).

Although molecular frictional effects impede the movement of molecules through a liquid, an effect that increases with the size of the molecule, the major factors in electrophoresis are the charge carried by the molecule and the voltage applied. The nature of the charge (positive or negative) will determine the direction of migration while the magnitude of the charge will determine the relative velocity.

This charge, although initially due to the ionization effects mediated by the pH of the buffer, will be appreciably modified by other features of the buffer composition. For a colloidal particle in suspension, e.g. a protein, its charged surface is surrounded by a layer of oppositely charged ions derived from the solution (Figure 3.23). There is an immobile, fixed layer of ions adsorbed on to the surface and a diffuse, mobile layer which becomes more mobile with increasing distance from the surface of the colloid. The charge developed by the molecule is due to its chemical nature and the pH of the buffer and is known as the electrochemical potential. For colloids, this is reduced as the salt concentration in the buffer increases, due to the partial neutralizing effect of the adsorbed layer of ions. The net resulting charge is known as the zeta potential and is the determining factor in electrophoretic mobility. The combined effect of the fixed and mobile layers of ions also increases the size of the particle and reduces its mobility even further.

The electrophoretic mobility of an ion is inversely related to the ionic strength of the buffer rather than to its molar concentration. The ionic strength (¡x) of a buffer is half the sum of the product of the molar concentration and the valency squared for all the ions present in the solution. The factor of a half is necessary because only half of the total ions present in the buffer carry an opposite charge to the colloid and are capable of modifying its charge:

The rate of movement of a charged particle is also related to the voltage gradient applied across the supporting medium, which is quoted in volts per centimetre (i.e. the distance between electrodes). The current generates heat,

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