Q.4.(c) Elaborate factors affecting electrophoretic mobility.
Ans.4. (c) Electrophoretic Mobility
The migration rate ʋ of an ion (cm/s) in an electric field is equal to the product of the field strength E (V cm-1) and the electrophoretic mobility µe (cm2V-1s-1). That is,
ʋ = µeE
Electrophoretic mobility, µe = E/ʋ
The electrophoretic mobility is in turn proportional to the ionic charge on the analytic and inversely proportional to frictional retarding factors.
Factors affecting Electrophoretic Mobility:
(1) Electrolysis- Electrophoresis is accompanied by electrolysis which causes microscopic bubbles to accumulate on the electrodes. When a bubble is formed. The electrical contact with buffer is lost. The resistance of the electrode gradually builds up until, by coalescing, the bubbles become large enough to break away from the surface and as a result, contact with buffer is restored. The variations in resistance due to formation and breaking away of buffers are negligible for electrodes of large areas, because only a fraction of total area is obscured at any given time. The variation in resistance with small electrodes cannot be neglected because almost the entire surface of the electrode may be covered before the bubbles become large enough to break away. These fluctuations in resistance may cause the particles to migrate at erratic rates and may also cause reduction in mobility.
(2) Ionic Strength- The activity coefficients of ionic substances in solution are influenced by the concentration of the solution and by the valency of the ions. When the solution is a buffer, the weakly ionized component makes virtually no contribution to the ionic strength of the solution. Thus, when pH of a buffer changes, the ionic strength must also change. Hence it is necessary to take pH into consideration when the ionic strength of a buffer is calculated. It is sometimes desirable to measure electrophoretic behavior of a substance at different pH values, while maintaining the same ionic strength, by adding enough neutral salt to the solution to make up for the decrease in ionic strength of the buffer because of change in pH. The electrophoretic mobility is approximately proportional to the reciprocal of the square root of ionic strength. Since mobility increases with diminishing ionic strength, it is often possible to shorten the time required for electrophoretic separations by making use of a dilute buffer.
(3) pH and other Chemical Characteristics- The electrophoretic mobility is greatly affected by the pH of a buffer, particularly when the sample is either a weak acid or a weak base, because the pH establishes its degree of ionization. In case of amphoteric substances such as proteins, the direction of migration depends upon whether the buffer pH is above or below the isoelectric point of the sample substance. Better separations can be achieved using either strongly alkaline or strongly acidic buffers. The optimum pH range for separating proteins is 8.6-9.2 pH. Best results are obtained between these ranges of pH. The ideal substance for buffering to pH 8.6 would exist half as the acid and half as the salt at this pH, which would require a dissociation constant of 2.5 X 10-9.
(4) Electro-osmosis- during electrophoresis, there is often a flow of water under the influence of the voltage gradient. This is called electro-osmosis, the rate of which is influenced by the species and concentration of ionic solutes in water. This process causes a passive displacement of the entire sample with respect to the electrophoretic bed. When mobility measurement becomes important, it is necessary to make corrections for this motion by observing the displacement of some uncharged solute.
(5) Interaction with supporting medium- Electrophoretic migration is slower in stabilizing medium than in free solution. If the supporting medium has ionic side chains (e.g., sulphate groups in agar or carboxylate groups in paper), these can interact with the particles being separated by electrophoresis. Ionic side chains are usually undesirable because they provide trailing. Most protein molecules have a number of ionic side chains; some of them positive and some negative and the direction of migration depend upon the net charge. For example, a molecule having six negative side chains and five positive side chains of either charge on the stabilizing medium may affect their mobility’s.
(6) Heat- The unavoidable electrical heating that accompanies the process of electrophoresis has a number of adverse effects. In free solution electrophoresis, heat causes convection currents. As a result, electrophoretic pattern is disrupted. Supporting media are intended to prevent this by impeding liquid flow. The adverse effects of heat are most evident in gel beds, where convection is virtually eliminated.
A strip having a rectangular cross section has four surfaces from which evaporation can occur. A molecule of liquid at the edge of the strip has a chance to evaporate from any of the three surfaces, while a molecule at the Centre can evaporate from only two surfaces. Thus there is an increased rate of evaporation from the edges and this causes the buffering salt to be concentrated. In addition to evaporation from edges, the effects of diffusion and solvent flow cause concentration gradient that diminishes towards the Centre of the paper.
Since mobility decreases with increasing ionic strength, migration is slower at the edges than it is in the Centre of the strip and each zone assumes the shape of a V pointing in the direction of migration. In gels, similar V-shaped patterns occur even when there is no evaporation. Since mobility increases with heat, the central portion of each zone migrates in advance of its edges.
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