Nature of the Electrophoretic System

As well as the characteristics of the substances to be separated, there are several parameters relating to the electrophoretic system itself that have a pronounced effect on the electrophoretic mobilities of the molecules or ions. These parameters are as follows.

1. The ionic composition of the electrophoresis buffer.

2. The temperature.

3. The pH of the electrophoresis buffer.

4. The applied voltage.

5. In the case of zone electrophoresis, the type of support medium chosen, and if the support medium is gel, its pore size.

Ionic composition of the electrophoresis buffer

A charged macromolecule becomes surrounded by an ionic atmosphere of opposite charges because of interactions between ionizable groups on the surface of the charged molecule and ions in the electrophoresis buffer. As a result, both its net charge and its elec-trophoretic mobility are decreased. This effect is quite pronounced in the electrophoretic separation of proteins, since different proteins have different amino acid side chains which interact to varying degrees with the ions in the solutions used.

In order to minimize these 'counterion' effects it is advisable to use an electrophoresis buffer with as low an ionic strength as possible. However in some cases, such as with polypeptides and polynucleotides, elec-trophoresis has to be carried out in solutions of high ionic strength, otherwise these macromolecules will not be soluble. It therefore becomes necessary to choose a suitable salt concentration.

Temperature Temperature plays a pronounced effect on electrophoresis. In an electrophoretic run, heat (Joule's heat) is generated and may affect the electrophoresis in a number of ways.

1. Diffusion. An increase in temperature causes an increase in the diffusion of migration zones of charged molecules. If the electrophoresis takes a long time (several hours) diffusion effects become more significant.

2. Evaporation. It is customary to perform elec-trophoresis in a closed system to avoid loss of water by evaporation, which increases with temperature. This evaporation results in the drying out of the supporting medium and also leads to an increase in the ionic strength of the buffer during the analysis.

3. Viscosity. In gel electrophoresis an increase in temperature can change the viscosity of the medium. Since this takes place during the electrophoretic run, the interpretation of the results may become complex.

4. Distortion of zones. During an electrophoretic run, particularly in column gels if cooling is inadequate, the portions of the migration zones in the warmer parts of the gel move faster than those in the cooler parts. This difference in migration speeds produces curved bands. This may result in the overlap of neighbouring zones and consequently in poor resolution.

5. Convection currents. During an electrophoretic run the warmer solution in the centre of the apparatus has a lower density than the cooler solution close to the walls. This density gradient induces convection currents in the solution. Since water has its maximum density at 4°C, and the smallest variations in density of aqueous solutions are observed around this temperature, it is advisable to perform electrophoresis at a temperature as close as possible to 4°C. However, the viscosity of an aqueous solution increases as the temperature is lowered, which may result in an increase in the frictional resistance to the migration of the charged molecules. If the temperature is maintained at 4°C the electrophoretic mobilities of the charged molecules will be relatively low. It is therefore necessary to choose an optimal temperature for a particular electrophoretic run and maintain it throughout the course of analysis.

pH of the electrophoresis buffer pH has a marked effect on the nett charge on a protein molecule. At a definite pH value, i.e. at the isoelectric point, the nett charge on the molecular is zero. Molecules acquire a nett positive charge at pH values below their isoelectric points. At pH values above their isoelectric points, they acquire a nett negative charge. Thus different molecules (e.g. proteins) at any particular pH value will have different nett charges. To optimize the separation of a mixture of (protein) molecules the buffer pH must be chosed on the basis of the nett molecular charges. For example in an electrophoretic run two or more proteins may migrate together to give only one band. If the analysis is done at a different buffer pH value it may result in the appearance of extra protein bands, indicating the presence of other proteins in the sample.

Applied voltage In electrophoresis the applied voltage plays an important role. The migration velocity of a molecule is proportional to the field strength across the medium. The higher the applied voltage, the larger the field strength across the medium, and the faster a molecule will migrate. Thus, the charged molecules will migrate more quickly with increasing voltage. This saves time and reduces the diffusion of migrating molecules. However, with increasing voltage the current also increases, resulting in greater power generation (the power increases as the square of the current).

Some of this power is dissipated as heat (Joule's heat). The heat generated can have serious effects on an electrophoretic analysis, as discussed previously. It is therefore necessary to select a definite value of applied voltage. The voltage (or current) should be large enough to allow rapid migration of charged molecules, but not so large as to generate excessive heat.

Support medium In zone electrophoresis different types of support media are used. The selection of the most suitable support medium for a particular zone electrophoretic analysis is based on the following considerations.

1. Sample quality.

2. Size of the molecules - whether they are small or large. If a sieving gel has to be used its pore size (concentration) is chosen so as to suit the molecular size under study.

3. The time required for analysis.

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