General Properties of Electrophoresis

Read this article to learn about the general properties of electrophoresis.

The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any pH, exist in solution as electrically charged species either as cations (+) or anions (-).

Under the influence of an electric field charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

In order to understand fully how charged species separate, it is necessary to look at some simple equations related to electrophoresis. When a potential difference (voltage) is applied across the electrodes, it generates a potential gradient, E, which is applied voltage, V, divided by the dis­tance, d, between the electrodes. When this potential gradient E is applied, the force on a molecule bearing a charge of q coulombs is Eq newton’s.

It is this force that derives a charged molecule towards an electrode. However, there is also a factional resistance that retards the movement of this charged molecule. This frictional force is a measure of the hydrodynamic size of the molecule, the shape of the molecule, the pore size of the medium in which electrophoresis is taking place and the viscosity of the buffer.

The velocity, v, of a charged molecule in electrical field is, therefore, given by the equation:

v = Eq/f,

where f is the frictional coefficient.

More commonly the term electrophoretic mobility (µ) of an ion is used, which is the ratio of the velocity of the ion to field strength (v/E). When a potential difference is applied, therefore, molecules with different overall charges will begin to separate owing to their different electro­phoretic nobilities.

Even molecules of similar charges will begin to separate if they have different molecular sizes, since they will experience different frictional forces. As will be seen below, some forms of electrophoresis rely almost totally on the different charges on the molecules to effect separation, whilst other methods exploit differences in molecular size and, therefore, encourage frictional effects to bring about separation.

In short, electrophoretic mobility can be given as:

Mobility = (voltage) (charge)/ (frictional coefficient)

Provided the electric field is removed before the molecules in the sample reach the electrodes, the components will have been separated according to their electrophoretic mobility. Electrophore­sis is thus an incomplete form of electrolysis. The separated samples are then located by staining with an appropriate dye or by autoradiography if the sample is radiolabelled.

The current in the solution between the electrodes is conducted mainly by the buffer ions, a small proportion being conducted by the sample ions. Ohm’s law expresses the relationship be­tween current (I), voltage (V) and resistance (R):

V/I = R.

It, therefore, appears that it is possible to accelerate an electrophoretic separation by voltage, which would result in a corresponding increase in the current flowing. The distance migrated by the ions will be proportional to both current and time. However, this would ignore one of the major problems for most common forms of electrophoresis, namely the generation of the heat.

During electrophoresis, the power (W, watts) generated in the supporting medium is given by:

W = I²R.

Most of this power generated is dissipated as heat. Heating of the electrophoretic medium has the following effects:

i. An increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples.

ii. The formation of convection currents, which leads to mixing of the separated samples.

iii. Thermal instability of the samples that are rather sensitive to heat. This may include denaturation of the proteins (e.g., the loss of enzyme activity).

iv. A decrease of buffer viscosity, and hence a reduction in the resistance of the medium.

If a constant voltage is applied, the current increases during electrophoresis owing to the de­crease in the resistance and the rise in current increases the heat output still further. For this reason, workers often use a stabilized power supply, which provides constant power and thus eliminates fluctuations in heating.

Constant heat generation is, however, a problem. One may think that electrophoresis may be run at a very low power (low current) to overcome any heating problem, but this can lead to poor separation as a result of the increased amount of the diffusion resulting from a long separation times. Compromise conditions, therefore, have to be found with reasonable power settings, to give acceptable separation times, and an appropriate cooling system, to remove liberated heat.

While such systems work fairly well, the effects of heating are not always totally eliminated. For example, for electrophoresis carried out in cylindrical tubes or in slab gels, although heat is generated uni­formly through the medium, heat is removed only from the edges, resulting in a temperature gradient within the gel, the temperature at the centre of the gel being higher than that at the edges.

Since the warmer fluid at the centre is less viscous, electrophoretic mobility are, therefore, greater in the central region (electrophoretic mobilities increase by about 2% for each 1°C rise in the tempera­ture), and electrophoretic zones develop a bowled shape, with the zone centre migrating faster than the edges.

A final factor that can effect electrophoresis separation is the phenomenon of electroendoosmosis (also known as electro-osmotic flow), which is due to the presence of charged groups on the surface of the support medium. For example, paper has some carboxyl groups present, agarose (depend­ing on the purity grade) contains sulphate group and the surface of the glass walls used in capillary electrophoresis contains silanol (Si-OH) groups.

Mechanism of electroendoosmosis has been ex­plained in details in the section covering capillary electrophoresis, although the principle is the same for any support medium that has charged groups on it. In short in a fused silica capillary tube, above a pH value of about 3, silanol groups on the silica capillary walls will ionise, generating negatively charged sites. It is these charges that generate electroendoosmosis.

The ionized silanol groups create an electrical double layer, or region of charge separation, at the capillary wall/electro­lyte interface. When a voltage is applied, cations in the electrolyte near the capillary wall migrate towards the cathode, pulling electrolyte solution with them. This creates a net electrosmotic flow towards the cathode.

However, the introduction of the use of gels as a support medium led to a rapid improvement in methods for analyzing macromolecules. The earliest gel system to be used was the starch gel and, although this has some uses, the vast majority of electrophoretic techniques used nowadays involve either agarose gel or polyacrylamide gel.