The supporting medium
The effects of convection currents (resulting from the heating effect of the applied electric field) and the diffusion of molecules within the buffer solution can be minimized by carrying out the electrophoresis in a porous supporting medium. This contains buffer electrolytes and the sample is added in a discrete location or zone. When the electric field is applied, individual sample molecules remain in sharp zones as they migrate at different rates. After separation, post-electrophoretic diffusion of selected molecules (e.g. proteins) can be avoided by 'fixing' them in position on the supporting medium, e.g. using trichloracetic acid (TCA).The heat generated during electrophoresis is proportional to the square of the applied current and to the electrical resistance of the medium: even when a supporting medium is used, heat production will lead to zone broadening by increasing the rate of diffusion of sample components and buffer ions. Heat denaturation of certain sample types may also occur, e.g. proteins. Another problem is that heat will reduce buffer viscosity, leading to a decrease in resistance. If the electrophoresis is run at constant voltage, Ohm's law dictates that as resistance falls, the current will increase, leading to further heat production. This can be avoided by using a power pack that provides constant power. In practice, most electrophoresis equipment incorporates a cooling device; even so, distortions of an electrophoretic zone from the ideal 'sharp, linear band' can often be explained by inefficient heat dissipation.
Supporting media can be sub-divided into:
- Inert media - these provide physical support and minimize convection; separation is based on charge density only (e.g. cellulose acetate).
- Porous media - these introduce molecular sieving as an additional effect: their pore size is of the same order as the size of molecules being separated, restricting the movement of larger molecules relative to smaller ones. Thus, separation depends on both the charge density and the size of the molecule.
Where necessary, EOF can be avoided by using supporting media such as agarose or polyacrylamide, but it is not always a hindrance to electrophoretic separation. Indeed, the phenomenon of EOF is used in the high-resolution technique of capillary electrophoresis