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
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
With some supporting media, e.g. cellulose acetate, a phenomenon called
electro-endosmosis or electro-osmotic flow (EOF) occurs. This is due to the
presence of negatively charged groups on the surface of the supporting
medium, attracting cations in the electrophoresis buffer solution and creating
an electrical double layer. The cations are hydrated (surrounded by water
molecules) and when the electric field is applied, they are attracted towards
the cathode, creating a flow of solvent that opposes the direction of
migration of anionic molecules towards the anode. The EOF can be so great
that weakly anionic molecules may be carried towards the cathode.
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