Electrophoresis using a constant potential across the separation field.

Reproducibility of electrophoretic runs depends on the maintenance of a smooth and constant voltage gradient within the supporting medium or separation chamber. Power packs to supply constant current or constant voltage as measured at the pack are readily available, but if such a pack is used with constant voltage, the proportion of the voltage drop available for the electrophoretic separation will be affected not only by changes in the resistance in the separation field, but also by changes in the external circuit, which will be certain to occur at the electrodes, separation membranes, or buffer reservoirs. For this reason, it is usual to work with constant current, since the voltage drop in the separation field will then be independent of the resistance changes in the external circuit. Brattsten (1955) demonstrated that the effect of temperature variation on migration velocity may be greater where constant voltage is used and recommended constant current. He was using a system without a supporting medium. Where supporting media are used there are many variables in addition to temperature. Under constant current conditions any change affecting the resistance within the supporting medium or separation chamber will cause a variation in the voltage gradient and consequently a lack of reproducibility from one run to another. In continuous electrophoresis, either with or without a supporting medium (as in the Elphor VaP apparatus), such variation leads to a reduction of the efficiency of separation. In micro-immunoelectrophoresis in agar the thickness of the agar and the buffer retention might vary from one batch of slides to the next. Supporting media, such as cellulose acetate, cellogel, or paper which have to be 'blotted' to remove surplus buffer, do not provide exactly reproducible conditions; ionic pile up at membranes or edges of supporting media will also affect reproducibility, and the heat produced during a run may cause progressive changes in supporting media. All these effects are even more pronounced with