Isoelectric Focussing

Substances (e.g. proteins) that contain both acid and basic groups are called amphiprotic and typically have an isoelectric point, which is a condition where the molecule has no charge. The isoelectric point occurs at a specific pH. This pH at which molecules have no charge has been given the term the pI. It follows that under electrophoretic conditions (where the pH gradient will exist superimposed on an electric field gradient) the molecule will remain stationary at their isoelectric point. An ionisable molecule that has no charge it is called a ‘zwitter ion’. If a zwitter ion is placed in a medium of higher pH the zwitter ion will loose a proton and become negatively charged and, as a consequence, will then migrate towards the anode. Conversely, if the pH becomes lower the zwitter ion will assume a positive charge and migrate towards the cathode. Consequently, if the potential gradient is accompanied by a pH gradient between the electrodes of an electrophoretic system then an interesting and useful effect called isoelectric focussing will become apparent.

In practice a buffer gradient is set up in the electrophoretic system so that the pH of the electrolyte will be highest at the cathode and lowest at the anode (the procedure for achieving this buffer gradient will be discussed later. On the introduction of a mixture of amphiprotic substances all the substances will acquire a different charge, the nature of which will depend on their respective pI values. As a consequence, all the different substances will have differing mobilities. The introduction, for example, of a protein into an environment at a pH higher than its pI it will cause it to become negatively charged and migrate in the direction of decreasing pH. The protein will finally reach a point where the pH is the same as its pI upon which. It’s charge will become zero, migration will cease and the molecule will become stationary.

Extending the argument it is seen that all the different substances will migrate towards that point where the pH of the environment is equal to their pI’s and, as a consequence, will form sharp, focussed bands As a result of this self focussing effect, the electrophoretic system will have a very high resolving power and in fact, it has been shown that substances whose pI values differ by only 0.02pH can be successfully separated.