Isoelectric Focussing

 

Amphiprotic substances (e.g. proteins), which contain both acid and basic groups in their molecules, characteristically have an isoelectric point where the molecule has no net charge and this occurs at a specific pH.  The pH at which this occurs for a specific substance is given the term pI. Consequently under electrophoretic conditions (where it will exists in a gradient electric field) the molecule will remain stationary. When the molecule has no net charge it is called a ‘zwitter ion’. If the molecule is placed in a medium of higher pH the molecule will loose a proton and become negatively charged and will then migrate towards the anode. If the pH becomes lower the molecule will assume a positive charge and migrate towards the cathode. It follows that if the potential gradient is accompanied by a pH gradient from electrode to electrode then an interesting and useful effect called isoelectric focussing can be observed.

 

A buffer gradient is set up so that the pH will be highest at the cathode and lowest at the anode (the procedure for achieving this buffer gradient will be described in due course). If a mixture of amphiprotic substances is now introduced into such a system 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. If on introduction, for example, a protein exists in an environment at a pH higher than its pI it will become negatively charged and migrate in the direction of decreasing  pH. And finally reach a point where the pH is the same as its pI whereupon its net charge will become zero and migration will cease and it will become stationary.

 

As a result all the different substances will migrate towards the point where the pH of the environment is equal to their pI’s and will form sharp, focussed bands Due to this self focussing effect, the system can have a very high resolving power and in fact substances whose pH differ by only 0.02pH can be successfully separated.