Introduction

 

Isotachophoresis is one form of the separation technique called electrophoresis. However, electrophoresis also includes systems such as zone-electrophoresis, moving boundary electrophoresis and iso-electric focusing. Electrophoretic migration, beitmay zone electrophoresis or other electrophoretic techniques depends on the interaction between a charged particle (or molecule) and an applied external electric field.

 

Under the influence of an electric field (E) a charged particle or molecule will move at a velocity (v) according to the simple equation,

 

 

Where (m) is the mobility of the particular charged species.

 

As the mobility of the each charged moiety will be different, their respected velocities will also be different consequently, in a given time, they will travel different distances and, thus, be separated. (This separation procedure will apply to moving boundary electrophoresis, zone electrophoresis and isotachophoresis but not to iso-electric focusing.

 

In the technique of iso-electric focusing the separation does not depend on the different mobilities of the components of the mixture but on the difference between their (pI) values. The (pI) value of a substance is that specific pH at which its molecules have no net charge. If the molecules are placed in a medium of higher pH they will loose a proton and become negatively charged and will then migrate towards the anode. If the pH becomes lower the molecules will assume a positive charge and migrate towards the cathode. Consequently, when pH = pI and under electrophoretic conditions where they exist in an electric field, the molecules will remain stationary. Under such circumstances when the molecules have no charge they arecalled a ‘zwitter ion’.

 

In practice a buffer gradient is set up so that the pH will be highest at the cathode and lowest at the anode. 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 charge will become zero, 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.