The Basic Principal of Isotachophoresis

 

The name isotachophoresis is derived from the three Greek terms, iso that means equal, tachos that means speed and phoresis that means migration. Thus, liberally translated isotachophoresis means migration at equal speeds. Consider a situation where a tube is connected to two reservoirs; one reservoir and the tube is filled with an electrolyte buffer containing fast moving ions, the leading electrolyte (i.e. an elecetrolyte containing ions of high mobility); the leading electrolyte will also control the pH at which the separation is to be carried out; the other reservoir is filled with an electrolyte buffer containing very slow moving ions, the terminating electrolyte (i.e. ions with small mobility). In addition, the fast moving ions must have higher mobility than any of the ions in the sample and the slow moving ions in the terminating electrolyte must have lower mobility than any of the ions in the sample. If the system is set up to separate anionic species then the reservoir containing the leading electrolyte will contain the anode and the reservoir containing the terminating electrolyte will contain the cathode.  

 

Employing a simple sample-tap procedure, let a sample containing different ions be placed between the fast and slow moving electrolytes. When an electric field is set up and a current passed through the system, initially a uniform electric field will be formed across the sample and the individual ions will separate from one another according to their different migration rates (i.e. on their differing mobilities) as would be expected from normal electrophoretic procedure. The pH is controlled by the counter ion of the leading electrolyte, which travels in the opposite direction to the ions in the sample. As, initially, the ions travel at different speeds a separation will start to occur. However, the faster ions in the sample will create a lower field around them and conversely the slower moving ions will create a higher field across around them. This means that the faster moving ions will be retarded by the weaker field around them and the slower moving ions will be accelerated by the stronger field around them.  Finally, all the constituents of the sample will be separated and migrate at the same rate determined by a constant value for the product of the ion mobility and its respective field associated with it.

 

The system is self sharpening as, if an ion diffuses out of its band into a higher or lower field band, then it will either be de-accelerated and so forced backed into its original band or accelerated from the higher field band back into it original faster band. When equilibrium has been established and all the ions are migrating at a constant rate the bands can be easily identified, as there will be a sharp electric field difference at the boundary between each band. As will be discussed in due course, space marker molecules can be mixed with the sample to physically separate the individual constituents of the sample from one another.

 

An isotachophoretic separation is depicted diagrammatically in figure 32. As already defined for a steady state separation all ions will have identical migration velocities, which will be defined by the migration rate of the ions of the leading electrolyte. Consider the zone (L) containing the leading electrolyte anions, and the zones for the anions (A), (AB, and (C).

 

Figure 3. An Isotahcophoretic Separation of a Three Component Anion Mixture

 

        Then ,                             

 

Where, ( ), (), (), (), and () are the migration velocities of the leading electrolyte anion, substance (A) anion, Substance (B) anion, substance (C) anion and the anion of the terminating electrolyte respectively.

 

              or,                                           

 

Where, (), (), (), (), (), (), (), (), (), (), are the mobilities and the electric field associated with the leading electrolyte anion, substance (A) anion, substance (B) anion, substance (C) anion and the anion of the terminating electrolyte respectively.

 

The latter equation has been given the term the ‘isotachoporetic condition’

 

The anions arrange themselves under the influence of the applied field in the order of their decreasing mobilities and consequently electric field strength also increases with decreasing ion mobility. Thus, for constant current flow the energy evolved will also increase as the ion mobilities decrease.  As a consequence, more heat will be generated in those areas where the ionic mobilities are small and consequent each band that represents ions of a particular mobility will assume a particular temperature and produce a stepped temperature stepped of the form shown in figure 4. 

 

 

Figure 4. Temperature Profile of an Isotachophoretic Separation