Page 57 - Tandem Techniques

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At the (pI) the ampholyte will not migrate in an electric field as it will be existing probably as a
zwitterion (an internal salt) with no charge. A separation due to isoelectric focusing is depicted in
Figure 1.11. Using a mixture of ampholytes (usually polyamino polycarboxylic acids) contained in the
capillary tube, the anode vessel containing and acid solution, and the cathode vessel filled with an
alkaline solution, a (pH) gradient will be formed along the capillary tube on the application of an
electric field. When this gradient is stabilized, the sample is introduced into the center of the electrically
arranged ampholytes. Each substance will migrate under the applied field until it reaches the position
where the (pH) is equal to its (pI) and, as at that point it will no longer be charged, it will come to a halt.
If any solute tends to move out of the isoelectric point, it will immediately ionize and become charged
again and under the electric field forced back into its isoelectric position; i.e. it is focused. As a result, a
series of bands of solutes are formed in the capillary tube in the order of their increasing (pI) values. It
is clear that the resolution of such a system can be extremely high and separation will depend on all the
substances having sufficiently different isoelectric points.

Electro-osmotic Flow (Electro-endosmosis)

The movement of a liquid, when in contact with a charged surface, situated in a strong electric field is
called electro-endosmosis. The flow of liquid through a silica tube under electro-endosmosis is of 'plug'
form and does not exhibit the parabolic velocity profile that normally occurs in Newtonian flow. It is
interesting to note that there is very little band dispersion due to resistance to mass transfer when the
flow is electro-osmotically driven.

Figure 1.12
Newtonian Flow and Electro-endosmosis