High performance capillary electrophoresis - T.E.A.M.

Modes
3.5 Capillary
isotachophoresis
Capillary isotachophoresis (CITP) is a “moving boundary”
electrophoretic technique. In ITP, a combination of two
buffer systems is used to create a state in which the separated
zones all move at the same velocity. The zones remain
sandwiched between so-called leading and terminating
electrolytes. In a single ITP experiment either cations or
anions can be analyzed.
For anion analyses, for example, the buffer must be selected
so that the leading electrolyte contains an anion with an
effective mobility that is higher than that of the solutes.
Similarly, the terminating anion must have a lower mobility
than that of the solutes. When the electric field is applied
the anions start to migrate towards the anode. Since the
leading anion has the highest mobility it moves fastest,
followed by the anion with the next highest mobility, and so
on. In ITP the individual anions migrate in discrete zones,
but all move at the same velocity, as defined by the velocity
of the leading anion. The separation process was illustrated
in figure 21c.
The steady-state velocity in ITP occurs since the electric
field varies in each zone. The field is self-adjusting to
maintain constant velocity (that is, velocity = mobility x
field), with the lowest field across the zone with highest
mobility. This phenomenon maintains very sharp boundaries
between the zones. If an ion diffuses into a neighboring
zone its velocity changes and it immediately returns to its
own zone.
Another interesting feature of ITP is the constant concentration
in each zone, determined by the concentration of the
leading electrolyte. Since ITP is usually performed in
constant current mode, a constant ratio must exist between
the concentration and the mobility of the ions in each zone.
Zones that are less (or more) concentrated than the leading
electrolyte are sharpened (or broadened) to adapt to the
proper concentration. The solute-concentrating principle of
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