Frans_M_Everaerts_Isotachophoresis_378342.pdf

378 COUNTER FLOW OF ELECTROLYTE
11
I , I
I
I I
1 1
B
6
1 A
I
,
I
,
v,
I
Fig.17.1. Diagram to illustrate the 'concentration' (a) and 'dilution' (b) principles of isotachophoretic
analyses.
17.2. EXPERIMENTAL
If the disturbance caused by the counter flow ofelectroIyte is to be studied, a
scanning detector is needed or dyes must be applied. We studied the disturbance by
using the dyes amaranth red, bromophenol blue and fluorescein. Acetate and glutamate
were found to be suitable for spacing the dyes. The separations were carried out in the
operational system at pH 6 (Table 12.1) and the results are shown in Fig.17.2 and 17.3.
The photographs in Fig.17.2 were obtained in a fluoroethylene polymer (FEP)
narrow-bore tube of I.D. approximately 0.5 mm, while the isotachopherograms in
Fig.17.3 were obtained with a conductivity detector (a.c. method) that had a probe made
almost completely of Perspex (see Chapter 6) with I.D. 0.4 mm. It can be seen that the
optimal sharpness of zones is obtained if a small counter flow of electrolyte is applied.
This optimal counter flow depends on, amongst other things, the viscosity of the
electrolyte, the diameter of the bore, the temperature inside the bore and the material
of which the narrow bore is made. This is why the recording of the zones by the a.c.
method (Fig.17.3) indicates another optimum for the sharpness of the zone boundaries,
~ ~ ~.
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Fig.17.2. Results of experiments carried out in the operational system at pH 6 (Table 12.1) to show
the disturbance of the profiles by a counter flow of electrolyte (indicated in percentages). In (a) a
free solution was applied, while in (b) an electrolyte in which the viscosity was increased by addition of
2% of hydroxyethylcellulose (purified by shaking it with a mixed bed ion exchanger) was used; the
viscosity of the solution was approximately 100 cP. 1 = Chloride; 2 = amaranth red; 3 = acetate;
4 = bromophenol blue; 5 = glutamate; 6 = fluorecein; 7 = MES. It can clearly be seen that the zone
boundaries are first sharpened, and then are disturbed. The disturbance is a function of the viscosity.
In the two photographs in the bottom right-hand corner, two examples are given of the disturbance
of the zune boundary as a function of the effective mobilities of the consecutive zones: (a) shows the
boundary amaranth red/MES and (b) shows the boundary amaranth red/glutamate (loo%* counter
flow of electrolyte).