Frans_M_Everaerts_Isotachophoresis_378342.pdf

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DETECTION SYSTEMS
Fig.6.49. Isotachopherogram in the operational system at pH 4, with HCI (0.01 N) and e-aminocaproic
acid as the counter ion. The terminating ion was glutamate. While the UV detector (below) indicates
only one of the two UV-absorbing components injected, the conductimeter (above) shows the separa-
tion of the two components. In the trace derived from the conductimeter, a further component is
determined, which is an impurity from the electrolytic system. The current was stabilized at 80 PA.
R = Increasing electric resistance;A = increasing UV absorption.
The influence of variations in the direct driving current were small in the range studied.
In order to prevent the already discussed electrode reactions, this driving current has to
be limited to 150 PA. However, experiments at 300 pA showed that the standard mixture
of anions was separated in a few minutes, although the resolution decreased, The main
reason for this effect must be that high current densities increase the radialnon-uniformity
of the temperature profile inside the narrow-bore tube. This causes an increased parabolic
profile, especially for the zones that are situated towards the rear side. Also, the electro-
endosmotic profile increases.
The use of the dilution method of concentration determination improves the resolu-
tion of the UV detector (Fig.6.33) about 50-fold. This factor depends on the molar
extinction coefficients of the ionic species involved. The disadvantage of this method of
detection, as can be seen in the determination of salicylic acid in Fig.6.32, is that a small
change in the pH of the leading electrolyte may change the effective mobilities between
the two ions forming the mixed zone in such a way that an enrichment of one of the
components is soon detectable in the UV trace*. This sometimes makes an accurate
determination of the step height in the UV trace (quantitative determination) less
accurate. If a W-absorbing ion can be sandwiched between two non-W-absorbing ions,
minimal amounts of the UV-absorbing ion can be measured quantitatively because one
can make use of the parabolic profile of the consecutive zones. The W-absorbing ion can
be determined, in spite of its small zone length (e.g., less than 0.01 mm), because the
*A similar effect can be expected if the pK, values of the components involved differ much.