Frans_M_Everaerts_Isotachophoresis_378342.pdf

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CHOICE OF ELECTROLYTE SYSTEMS
Fig.5.10. Isotachopherograms for the separation of phospate and salicylate. The leading electrolyte
was 0.01 N hydrochloric acid (pro analysi grade), adjusted to a pH 4 by the addition of recrystallized
p-alanine; the terminating electrolyte was glutamic acid (0.01 N), adjusted at pH 4 by the addition of
Tris. The electric current was stabilized at 70 PA. The detection was performed with a linear
conductivity detector (a.c. method) and a UV absorption detector (256 nm), both of which are
described in Chapter 6. In the left-hand experiment 10 nmole of phosphate, in the centre experiment
10 nmole of phosphate plus 10 nmole of salicylate and in the right-hand experiment 10 nmole of
salicylate was introduced. Attention should be paid to the difference in step heights, as obtained
in the linear traces of the W absorption detector. t = Time; R = increasing resistance; A =
increasing UV absorption. I = Chloride; 2 = salicylate; 3 = glutamate; 4 = phosphate.
is 3.1, we can separate them according to their pK values. In Fig.5.11 this separation is
shown for three different pH values. The left-hand isotachopherogram shows the
separation with a leading electrolyte consisting of 0.01 N hydrochloric acid plus 0-alanine
at a pH of 3.2 and glutamate as terminator. In the centre as shown the mixed zone as in
Fig.5.10 and on the right is shown a separation at a pH of 7 with a leading electrolyte
consisting of 0.01 N hydrochloric acid plus imidazole and glutamate as terminator. It can
be seen in Fig.5.11 that the problem is not whether the ionic species can be separated,
but how to recognize a mixed zone. The UV detector can provide the solution. The UV
signals for zones with only one ionic species of the sample show sharp step heights and
each sample zone has its own characteristic step height in a particular electrolyte system.
However, when mixed zones are present, a typical form is often shown as can be seen in
Fig.5.10 (centre), while its average step height lies between those of the pure sample zones,
depending on the concentrations of the sample ionic species present in the mixed zones.
Example H Suppose we wish to separate anionic species with ionic mobilities of about
30, but with pK values of 2, 3, 5,7 and 8.
In this instance, of course, we have to choose a separation according to pK values.
Thus we have to choose a leading electrolyte with a pH lying between the pK values of
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