Frans_M_Everaerts_Isotachophoresis_378342.pdf

Fig.5.9. Isotachopherograms of the separation of 1 pl of a mixture of chlorate (0.01 M), formate (0.0 1 M),
acetate (0.01 M) and glutamate (0.01 M). In both instances morpholinoethanesulphonic acid was
used as the terminator, which is the reason why the electric current was stabilized at 30 fiA. 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. R = Increasing resistance; t = time; A =increasing UV
absorbance. The time of analysis was 15 min. For a comparison with the isotachopherograms
obtained with a thermometric detector (e.g. Fig.5.8), it should be noted that the speed of the
recorder paper was five times higher in the isotachopherograms shown here. In the experiment shown
on the right, the buffering counter ion E-aminocaproic acid, which shows no UV absorption, was
used as the buffer, added to 0.01 N hydrochloric acid (pro analysigrade) to a pH of 4.5. On the
left, an isotachopherogram is shown for the operational system consisting of creatine (as the buffering
counter ion), which has a molar extinction coefficient that is influenced by the pH. The creatine was
also added to 0.01 N hydrochloric acid to a pH of 4.5. Special attention should be paid to the
impurities, revealed by both the detectors, and of course the shift in the pH, as normally obtained in
an isotachophoretic separation but now shown in the linear W trace in the left-hand isotachophero-
gram [examine the pH of the zone of glutamate (S)]. The difference in step height, as obtained in
the linear trace of the conductivity detector, must be ascribed mainly to the difference in the counter
ion taken, rneff. and pK. 1 = Chloride; 2 = chlorate; 3 = formate; 4 = acetate; 5 = glutamate;
6 = morpholinoethanesulphonate.
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