Frans_M_Everaerts_Isotachophoresis_378342.pdf

260 PRACTICAL ASPECTS
is important and the ratio ZJZC is well chosen in order to show the effect as illustrated in
Fig.9.5. Because the buffer capacity of the creatinine is not sufficient, a front of H+ moves
towards the cathode and hence reaches the conductivity detector first. It increases the
conductivity of the leading electrolyte and is therefore recorded as a dip. If the H+ ions
reach the UV absorption detector, it isindicated by a change in absorption of the creatinine.
Shortly after this moving pH front has passed the UV detector, the glutamate reaches the
detector, already adjusted to the ‘new’ leading electrolyte (mixed zone). For this, the pH
of the glutamate is less than 4, which is abnormal if the conditions are chosen well (see
Fig.S.9). It will be clear that the conductivity as measured under these circumstances may
differ from experiment to experiment, because the disturbance is not exactly reproducible.
By changing the length of narrowbore tube between the anode compartment and the set
of detectors, another electropherogram could easily be obtained in which the glutamate
has passed the UV detector before the H‘zone.
9.2.3. Disturbances due to the presence of hydrogen and hydroxyl ions in buffered systems
In sections 9.2.1 and 9.2.2, disturbances due to the presence of H’ and OH- at high
and low pH for anionic and cationic species in unbuffered systems have been described.
Disturbances can also be expected sometimes in buffered systems, especially at low pH
for cationic species and at high pH for anionic species. The disturbances arise because at
these low and high pH values the H+ and OH- ions, respectively, are present in such large
amounts that they can carry the electric current and hence low step heights are obtained
that are almost identical for all ionic species. Under such conditions, the isotachophoretic
condition is no longer valid, the zones can release and a type of zone electrophoresis is the
result. Some examples are given below for cationic species.
In section 4.3.3, we mentioned that sometimes no real values for pH, could be
obtained because the isotachophoretic conditions were lost at low pH in cationic and at
high pH in anionic separations. This can be caused because the pH increases in anionic
separations and decreases in cationic separations until values at which ‘water’ acts as a
background electrolyte. This phenomenon was observed when analyzing nucleic bases,
which have low mobilities and low pK values. The step heights of some substances have
been determined with a leading electrolyte consisting of a mixture of potassium acetate
and acetic acid at different pH values (Table 9.1).
In Table 9.1 it can be seen that at low pH of the leading electrolyte (in the sample
zones the pH is even lower) all substances have the same step heights; some substances
have double peaks. At higher pH, the substances have different step heights but the
differences are too small to separate all of them together. Moreover, the step heights are,
in fact, step heights of mixed zones of the substances obtained at a high concentration of
H+, as the pH in the sample zone can be decreased substantially. It can be concluded that
substances with low pK values and low mobilities cannot be separated at low pH.
Some experiments were also carried out with amino acids, and similar results have
*I,= Length of narrow-bore tube between the point of injection of the sample and the detector;
2, = length of narrow-bore tube between the semipermeable membrane in the counter electrode com-
partment and the detector.