Frans_M_Everaerts_Isotachophoresis_378342.pdf

204 INSTRUMENTATION
varying the concentration of the sample or by inserting a small piece of insulating material
in the bore of the tap. The latter procedure is very complicated.
Introduction of the sample with aid of a syringe seems to be the most commonly used
technique, because the sample size can be vaned quickly and usually a smaller amount
of sample is required. However, if the sample is introduced in the leading electrolyte,
mixed zones can be expected between the leading ion and the fastest moving ion of the
sample. If the sample is injected in the terminating electrolyte, ionic species with a low
pK value (cationic separation) or a high pK value (anionic separation) can be retarded so
much that considerable amounts of these ionic species can be missed or even lost.
Reproducible quantitative results can hardly be expected. Particularly if experiments are
carried out at low concentrations (0.001 N), special care must be taken in selecting the
concentration and the pH of the terminating electrolyte. If the sample is mixed with a
terminating electrolyte that has too high a concentration or an incorrect pH, both the
qualitative and quantitative results will be poor. In addition, the influence of impurities
in the electrolyte may play an important role, but this is not influenced by the method
of sample introduction. More attention is devoted to this aspect in the Section
Applications.
Of course, if a syringe is used for sample introduction, some of the sample will always
be mixed with the leading and terminating electrolytes if the sample is introduced at
the boundary between these electrolytes, as this boundary is never well defined.
7.2.2. Four-way tap
The principle of the four-way tap is shown in Fig.7.1. The mechanism is shown in
four alternative positions. In position 1 the narrowbore tube is rinsed and can be filed
with the leading electrolyte, in position 2 the terminating electrolyte can be introduced
into the reservoir for the terminating electrolyte, in position 3 the sample tap can be
rinsed and filled with the sample and in position 4 the sample is sandwiched between the
leading electrolyte and the terminating electrolyte. The analysis can be performed with
the tap in position 4, in which case the connections must fit exactly, because no dead
volumes can be allowed (gas bubbles may stick to these connections and if the dead
volume is located between the narrow bore and the sample tap the time of analysis is
adversely influenced). The other connections are not important in this respect, because they
are used only for rinsing and filling the various compartments of the electrophoretic
equipment .
The tap initially applied by us was made of Pyrex glass, although any other insulating
material can be used. A combination of Kel-F and Arnite can be particularly recom-
mended. The average volume of the tap applied by us was 20-100 pl. The volume of the
tap was sometimes changed by inserting a piece of insulating material, but this procedure
proved to be very complicated if good qualitative and quantitative results were to be
obtained.