Frans_M_Everaerts_Isotachophoresis_378342.pdf

THERMOMETRIC MEASUREMENTS 215
vj c
Kcl= c*L"
(10.4)
where Kcd is the calibration constant and L* (sec) is the zone length as detected between
two successive signals obtained from the equipment if a zone boundary passes the detector. By
means of a computer program (Chapter 4), the actual concentrations of the ionic species
in the zone can be calculated in a well defined operational system. This means that once
the calibration constant is known, the concentration of all ionic species in a sample can
be calculated from the zone length. Not all calibration graphs for each ionic species have
to be measured separately.
In order to check the reproducibility and to determine simultaneously the calibration
constant, K,,, quantitative experiments were carried out in different electrolyte systems
with water as the solvent. The calibration constant is not a constant for all systems. Some
factors, such as variations in the concentration of the leading electrolyte, temperature
and changes in the current density, result in different potential gradients and hence affect
the migration speed in the system. This effect produces different zone lengths for the
same amounts of ionic species in different systems.
10.3. THERMOMETRIC MEASUREMENTS
10.3.1. Reproducibility
In order to estimate the reproducibility, the zone length of formic acid (injected volume
3 p1 of a 0.05 Nsolution) was measured ten times in different experiments. The leading
electrolyte was 0.01 Nhistidine and 0.01 N histidine hydrochloride. The pH of the
solution was thus 6.02. The current was stabilized at 70 FA. The terminator was 0.005N
glutamic acid (adjusted to pH 6 by addition of Tris).
The average zone length found was I* = 31 1 sec from ten experiments and the
average deviation was 4 sec. Owing to the asymmetry of the step response, the zone length
depends on the terminator used. Some experiments were therefore carried out with
the same sample but with a different terminator (acetic acid). The average zone length
then found was i* = 307 sec from five experiments and the average deviation was
3 sec. No significant differences were found when the values found in the experiments
with glutamic acid and acetic acid were compared. Glutamic acid was therefore used
as the terminating ion in the other experiments.
Later experiments showed that one has to be careful if glutamic acid is used as a
terminating ion because it is a strong acid and hence mixed zones can easily be formed,
which cannot be separated further.
10.3.2. Calibration constant
The calibration constant was determined from experiments carried out with histidine
(0.01 N)/histidine hydrochloride (0.01 N) (pH 6.02) as the leading electrolyte. The
current was stabilized at 70pA. All zone lengths are given in Table 10.1. The third