Frans_M_Everaerts_Isotachophoresis_378342.pdf

94 CHOICE OF ELECTROLYTE SYSTEMS
zone, such low effective mobilities are obtained that the potentials required rise above
the maximal potential of the stabilized d.c. power supply (this factor is of minor
importance for the buffer.)
Another limitation is due to the buffering capacity of the counter ions. A maximd
buffering capacity is obtained if pK + 1 pK - 1. Hence the buffer will have a
low buffering capacity if its pK value differs more than about 2 pH units from the pH
in a zone. The pH lies between the pK values of buffer and sample ionic species, so
that we cannot use a buffer when its pK value differs more than about 3 pK units from
that of the sample ionic species (this is valid when the pK is lower than the pK values
of the sample ions). In order to demonstrate this important effect, the relationship
between the pK values of ionic species in the sample and the pH values of their zones
is shown in Fig.5.1 for a pKB of 6 and a pH of the leading electrolyte, pH, of 5.75.
Fig.5.1 shows clearly that the buffer has a low buffering capacity if its pKB differs
more than about 3 pH units from the pK of the ionic species. In general, a buffer should
be chosen with a pKB less than 3 pH units lower than the pK value of the anionic species
to be separated.
After considering the limitations concerning demands for the buffering ions at a
chosen pH, the next problem is to choose the pH, value, and two approaches are
possible.
If no data such as pK values and mobilities are available, the experimental method must
be used. All step heights of the substances concerned have to be measured at different
pH, values and for several electrolyte systems, after which a pH, value can be chosen
for the optimal separation (maximal differences in step heights). A combination of
systems can be applied.
If all data are known, theoretically all effective mobilities can be calculated and from
the results obtained the pH, that shows maximal differences in effective mobilities can
be decided.
In order to demonstrate those two approaches to separations according to pK values,
eleven anionic species that cannot be separated according to mobilities in the system
histidine/histidine hydrochloride at a pH, of 6.02 (see Table 12.1) were selected. The
effective mobilities were computed with a computer program for five electrolyte systems
at different pH, values and all step heights were measured for the systems. In Table 5.7,
the conditions are given for the five electrolyte systems and Table 5.8 gives the calculated
effective mobilities (theoretical method) and measured step heights (experimental
method). In Fig.5.2 the step heights are plotted for the different systems. The results
indicate that the differences in effective mobilities (and step heights) are much greater
at lower pH values of the leading electrolyte, which permits better separations to be
achieved.
Some separations have been carried out. Fig.5.3A shows the electropherogram for
the separation of a mixture of trichloroacetate, P-chloropropionate, benzoate, crotonate,
paminobenzoate and trimethyl acetate at a pH, of 6.02. The terminator was glutamate,
and the leading ion was chloride. No complete separation could be achieved. In Fig.5.3B,
the separation is shown for the same mixture in system E (see Table 5.7) at a pH, of4.
Trimethyl acetate was used as the terminator and a complete separation could be easily
achieved. A thermometric detector (copper-constantan thermocouple) was used.