Chemistry Reference
In-Depth Information
(10.3)), one can note that there are two limiting conditions that can be accepted
in the method development:
(1)
The new species formed by complexation is not labile. In such a case, one
could prepare a mixture of the sample with the reagent to form the ionic
complex.
d
n
0
t
2
n
g
|
9
(2)
The formation and decomposition reactions of the complex are fast
enough when compared with the electrophoretic experiment, which
consumes some minutes to be concluded. In this case, Equation (10.3)
allows predicting that the effective mobility of the complex can be
controlled by the concentration of the complexing agent used in the
composition of the BGE.
A preliminary study suggested that the second condition is obeyed. Thus, the
next step is the choice of the cinnamate, which presents an appropriate stability
constant and attends some practical requirements.
Although chlorogenic acid is the original complex found in plants, one can
argue that it is not a good candidate to be included in the BGE of a new
electrophoresis method, because it is too expensive for routine analysis. Of
course, the reagent consumption is an advantageous feature of CE, and
chlorogenic acid could be used despite its cost. However, the study by Horman
and Viani clearly shows that the quinic moiety is not essential to the complex
formation. For instance, the association constants for chlorogenate and
caffeate are, respectively, 16.9 and 12.2 kg mol
21
(Horman and Viani 1972).
Thus, caffeic acid seems to be natural choice, because it is the cinnamate
moiety in the chlorogenic acid structure.
The experiments with caffeic acid were carried out with a homemade CE-
C
4
D equipment (da Silva and do Lago 1998; da Silva et al 2002) with a 60 cm
long fused silica capillary and 75 mm of inner diameter. The voltage applied
was +25 kV, which results in an electric field of 417 V cm
21
. This is the average
electric field that allowed the species to migrate from the injection point to the
detector positioned 50 cm downstream. The samples were hydrodynamically
injected at 10 cm H
2
Oby30s.
Figure 10.1 shows an electropherogram obtained for a synthetic sample of
caffeine (500 mg L
21
) using caffeate as the complexing agent. The BGE was
composed by 30 mmol L
21
caffeic acid and pH adjusted to 8.0 with Tris. The
carboxylic group of caffeic acid has pK
a
4.44, while the phenolic groups have
pK
a
values of 7.6 and 11.85 (Jovanovic et al 1994). Thus, the carboxylic group
should be completely dissociated at pH 8.0, while one of the phenolic groups
has a partial negative charge. Thus, the complexing agent should have a net
negative charge about 1.5.
The remarkable transient observed on the electropherogram from
Figure 10.1 (between 5 and 6 min) is related to the EOF transportation of
the region occupied originally by the sample at the end of the capillary. The
cationic species migrate faster and so should be recorded before this time. On
the other hand, anionic species have migration times greater than it. Thus, the
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