Chemistry Reference
In-Depth Information
time difference (Dt
mig
) and large t
mig2
: Dt
mig
ratio, see Figure 9.2 (B) (Amini et
al 2008). These requirements were met under the chosen separation conditions,
i.e.,S-b-CD at 15 mg mL
-1
dissolved in 50 mM phosphate buffer, pH 7.0.
Higher S- b -CD concentrations resulted in increased resolution between the
peaks but also in excessive analysis time, high current and reduced sample
capacity.
In the presence of S-b-CD, the IM and caffeine were separated due to the
difference in their effective mobilities (Dm
eff
). In contrast to the non-specific
mobility of the EOF, the effective mobility is a selective mobility. Eqn (1)
shows that the analyte with stronger interactions with the negatively charged
CD migrates slower. As evidenced by the electropherogram in Figure 9.2 (B),
the CD had stronger influence on the migration time of acetaminophen than
caffeine.
The negatively charged CD migrates electrophoretically towards the anode,
i.e., opposite to the direction of the EOF. However, the caffeine and the IM
zones migrated towards the detection site near the cathodic end of the capillary
by a combination of the electrophoretic mobility of S-b-CD and the bulk EOF.
The contribution of each of these two mobilities to the overall mobility is
described by eqn (1).
The separation method was further optimized by addition of 10%
acetonitrile and 20 mM NaCl to the BGE, as well as by reducing the capillary
temperature to 20
o
C. It has been suggested that addition of acetonitrile at
appropriate concentration, e.g. 10%, disrupts the complex between caffeine
and polyphenols present in the extract samples (Larger et al 1998; Collier 1972)
thereby solubilizing the polyphenols, which results in the sharpening of the
caffeine peak. Acetonitrile changes the apparent electrophoretic mobility (m
app
)
of the analytes by reducing the EOF as well as their effective electrophoretic
mobility (m
eff
) by affecting the CD-analyte interactions.
In the presence of S-b-CD the apparent mobility (m
app
) of the analytes is
determined by the vector sum of the effective electrophoretic mobility (m
eff
)
and the electroosmotic mobility (m
eo
), eqn (1).
d
n
0
t
2
n
g
|
4
m
app
5 m
eo
- m
eff
(1)
and
m
eff
5 dm
ep(S-b-CD)
(2)
where d is a coefficient indicating the affinity of the analyte for the CD, see eqn
(3). The effective mobility of the analyte is equal to the product of d and
m
ep(CD),
eqn (2). The negative sign in eqn (1) indicates anodic electrophoretic
mobility of the S-b-CD. The value of d is small since the apparent migration
mobilities were only 5 to 10% smaller than the electroosmotic flow, indicating
that the EOF had a huge contribution to the apparent mobility of the analytes,
i.e., the mobility was predominately determined by the cathodic EOF.
d 5 [Analyte-S-b-CD] / C
(3)
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