33
for each peak to travel between the two windows was used to get an accurate measure of the velocities and mobilities under steady state conditions. Figure 6 shows an example of primary data.
Equation 3 was used to determine the effective mobilities, eff
, of the charged species
lidocaine and benzoate, with the 2nd peak (phenylmethanol) used as neutral marker [3,4]
(3)
Figure 3. Absorbance at 214 nm from Agilent 7100 detector at the first window; 20 mbar, 20 kV for 3.5 min. Analyte migration order lidocaine, phenylmethanol, benzoate. Inset: UV spectrum for the 3rd peak (benzoate).
detection on an Agilent CE system [9]
. Use of the ActiPix
Figure 4. ActiPix D100 traces of 214 nm absorbance at window 2 (red) and window 3 (blue). Overlay of 9 consecutive runs with injection of 34 nL of the sample solution. Run sequence: (i) 20 mbar, 20 kV, 3.5 min; (ii) 0 mbar, 0 kV, 0.5 min; (iii) 50 mbar, 15 min.
Hydrodynamic radius / nm
Analyte Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Run 9
Average Rh STD DEV RSD (%)
lidocaine phenylmethanol benzoate 0.509 0.502 0.510 0.501 0.500 0.500 0.498 0.497 0.499 0.502
0.329 0.325 0.318 0.318 0.320 0.320 0.319 0.321 0.319 0.321
0.0045 0.89
Average ∆τ/ s 6.99 STD DEV ∆τ/ s 0.039
0.0035 1.1
5.59 0.041 Table 1 Repeatability and precision using Taylor dispersion analysis.
D/10-10 eff/10-8
/10-8 m2 f m2 z V-1 s-1 s-1 m2 V-1 s-1
lidocaine phenylmethanol benzoate 5.02 1.29 0.77 1.68 0.86
7.68 0 0 0 0
7.13
-2.40 1.00
-2.40 -0.86
Table 2 . Diffusion coefficient, effective mobility, fraction in ionic form, mobility and charge for lidocaine, phenylmethanol and benzoate at 23o buffer, ionic strength 0.13 M
C in a pH 7.5 phosphate
0.361 0.355 0.359 0.349 0.346 0.367 0.363 0.346 0.344 0.354
0.0083 2.4
5.87 0.076
D100 detector for Taylor dispersion analysis has the inherent benefit that variance contributions of CE dispersion and injection are automatically removed when variances at the second and third windows are combined as in Figure 2. An additional benefit is that all diffusion coefficient and size measurements are made in a single run, unlike other cases where a sequence of runs at different pressures was required to separate out the different variance contributions [6-10]
.
Mobility: voltage + pressure drive between 2nd and 3rd windows Whilst mobilities may be estimated from the times at the first window, the accuracy is compromised by the short length to the detector, contributions from pressure and voltage start-up characteristics, and Joule heating in the capillary [3]
. An
alternative approach is to use the difference in times between the second and third windows during pressure assisted CE. A series of pressure assisted (30 mbar) CE runs were carried out using a range of applied voltages (5, 10, 15 and 20 kV) to enable the electrophoretic mobility at zero power to be
determined. The time taken
where ∆lis the distance between windows 2 and 3, Lthe total capillary length, Vthe applied voltage, ∆tthe time difference for the ionic analyte and ∆t0 the neutral marker.
the time difference for
Figure 7 shows the extrapolations of measured effective mobilities to zero power.
The effective mobility is converted to the electrophoretic mobility, , using the relationship eff
.
= f, where f is the fraction of
the analyte in the ionic form. For benzoic acid, which is fully dissociated at pH 7.5, f= 1 and = eff
. For lidocaine, which is partially in the
ionic form at pH 7.5, fand are determined as described in the next section.
Determination of analyte charge The analyte charge, z, is obtained by combining results for electrophoretic mobility at zero power and diffusion coefficient, D, using Equation 4[1]
(4) where eis the electronic charge.
Table 2 lists diffusion coefficient, effective mobility, fraction in ionic form, electrophoretic mobility and charge for each of the three analytes lidocaine, phenylmethanol and benzoate. Values of f given in the third row were calculated from pKa
values and the composition of the
background electrolyte. Benzoic acid (pKa 4.2) is fully dissociated at pH 7.5. The composition of the pH 7.5 BGE was calculated using methodology described elsewhere [16,17] H2
to be 9 mM H2 PO4 2-, 91 mM Na+ PO4 - and to have ionic
strength 0.132 M. Using the same calculation method, from the pKa
for lidocaine of 7.94 at
a fraction 0.77 is in the acid (ionic) form at this pH and ionic strength.
23o C [18]
The electrophoretic mobility of benzoate, -2.40×10-8
m2 V-1 s-1 , giving o = m2 m2 V-1 V-1 s-1 (Table 2) was corrected
to zero ionic strength using the procedure of Nhujak and Goodall [19] -3.30×10-8
agreement with the literature value o -3.29×10-8
s-1 [20]
. This is in very good =
, demonstrating the
, 41 mM
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