32


December 2010


which drives the component peaks past the second and third capillary windows. It should be noted that a capillary with 75 m ID was used since this is optimal for Taylor dispersion experiments with small molecules.


Figure 1. Integration of the ActiPix D100 UV imaging system and the Agilent 7100 CE system.


sample at 254 nm is recorded at the second and third detection window in order to characterise band broadening. Whilst the area of the peak is the same, the widths of both peaks are different: the signal from the third window has a greater width and lower amplitude due to Taylor dispersion, as shown in Figure 2.


Figure 2. Schematic of ActiPix sizing cartridge and peak broadening in pressure-driven flow between second and third windows.


outside of the capillary. The capillary was re-equilibrated with BGE prior to starting data collection.


Results and discussion


For this application the capillary used has three windows. The first is at 8.4 cm from the end of the 101 cm length capillary and is used with the Agilent 7100 diode array detector following short-end injection in the CE system. The capillary then passes twice through the ActiPix D100 cartridge (see Figure 2), with the second and third windows positioned at 35 cm and 75 cm respectively. A hydrodynamic injection of a few nL is made by the CE system and a voltage is applied to separate the sample into its various components by electrophoresis. The applied voltage is removed before any of the components reach the first ActiPix detection position. A pressure is then applied to induce flow driving the sample components past the two ActiPix detection positions. UV absorption of the


Separation: voltage + pressure drive to 1st window Pressure assisted capillary electrophoresis (20 mbar, 20 kV) for 3.5


min separates the components in the sample mixture. Pressure assisted CE [13-15]


is used


rather than CE alone for this separation stage, in order to assist mobilization of one of the analytes, benzoate, which has a very low net mobility at the pH of the experiment. Figure 3 shows absorbance at 214 nm output from the Agilent 7100 detector at the first window, and the UV spectrum for the 3rd peak (benzoate ion). It can be seen that there is baseline separation of all peaks.


Taylor dispersion: pressure drive between


2nd and 3rd windows When the pressure and voltage are turned off at 3.5 minutes, the fastest component is positioned just before the second window. Joule heating raises the temperature inside the capillary while the voltage is applied; a 0.5 minute rest allows thermal equilibration to ambient temperature. Taylor dispersion analysis on the separated mixture is then carried out by applying pressure (50 mbar)


Figure 4 shows an overlay of primary data obtained from 9 consecutive runs. Excellent concordance is seen between consecutive runs, demonstrating the high reproducibility of the technique. The following method is used to determine diffusion coefficient and size using TDA. Each peaks measured at the second window is convoluted with a Gaussian function; the width of the Gaussian is adjusted in the software algorithm in order to obtain the best fit between the result of the convolution and the corresponding peak measured at the third window. An example of the fitting for the third peak, benzoate, is shown in Figure 5. The standard deviation of the Gaussian convolution function, ∆τ, is used together with the measured difference in peak centre times at the second and third windows, ∆t, to calculate the diffusion coefficient, D, using Equation 1


(1)


where r is the capillary radius. The hydrodynamic radius, Rh


, is calculated


using Equation 2, which is obtained by combining equation 2 with the Stokes Einstein expression linking diffusion coefficient and hydrodynamic radius.


(2) where kB is the Boltzmann constant, Tthe


absolute temperature and  the viscosity of the solution. For dilute solutions used in these experiments, the viscosity of the solution may be assumed to be that of water at that temperature. Further details concerning equations 1 and 2 and data analysis are available elsewhere [11,12]


.


Table 1 gives a summary of the peak width increase and hydrodynamic radii obtained from 9 consecutive runs with 8 nL injection of the sample mixture containing 0.2 mM lidocaine, 1 mM phenylmethanol and 0.2 mM sodium benzoate dissolved in the BGE. The increase in peak width can be measured very reproducibly, for example the average lidocaine peak width increase (∆τ) is 6.99 s with a standard deviation of only 39 ms for the 9 runs. This translates to RSD for the diffusion coefficient and hydrodynamic radius of 0.9%. The precision in Taylor dispersion measurements for separated components in a mixture is considerably better than that from using a method with single point


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