Anal. Chem. 1988, 60, 2642-2646
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Optimization of Sensitivity and Separation in Capillary Zone Electrophoresis with Indirect Fluorescence Detection Werner G . K u h r a n d Edward S. Yeung*
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
Laser-lnduced lndlrect fluorescence detection can be USBd as a general detector In capillary zone electrophoresis. Indlrect fluorescence detection, where a fluorophore Is used as the prlnclpal component of the electrophoretic buffer, allows the visuailzatlon of nonfluoresclng Ions through charge dlsplacement of the fkrorophore. StaMatatkn of laser power Improves the dynamlc reserve to lo3 wlthout the compllcatlon of double-beam correction techniques. A modlflcatlon of the CZE power supply allows the use of dllute solutlons and narrow bore caplllary tublng (300 kHz) noise from the stabilization feedback circuit. Simal is in arbitrarv units. noise is reDorted as root mean sauare. ~~
reduction of background noise in the indirect signal. Both peak broadening (possibly resulting from adsorption/desorption of ions) and long-term drift were significantly reduced by the deactivation of the column surface. This allowed the use of more dilute buffer solutions (e.g. 50 pM salicylate) and provided a much more stable background fluorescence in all instances (Figure 5-7). Laser Power Stabilization. The stability of the background fluorescencewill be dependent on the stability of the light source and the intensity of fluorescence, although shot noise is minimal when a laser is used as the light source and high fluorophore concentrations are employed (21). As noted previously, laser flicker noise is limiting in this case (22). Although previous work has shown that high-frequency modulation with a double-beam arrangement can improve
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
stability (20,21),the dynamic reserve can also be increased to over lo3 by feedback control of the intensity of the laser light with a commercially available laser power stabilizer (Table IV). This simple system facilitates the use of very small detection volumes (less than 3 pL) since the stabilizer produces only minimal degradation of the optical quality of the laser beam. It does, however, reduce the laser power by 40%. By use of short focal-length quartz optics, it is still possible to focus to a spot less than 15 pm in diameter. This increase in dynamic reserve, when combined with the small detector volume, results in very high mass sensitivity for the indirect fluorescence detector. An electropherogram of 1.0 fmol each of four nucleotide 5’-monophosphates is shown in Figure 5. The detection limit for this system (top) is approximately 70 amol injected (SIN,, = 3). This level of sensitivity is competitive with even the most sensitive direct detection methods in CZE. Electrochemical detection (6) and direct laser fluorescence (14, 17) also have subfemtomolar sensitivity but are only useful for a small number of analytes. Derivatization of samples may increase the usefulness of those techniques, but it is not always appropriate or easy to implement. Other, more general, detection techniques such as conductivity or electric field gradient (7-10) or UV absorbance (2,10-13)methods lack the sensitivity necessary to be broadly applicable in CZE. An additional consequence of an improved dynamic reserve is a dramatic improvement in separation efficiency. As shown by Lukacs and Jorgenson (31),separation efficiency in CZE is very dependent on the ratio of analyte to buffer concentrations. Since an increase in dynamic reserve allows the use of more dilute samples, there is a consequent decrease in sample loading (Figure 5). This is extremely important in CZE with indirect detection, since the major ionic component in the electrophoretic buffer must be the fluorophore used to provide the signal. As shown in Figure 5, when the ratio of buffer to analyte exceeds 100, the separation efficiency is over 300 000 theoretical plates for thymidine 5’-monophosphate. It should be noted that the widths in Figure 5 are comparable to the injection peak width (Figure 4). The separation efficiency achieved is therefore comparable to those reported by other workers, after accounting for injection broadening. The generality of this detection scheme can be demonstrated by the study of several different types of samples. We have already shown the applicability of this technique to the separation and detection of underivatized amino acids (22). Strong anions have been studied previously with indirect fluorescence in liquid chromatography (20,21). Visualization in CZE should be analogous. As shown in Figure 6, injection of 3 fmol of bicarbonate and iodate results in well-defined peaks. Injection of 20 pg of lysozyme (molecular weight 14OOO) reveals that proteins can be easily visualized with indirect detection even at a relatively low pH (Figure 7). Because there
can be a number of anionic amino acids per protein molecule, the detection limit for this protein (100 amol, SIN,, = 3) may be improved substantially by adjusting the pH to one above the PI of the protein, where more groups will be ionized.
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RECEIVED for review June 7,1988. Accepted August 30,1988. The Ames Laboratory is operated by Iowa State University for the U.S. Department of Energy under Contract No. W7405-Eng-82. This research was supported by the Director of Energy Research, Office of Health and Environmental Research.
