Fluorescence detector capillary electrophoresis

Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done on-column before the solutes elute from the capillary tube and additional band broadening occurs. 

Solutes that do not absorb UV/Vis radiation or undergo fluorescence can be detected by other detectors. Table 12.8 provides a list of detectors used in capillary electrophoresis along with some of their important characteristics. 

Wu S, Dovichi NJ (1989) High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis. J Chromatogr 480 141-155... 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Capillary electrophoresis coupled with a laser-induced fluorescence (LIF) detector has also been applied for the analysis of copper chlorophyll in olive oils. Samples were... 

Fig. 3.161. (A) Zone electrophoresis patterns of FITC-labelled transferrin samples by fluorescence detection. The unbound dye (providing a main peak and several minor ones) was not removed from the samples. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length 41 cm) X 75 pm i.d. injection of samples 100 mbar x s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut-off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 13 pm (1 mg/ml) Tf and (a) 0.01 mM FITC, (b) 0.1 mM FITC, and 1 mM FITC. (B) Zone electrophoresis patterns of an FITC-labelled transferrin sample by simultaneous fluorescence (upper trace, left axis) and UV detection (lower trace, right axis). The unbound dye shows several peaks with both detections. Experimental conditions background electrolyte, 100 mM borate buffer, pH 8.3 voltage, 20 kV capillary 59 cm (effective length fluorescence 41 cm, UV 50.5 cm) X 75 pm i.d. injection of samples 100 mbar X s 20°C detection with fluorescence detector (240 - 400 nm, broadband excitation filter and a 495 nm cut off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 6.5 pm (0.5 mg/ml) Tf and 0.1 mM FITC. Reprinted with permission from T. Konecsni et al. [199].

Figure 1.8 Use of capillary electrophoresis for separating the diastereomers quinine (QN) and quinidine (QD) (H-QN is hydroquinine, QD-N-OX is quinidine iV-oxide, H-QD is hydroquinidine, 3-OH-QD is 3-hydroxyquinidine, and asterisk is an unidentified impurity). Reprinted from [17], copyright 2001, with permission from Elsevier. (Capillary 47 cm X 75 pm i.d. (40 cm to detector) (Polymicro Technologies) running buffer 50 mM phosphoric acid containing 15 mM /3-cyclodextrin adjusted to pH 2.5 with NaOH voltage 7 kV current 21 pA injection at 0.5 psi for 4 s detector fluorescence (HeCd laser) excitation 325 nm, emission 450 nm.)...

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 Jim capillary, for example, there is 0.03 LL/cm capillary length. This is 1 /100 to 1 /1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or fluorescence (47,48). The conductivity detector claims detection limits down to 1 O molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace and residue analysis). 

Musenga et al. [55] described a capillary electrophoresis method for determination of vigabatrin in human plasma after precolumn derivatization with 6-carbox yfluorescein-N-s ucc i n i m i d i d yl ester. Optimal separation and detection were obtained with 50 mM borate buffer (pH 9.0) containing 100 mM N-methylglucamine with laser-induced fluorescence detector (Aexc = 488 nm). The assay was rectilinear over the concentration... 

Webster, J.R., Burns, M.A., Burke, D.T., Mastrangelo, C.H., Monolithic capillary electrophoresis device with integrated fluorescence detector Anal. Chem. 2001, 73(7), 1622-1626. 

Unlike the case with UV detection, few commercial HPLC fluorescence detectors may be suitably modified to allow efficient use with small capillaries.48 Consequently, custom-built systems are the rule when fluorescence detection is employed with capillary electrophoresis. Because of superior focusing capabilities, which allow the excitation energy to be more effec-... 

Despite the availability of several detectors that have been successfully adapted for capillary electrophoresis, only ultraviolet and noncoherent light fluorescence are currently configured in the capillary electrophoresis instruments. Presently, the most common way to enhance detection of... 

Toulas, C., and Hernandez, L. (1992). Applications of a laser-induced fluorescence detector for capillary electrophoresis to measure attomolar and zeptomolar amounts of compounds. LC-GC 10(6), 471-476. 

Detectors used in the initial experiments with capillary electrophoresis were simple absorbance and fluorescence detectors that had been adapted from HPLC equipment. However, it soon became apparent that these instruments yielded poor... 

Figure 12.4. Laser-induced fluorescence detector for CZE.4 [Reprinted, with permission, from Y.-F. Cheng and N. J. Dovichi, Science 242, 1988, 562-564. Subattomole Amino Acid Analysis by Capillary Zone Electrophoresis and Laser-Induced Fluorescence . Copyright 1988 by A A AS.

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