Indirect Fluorescent Detection

For other applications using indirect fluorescent detection, see the Appendix. 

FIGURE 7.9 MEKC separation with indirect LIF detection of explosives from spiked soil samples (a) soil blank, (b) soil containing 1 ppm of each analyte, and (c) soil containing 5 ppm of each analyte. Analytes (1) TNB, (2) DNB, (3) NB, (4) TNT, (5) tetryl, (6) 2,4-DNT, (7) 2,6-DNT, (9) 2-Am-2,6-DNT, (10) 4-Am-2,6-DNT. Conditions run buffer, 50 mM borate, pH 8.5, 50 mM SDS, 5 pM Cy7 separation voltage, 4 kV separation distance, 65 mm [620], Reprinted with permission from the American Chemical Society. 

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Richmond, M. D. and Yeung, E. S., Development of a laser-excited indirect fluorescence detection for high-molecular weight polysaccharides in capillary electrophoresis, Anal. Biochem., 210, 245, 1993. 

Lovdahl, M. J. and Pietrzyk, D. J., Anion-exchange separation and determination of bisphosphonates and related analytes by post-column indirect fluorescence detection,. Chromatogr A, 850, 143, 1999. 

Marti, V., Aguilar, M., and Yeung, E. S., Indirect fluorescence detection of free cyanide and related compounds by capillary electrophoresis, ]. Chromatogr. A, 709, 367, 1995. 

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. 

The high sensitivity and selectivity of fluorescence detection make this the obvious choice for improving detection of proteins. Three approaches have been used direct detection of intrinsic protein fluorescence, indirect fluorescence detection, and protein derivatization for fluorescence detection. 

A simple alternative to direct detection of intrinsic protein fluorescence detection is the technique of indirect fluorescence detection proposed by Kuhr and Yeung.20 In this approach, the analysis buffer contains a fluorescent anion that produces a high background fluorescence signal. Nonfluorescent analyte anions displace the fluorescent species, producing a zone of reduced signal. Sensitivity in indirect fluorescence detection is determined by the dynamic reserve (ratio of signal... 

Several publications describe electrochemical detection techniques, and some authors report indirect fluorescence detection and mass spectrometry detection. 

Figure 8.15 Electrophoretic separation of biogenic amines using a counterionic Indirect Fluorescence Detection (IFD) system, (a) putrescine, (b) histamine, and (c) tryptamine [127],...

Munro, N.J., Huang, Z., Finegold, D.N., Landers, J.R, Indirect fluorescence detection of amino acids on electrophoretic microchips. Anal. Chem. 2002, 72, 2765-2773. 

Sirichai, S., de Mello, A.J., A capillary electrophoresis microchip for the analysis of photographic developer solutions using indirect fluorescence detection. Analyst 2000, 125(1), 133-137. 

An alternative to derivatization of nonfluorescent compounds is to perform indirect fluorescence detection. The procedure is performed on-column, by incorporating a fluorescent ion into the electrolyte. When ionic analytes interact with the fluorophore, the result is either displacement of the fluorophore or ion pairing with it.47 Kuhr and Yeung51 explored indirect laser-induced fluorescence detection using 1.0 mM salicylate as the fluorophore in the electrolyte they analyzed 10 amino acids and obtained detection limits on the order of 10 5 M. 

Chao YC, Whang CW. 1994. Capillary zone electrophoresis of eleven priority phenols with indirect fluorescence detection. J Chromatog 663 229-237. 

Hogan, B.L. Yeung, E.S. Determination of intracellular species at the level of a single erythrocyte via capillary electrophoresis with direct and indirect fluorescence detection. Anal. Chem. 1992, 64 (22), 2841-2845. 

While the type of available eluent depends upon the detection method being applied in anion exchange chromatography, a corresponding classification is not necessary in cation exchange chromatography. For the separation of alkali metals, ammonium, and small aliphatic amines, mineral acids such as hydrochloric or nitric acid are typically used as eluents, independent of whether the subsequent conductivity detection is performed with or without chemical suppression. The concentration range lies between 0.002 mol/L and 0.04 mol/L. Bachmann et al. [141] employed cerium(III) nitrate in very low concentrations as the eluent for the indirect fluorescence detection of alkali metals. 

Fig. 6-23. Analysis of monovalent cations with indirect fluorescence detection. — Separator column 100 mm x 3.2 mm I.D. ION-210 eluent 10-5 mol/L cerium(III) sulfate flow rate 1 mL/min detection indirect fluorescence injection volume 20 gL solute concentrations 0.16 ppm sodium (1), 0.15 ppm ammonium (2), 0.21 ppm potassium (3), 0.71 ppm rubidium (4), and 1.2 ppm cesium (5) (taken from [41]).

WG Kuhr, ES Yeung. Indirect fluorescence detection of native amino acids in capillary zone electrophoresis. Anal Chem 60 1832-1834, 1988. 

Many reported methods for BA and AA involve pre- or postcolumn (or capillary) derivatization of these compounds before detection using 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde," 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein, l,2-naphthoquinone-4-sulfonate," < -Phthalaldehyde (OPA), fluorescamine, and many other procedures reported in Table 30.2. OPA is disadvantageous in that it reacts only with primary amines, and the fluorescent derivatives are associated with significant instability. Dabsyl- and dansylchloride are better in this respect as they react with both primary and secondary amino groups, and provide stable derivatives. Indirect UV, indirect fluorescence detection, conductivity," and electrochemical detection have been utilized after CE separation as well as mass spectrometry Kvasnicka et al. " developed a direct,... 

Indirect fluorescence detection is another possibility for nonfluorescent compounds. In this particular case, a fluorophore co-ion is part of the electrolyte system. The simultaneous determination of cations (EDTA complexes) and anions in pond water by indirect fluorescence detection based on the displacement of fluorescein is an example. The use of fluorescein at pH 9.5 as electrolyte was also reported in the determination of anionic organophosphoms herbicides [glufosinate, glyphosate, and its major metabolite aminomethylphosphonic acid (AMPA)] in groundwater, " with LOD in the range. 

The majority of CZE methodologies for pesticides employs simple buffers at varying pH,vo,98-100 Yoiatile electrolytes (usually aiming at CZE-MS applications), solvent modified buffers, and electrolytes for special detector schemes (indirect fluorescence detection, for instance). " Table 31.1 compiles the details of a few CZE methodologies applied to pesticide analyses, classifying the application by the pesticide class and the environmental compartment assessed. 

S.-J. Chen, M.-J. Chen and H.-T. Chang, Light-emitting diode-based indirect fluorescence detection for simultaneous determination of anions and cations in capillary electrophoresis, J. Chwmatogr. A, 1017, 215-224, 2003. 

S.Y. Chang and C.-H. Liao, Analysis of glyphosate, glufosinate and aminomethylphosphonic acid by capillary electrophoresis with indirect fluorescence detection, J. Chromatogr. A, 959, 309-315, 2002. 

Munro et al. showed separation and detection of amino acids on microchips using an indirect fluorescence detection method. Figure 36.10 shows application of this method to urine samples with no pretreatment other than dilution in the appropriate separation buffer. Abnormal amounts of amino acids can easily be detected in the two patient samples compared to the healthy control sample. An absorbance detection based approach was utilized for the clinical analysis of calcium ion in serum, which is important in the regulation of a number of physiological processes. Beads with an immobilized calcium reactive dye were placed into the detection region, and the samples mobilized past the beads using electrophoretic flow. While a true separation was not intended, the interference... 

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