Fluorimetric detection

The scintillators are a special type of fluorescence indicators they are employed for the fluorimetric detection of radioactively labelled substances. They are stimulated by ) -radiation to the emission of electromagnetic radiation and will be discussed in Volume 2. 

The pink color of the cholesterol begins to fade after 5 min while the color of the bile acids deepens [2]. The visual detection limit in visible light is 1 pg for cholesterol and 2 pg per chromatogram zone for bile acids [2], Fluorimetric detection is more sensitive by a factor of 1000 ... 

Figure 15.4 Separation of mixtures of beta-blockers by using micellar HPLC, employing the following mobile phases (a) 0.12M SDS, 5% propanol, 0.5% tiiethylamine (b) 0.06 M SDS, 15% propanol (c) 0.1 IM SDS, 8% propanol. Adapted from Journal of Chromatographic Science, 37, S. Carda-Broch et al., Analysis of urine samples containing cardiovascular drugs by micellor liquid chromatography with fluorimetric detection , pp. 93-102, 1999, with permission from Preston Publications, a division of Preston Industries, Inc.

S. Carda-Broch, I. Rapado-Maitinez, J. Esteve-Romero and M. C. Garcia-Alvarez-Coque, Analysis of urine samples containing cardiovascular drugs by micellar liquid cliromatography with fluorimetric detection , J. Chromatogr. Sci. 37 93-102 (1999). 

By using chloroformates instead of acid chlorides, the resultant urethanes are useful and stable derivatives. The chloroformate derivatives most commonly used are menthylchloroformate [6] and l-(9-fluorenyl)ethylchloroformate (FLEC) [7], which exhibits excellent properties for fluorimetric detection. 

Table 2.2 Summary of some examples of fluorimetric detection after merely heating silica gel layers after chromatography.

The visual detection limits for fluorimetric detection are substance-dependent and lie between 5 ng (adrenaline, noradrenaline) and 30 ng (homovanillic acid) substance per chromatogram zone. 

KAYALI-SAYADI M N, RUBIO-BARROSO S, CUESTA-JIMENEZ M P and PALO-DIEZ L M (1998) Rapid determination of polycyclic aromatic hydrocarbons in tea infusion samples by high-performance liquid chromatography and fluorimetric detection based on solid-phase extraction , 123, 2145-8. 

Dominguez, L. M. and Dunn, R. S., Analysis of OPA-derived amino sugars in tobacco by high-performance liquid chromatography with fluorimetric detection, /. Chromatogr. Sci., 25, 468, 1987. 

A drawback to conventional amino analysis by chromatography is the need for pre- or post-column derivatization to improve sensitivity. Ninhy-drin, the reagent originally applied for detection, has been increasingly displaced by other reagents such as phenylisothiocyanate,71 9-fluorenylethyl chloroformate,72 and o-phthaldialdehyde (OPA). OPA allows fluorimetric detection, which offers the potential for greater sensitivity.73 A limitation of OPA is that it doesn t derivatize secondary amines, so an additional reaction must be added for proline detection. And, as noted for amine analysis in section A5.4.2, such derivatization adds to the analysis time and may yield unstable products. 

Bachmartn, K., Boden, J., and Haumann, I., Indirect fluorimetric detection of alkali and alkaline earth metal ions in capillary zone electrophoresis with cerium (III) as carrier electrolyte, /. Chromatogr., 626, 259, 1992. 

C for 1 h. A 100 pL portion of the solution was injected onto a column (15 cmx 3.2 mm) of LiChroscob RP-18 (7 pm) for HPLC at room temperature, using acetonitrile-0.033 M phosphate buffer of pH 8.2 (1 2) containing 0.05% of ethyle-nediamine as the mobile phase (eluted at 1 mL/min). Fluorimetric detection involved excitation at 338 nm and measurement at 540 nm (or with a 430 nm cutoff filter). For 50 300 ng of drug injected on to the column, the coefficient of variation was 7-8%. The method permits a simple determination of (z>)-penicilla-mine in serum at therapeutic levels. 

Three anticonvulsant drugs including valproic acid were determined using different dyes as ion-pair reagents. Gentian violet was used for the spectrophotometric detection at 588 nm and acridine orange for the fluorimetric detection at 470 nm after excitation at 297 nm. Calibration graphs were linear for 5-50 pg/mL 2.5 0.50 pg/mL for the spectrophotometric and fluorimetric methods, respectively [15]. 

As a consequence of the previous considerations Kieber et al. [75] have developed an enzymic method to quantify formic acid in non-saline water samples at sub-micromolar concentrations. The method is based on the oxidation of formate by formate dehydrogenase with corresponding reduction of /3-nicotinamide adenine dinucleotide (j6-NAD+) to reduced -NAD+(/3-NADH) jS-NADH is quantified by reversed-phase high performance liquid chromatography with fluorimetric detection. An important feature of this method is that the enzymic reaction occurs directly in aqueous media, even seawater, and does not require sample pre-treatment other than simple filtration. The reaction proceeds at room temperature at a slightly alkaline pH (7.5-8.5), and is specific for formate with a detection limit of 0.5 im (SIN = 4) for a 200 xl injection. The precision of the method was 4.6% relative standard deviation (n = 6) for a 0.6 xM standard addition of formate to Sargasso seawater. Average re-... 

Water None Ion chromatography with postcolumn derivatization and fluorimetric detection (free cyanide) 0.1 ng/mL 94-96 Gamoh and Imamichi 1991... 

Gamoh K, Imamichi S. 1991. Postcolumn liquid chromatographic method for the determination of cyanide with fluorimetric detection. Anal Chim Acta 251(102) 255-259. 

C. Wersig, W, Finke, E. Handler, H-P Josel and E. Schmidt, Performance of commercial laser diodes in fluorimetric detection, in Advances in Fluorescence Sensing Technology (J. R. Lakowicz and R. B. Thompson, eds.), Proc. SPIE 1885, 389-400 (1993). 

Moncrieff J. (1989). Determination of pharmacological levels of harmane, harmine, and harmaline in mammalian brain tissue, cerebrospinal fluid, and plasma by high-performance liquid chromatography with fluorimetric detection. J Chromatogr. 496(2) 269-78. 

Chan ECY, Wee PY, Ho PY, Ho PC. 2000. High-performance liquid chromatographic assay for catecholamines and metanephrines using fluorimetric detection with pre-column 9-fl.uorenylmethyloxycarbonyl chloride derivatiza-tion. J Chromatogr B 749 179... 

Sun L, Hall G, Lau CE. 2000. High-performance liquid chromatographic determination of cocaine and its metabolites in serum microsamples with fluorimetric detection and its application to pharmacokinetics in rats. J Chromatogr B Biomed Sci Appl 745(2) 315-323. 

Kalen P, Strecker RE, Rosengren E, Bjorklund A. 1988. Endogenous release of neuronal serotonin and 5-hydroxyindoleacetic acid in the caudate-putamen of the rat as revealed by intracerebral dialysis coupled to high-performance liquid chromatography with fluorimetric detection. J Neurochem 51(5) 1422-1435. 

Organic solvents Detection wavelength (VIS, UV, or fluorimetric detection)... 

Fig. 10. Calibration graph for phenol with fluorimetric detection for 5 concentrations (I0" to lO" gl ) s indicates single injection and c indicates correlation chromatography. The numbers below the data points indicate the correlation time (minutes) (ppt = ng 1" ). The bars represent 3 x standard deviation of the integrated noise (confidence interval).

Wagner J, Hirth Y, Claverie N, Danzin C (1986) A sensitive high-performance liquid chromatographic procedure with fluorimetric detection for the analysis of decarboxylated S-ad-enosylmethionine and analogues in urine samples. Anal. Biochem 154 604-617... 

The first data confirming this oxidoreductive epimerization were obtained by measuring the enzyme activity in protein extracts from a tomato cell suspension [24], It could be demonstrated, using a very sensitive fluorimetric detection method, that two different enzymes were involved in this subpathway. No enzyme activity could be detected with 24-ep/-teasterone as substrate and NAD+ or NADP+ as electronacceptors. But using the proposed intermediate 3-dehydro-24-epi-teasterone as substrate, enzymatic conversion to 24-epi-teasterone was measured in a microsomal fraction of tomato cell cultures (Fig. (9)). 3-dehydro-24-epi-teasterone-reductase showed a specific activity of 361 fkat/mg protein with NADPH as the only accepted electrondonor. 

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