Fluorescent reagents

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Reagents which form a derivative that strongly absorbs UV/visible radiation are called chromatags an example is the reagent ninhydrin, commonly used to obtain derivatives of amino acids which show absorption at about 570 nm. Derivatisation for fluorescence detectors is based on the reaction of non-fluorescent reagent molecules (fluorotags) with solutes to form fluorescent... 

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

Another commercially available fluorescing reagent, "Fluorescamine", (4-phenylspiro(furan-2-(3H),T-phthalan)3,3 -dione) reacts directly with a primary amine in aqueous acetone at a pH 8-9. 

In order to derive a quantitative relation between emission Intensity as measured by EMI and actual metal content, cell samples were subjected to graphite furnace atomic absorption (GFAA) analysis (14). Atomic absorption experiments were performed both on cells which had been stained with a fluorescent reagent and on cells not exposed to a lumlnophore. After EMI analysis, 50 fiL of cell suspension were withdrawn from the 0.30 mL of sample used for EMI and were digested In 150 iiL of concentrated HNO3 for 90 minutes at 85° . These solutions were then diluted to 1/10 of their concentration with deionized water, and the 150 liL of these diluted... 

Nitrous fiunes reagent 225, 226 Nondestructive detection 42 ff -with fluorescent reagents 44 -with iodine 46 -with pH-indicator 45 Noradrenaline 76, 240, 393 — 396 Norephedrine 76 Norfenefnne 76 Norfenfluramine derivatives 45 1 l-Nor-d -THC-9-carboxy lie acid 289,292 Novonal 339 Nucleosides 364... 

Reagent pump (post-column) Merck-Hitachi 655A-13, equipped with one reactor containing a 10-m Teflon loop (thermostated at 95 °C) for hydrolysis which is connected to a 50-p.L Teflon loop (at ambient temperature) for fluorescence reagent addition... 

The residues from a cortisol assay procedure (5 cc dichloromethane, 2.5 cc of a fluorescent reagent in 15 85 ethanol-sulfuric acid) were added to a 500 cc bottle and screw capped. After a 90 s delay, the bottle burst violently and brown fumes were seen. It was surmised that a nitrate or nitrite contaminant in the bottle had... 

Rhodamine B 67 is frequently used in the quantitative determination of DNA or RNA and fluorescent labeling for DNA [190-192]. This dye was assembled onto the surface of a quartz substrate by electrostatic interaction between the fluorescence reagent RB and y-aminopropyltriethoxysilane (APES), and the Quartz/ APES/RB film was constructed (Fig. 1) [193]. 

Marnela et al. [57] used an amino acid analyzer using fluorescence detection to determine penicillamine in urine. Urine is analyzed on a Kontron Chromakon 500 amino acid analyzer containing a column (20 cm x 3.2 mm) of AS70 resin in the Li (I) form. Buffers containing LiOH, citric acid, methanol, HC1, and Brij 35 at pH 2.60, 3.20, and 3.60 are used as mobile phases (0.4 mL/h). The fluorescence reagent is prepared by the method of Benson and Hare. Detection is at 450 nm (excitation at 350 nm). The analyte response is linear from 0.025 to 10 mM, with a limit of detection of 25 pM. 

Wilchek, M., Spiegel, S., and Spiegel, Y. (1980) Fluorescent reagents for the labeling of glycoconjugates in solution and on cell surfaces. Biochem. Biophys. Res. Comm. 92, 1215. 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

There are potentially viable reagents available that may be employed for the derivatization of compounds either for enhancing UV/visible radiation (called chromatags) or for reaction of non-fluorescent reagent molecules (called fluorotags) with solutes to yield fluorescent derivatives. 

K. W. Street, Jr. andS. A. Krause, Anew metal sensitive fluorescence reagent, Anal. Lett. 19,735-745 (1986). 

Aboul-Enein and Al-Duraibi (1998) employed dansyl chloride in a fluorescence assay for PUT, SP, SPD, and their acetylated derivatives by ion-pair reverse-phase chromatography. This assay could be applied to the separation of free and acetylated polyamines in biological samples. Dansyl chloride has also been used as the fluorescence reagent in the determination of polyamines in urine by Molins-Legua and colleagues (1999). Derivatization was carried out within the C18 cartridges that were used during the SPE extraction procedure. Recoveries were 80-95% for all four polyamines analyzed and the hmit of detection was 10 ng/ml. 

A variety of particles have been utilized in flow cytometric phagocytosis assays, including latex microspheres (7,8,12), bacteria (10,13,16), zymosan (9,13), and baker s yeast (11). Most of these particles are commercially available as fluorescent reagents. However, microorganisms may also be directly fluoro-chrome-conjugated (see Notes 1 and 2). 

A sensor for halides, Ch, Br and I , using fluorescence quenching of either acridinium or quinidinium-based fluorescent reagents covalently bound to a glass support via carbodiimide has been described. Other examples are sensors for Na, K+ and Ca. ... 

Figure 15 Representative RP-HPLC Separation of the Tryptic/Chymotryptic Peptides of Bovine Thyrotropin p-Subunit bTSHfS Labeled In Situ with the Fluorescent Reagent 5-[(Iodoacetamidoethyl)amino]naphthalene-l-sulfonic acid (5-l-AEDANS) MI a b...

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