Fluorescence reagents

In Chapter 7, approaches for visualization of zones in chromatograms are discussed, including use of nondestructive and destructive dyeing reagents, fluorescence quenching on layers with a fluorescent indicator, and densitometry. In Chapter 8, additional detection methods, such as those used for biologically active and radioactive zones, as well as the recovery of separated, detected zones by scraping and elution techniques are covered. 

Pierce Chemical Company PO Box 117 Rockford, IL 61105 Cross-linking agents, protein modification reagents, fluorescent probes, biotin avidin systems, chromatography supports, kits for production of various conjugates... 

Coumarins Benedict reagent Fluorescent spots under long-wave 1... 

Monitoring of solubility and dissolution Gravimetric analysis, a protein assay method (involving color reaction with bicinchoninic acid) and UV absorbance measurements at 214 nm Chemical analysis using TNBS reagent Fluorescence spectrophotometry... 

Instrumentation and reagents. Fluorescence experiments were recorded by a Hitachi F-4500 fluorescence spectrophotometer and Perkin Elmer 1420 multilabel counter. QDs were purchased from Invitrogen. ImmunoPure F(ab )2 Preparation Kit that is used to prepare F(ab )2, was obtained from Pierce. 

When a sample is separated into its component compounds on a TLC plate, each compound exists as a round or oval spot. With few exceptions, such as the dyes in inks, lipsticks, foods, or gasolines, these spots are invisible. Three major methods are used to make these spots visible spray reagents, fluorescence, and charring. 

Constit. of Eichhornia crassipes and Narcissus tazetta. Rubber vulcanisation accelerator. Indicator used in the anal, of organometallic reagents. Fluorescent probe for detn. of detergent critical micelle concentrations. Prisms or needles (EtOH), leaflets (petrol). Mp 62°. 

Figure B2.3.9. Schematic diagram of an apparatus for laser fluorescence detection of reaction products. The dye laser is syncln-onized to fire a short delay after the excimer laser pulse, which is used to generate one of the reagents photolytically.

Figure C3.1.1. The basic elements of a time-resolved spectral measurement. A pump source perturbs tlie sample and initiates changes to be studied. Lasers, capacitive-discharge Joule heaters and rapid reagent mixers are some examples of pump sources. The probe and detector monitor spectroscopic changes associated with absorjDtion, fluorescence, Raman scattering or any otlier spectral approach tliat can distinguish the initial, intennediate and final...

When the ascending solvent-front has reached a convenient height, the strip is removed, the position of the solvent-front marked, and the paper strip dried. The positions of the various solutes, if they are coloured compounds, now appear as clear separate spots. Frequently however, the solutes are colourless, and the position of their spots must be determined by indirect methods, such as their fluorescence in ultraviolet light, or their absorption in such light (when the spots appear almost black), or by spraying the paper with a dilute solution of a reagent which will give a coloured insoluble derivative with the solutes. 

Most of the thiazoles studied absorb in the ultraviolet above 254 nm, and the best detection for these compounds is an ultraviolet lamp (with plates containing a fluorescent indicator). Other indicator systems also exist, among which 5% phosphomolybdic acid in ethanol, diazotized sulfanilic acid or Pauly s reagent (Dragendorff s reagent for arylthiazoles), sulfuric anisaldehyde, and vanillin sulfuric acid followed by Dragendorff s reagent develop alkylthiazoles. Iodine vapor is also a useful wide-spectrum indicator. 

Molecular fluorescence and, to a lesser extent, phosphorescence have been used for the direct or indirect quantitative analysis of analytes in a variety of matrices. A direct quantitative analysis is feasible when the analyte s quantum yield for fluorescence or phosphorescence is favorable. When the analyte is not fluorescent or phosphorescent or when the quantum yield for fluorescence or phosphorescence is unfavorable, an indirect analysis may be feasible. One approach to an indirect analysis is to react the analyte with a reagent, forming a product with fluorescent properties. Another approach is to measure a decrease in fluorescence when the analyte is added to a solution containing a fluorescent molecule. A decrease in fluorescence is observed when the reaction between the analyte and the fluorescent species enhances radiationless deactivation, or produces a nonfluorescent product. The application of fluorescence and phosphorescence to inorganic and organic analytes is considered in this section. 

Fluorescence Immunoassay. Basic FIA follows the same formats and approaches as EIA. The difference Hes in the indicator a fluotophote is used instead of an enzyme. This allows direct quantification of the indicatot—antibody—antigen complex, or free indicator-reagent, without the need for a substrate. 

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). 

Zirconium is often deterniined gravimetrically. The most common procedure utilizes mandelic acid (81) which is fairly specific for zirconium plus hafnium. Other precipitants, including nine inorganic and 42 organic reagents, are Hsted in Reference 82. Volumetric procedures for zirconium, which also include hafnium as zirconium, are limited to either EDTA titrations (83) or indirect procedures (84). X-ray fluorescence spectroscopy gives quantitative results for zirconium, without including hafnium, for concentrations from 0.1 to 50% (85). Atomic absorption determines zirconium in aluminum in the presence of hafnium at concentrations of 0.1—3% (86). 

Bromo-2-pyridyla2o)-5-diethylamiQophenol (5-Br-PADAP) is a very sensitive reagent for certain metals and methods for cobalt have been developed (23). Nitroso-naphthol is an effective precipitant for cobalt(III) and is used in its gravimetric determination (24,25). Atomic absorption spectroscopy (26,27), x-ray fluorescence, polarography, and atomic emission spectroscopy are specific and sensitive methods for trace level cobalt analysis (see... 

Fluorescence quenching methods wits ai omatic complexing reagents are often recommended for copper (II) determination in water. 

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