Reaction fluorescence

Chemical reactions can be studied at the single-molecule level by measuring the fluorescence lifetime of an excited state that can undergo reaction in competition with fluorescence. Reactions involving electron transfer (section C3.2) are among the most accessible via such teclmiques, and are particularly attractive candidates for study as a means of testing relationships between charge-transfer optical spectra and electron-transfer rates. If the physical parameters that detennine the reaction probability, such as overlap between the donor and acceptor orbitals. 

The DOOB reagent, which is formed by reaction of diphenylboric anhydride with salicylaldehyde, yields fluorescent reaction products with primary amines [1]. 

In general compounds with heteroatoms (N, O, S and P) are more amenable to fluorescence reactions" than pure hydrocarbons. Under the influence of the catalytic sorbents substances rich in Jt-electrons are formed, that conjugate to rigid reaction products that are fluorescent when appropriately excited. The formation of fluorescent derivatives is frequently encouraged by gassing with nitrogen or carbon dioxide. 

The following Tables 2.1 to 2.3 summarize some examples based exclusively on thermochemical reactions on the sorbent surface which lead to the formation of fluorescent reaction products. The derivatives formed frequently remain stable for weeks [6] and the fluorescence can frequently be intensified and/or be stabilized by treatment with viscous liquids (liquid paraffin, Triton X-100, polyethylene glycol etc.). 

Note The sodium acetate was added to the mobile phase solely to improve the separation. It had no detectable effect on the production of fluorescence during thermal activation, since the fluorescence reaction also occurred in the absence of sodium acetate. 

The mechanism of the fluorescence reaction has not been elucidated. See Volume 1 a Ninhydrin — Collidine Reagent for the ninhydrin reaction. 

Yamauchi, S., Nakai, C., Nimura, N., Kinoshita, T., and Hanai, T., Development of a highly sensitive fluorescence reaction detection system for liquid chromatographic analysis of reducing carbohydrates, Analyst, 118, 773,1993. 

Figure 4.10 Direct analysis of catecholamines in urine sample. Column, Asahipak ES-502C eluent, 75 mM succinic acid + 25 mM borate buffer (pH 6.10) containing 0.5 mM EDTA flow rate, 1.0 min-1 detection, fluorescence reaction detection Ex. 350 nm. Peaks-. 1, adrenaline-, 2, noradrenaline-, and 3, dopamine.

G. L. Picciolo and D. S. Kaplan, Reduction of fading of fluorescent reaction product for microphotometric quantitation, Adv. Appl. Microbiol. 30, 197-324 (1984). 

The OPA reagent for HPLC is prepared according to the method of Benson and Hare (21). The fluorescence reaction is performed in a 55 C water bath. OPA reacts with the guanidino group of TTX presumably to form a fluorescent product, l-alkylthio-2-alkylisoindole (22, 23). TTX is monitored at 453 nm with 332-nm excitation. Peak areas are calculated by a data processing system of the analyzer. 

All aminochromes undergo a slow spontaneous rearrangement in aqueous solution, although the rate is considerably affected by the structure of the aminochrome in question. The rearrangement of all aminochromes is however markedly catalyzed by alkali (see ref. 3 for early references). The structure of the fluorescent alkaline rearrangement product of adrenochrome was not ascertained until 30 years after the characteristic fluorescence reaction had been first reported,138 and the substance [i.e. adrenolutin, 5,6-dihydroxy-V-methylindoxyl (31), often formulated as 3,5,6-trihydroxy-V-methyl-indole (32), cf. refs. 3, 108] was finally isolated in crystalline form in 1949.139,140 Subsequently, A-ethyl-5,6-dihydroxyindoxyl (33) and... 

M Roth. Fluorescence reaction for amino acids. Anal Chem 43 880-882, 1971. 

Instruments of this type may also be used quite effectively to evaluate kinetics of time-dependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods ( milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods. 

We have used the polyamide fluorescence reaction in accelerated shelf life tests of fats and oils, either with the neat oils or with plates pre-dipped in cobalt chloride for acceleration. 

Polyamide microcrystalline powders form measurable polymer-bound fluorescent reaction products with malonaldehyde from oxidizing lipids and with reducing sugars. The compounds form on the terminal amine groups which appear to exist in zwitterionic fields with carboxylate anions, as revealed by titration with acid, alkali, or benzoquinones. 

Del Castillo P, Llorente AR, Gomez A, et al. 1990. New fluorescence reactions in DNA cytochemistry 2. Microscopic and spectroscopic studies on fluorescent aluminum complexes. Anal Quant Cytol Histol 12 11-20. 

T. Yokoyama and T. Kinoshita, HPLC determination of biotin pharmaceutical preparations by post-column fluorescence reaction in the thiamine reagent, J. Chromatogr., 542 365 (1991). 

Deveaux et al. [21] explained the fluorescent reaction of mefenamic acid by the formation of a substituted acridone after dissolving mefenamic acid in concentrated H2S04 and heating for 10 min at 100°C. The acridone exhibits an intense green fluorescence when excited by white light, and blue when excited by ultraviolet light. 

Reaction on solid phase (9.93) Fj = no fluorescence, F2 = weak fluorescence, F3 = strong fluorescence. Reaction in solution (soluble aryl bromide). 

Fig. 8.1. Three major categories of molecular dynamic processes or reactions that can be analyzed by FCS. (a) Reactions that lead to a significant [15] change in the translational diffusion coefficient of the fluorescent reaction partner, (b) Molecular dynamic processes or reactions that change the fluorescence brightness of the studied molecules, (c) Spectral cross-correlation [16], e.g., of reaction partners labeled with green (G) and red (R) emitting fluorophores that upon association move in concert and generate correlated fluctuations in the green and red emission range...

OPA has been known to give a fluorescent adduct with most primary amines in the presence of a thiol compound, but only with several biogenic amines such as histidine, histamine, and glutathione in the absence of a thiol compound in a neutral or alkaline medium. In the case of histidine, it gradually reacts with OPA alone in an alkahne medium, to give a relatively stable fluorescent adduct showing excitation and emission maxima at 360 and 440 nm, respectively. Hakanson et al. optimized these reaction conditions and showed that the fluorescence intensity due to histidine reached a maximum 10 min after initiation of the reaction at pH 11.2-11.5, at 40°C. This fluorescence reaction is relatively selective for histidine and has been used in a batch method for the assay of histidine. "... 

On the other hand, we revealed the mechanistic pathway of the OPA-induced fluorescence reaction of... 

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