Naturally fluorescent

Figure C1.5.12.(A) Fluorescence decay of a single molecule of cresyl violet on an indium tin oxide (ITO) surface measured by time-correlated single photon counting. The solid line is tire fitted decay, a single exponential of 480 5 ps convolved witli tire instmment response function of 160 ps fwiim. The decay, which is considerably faster tlian tire natural fluorescence lifetime of cresyl violet, is due to electron transfer from tire excited cresyl violet (D ) to tire conduction band or energetically accessible surface electronic states of ITO. (B) Distribution of lifetimes for 40 different single molecules showing a broad distribution of electron transfer rates. Reprinted witli pennission from Lu andXie [1381. Copyright 1997 American Chemical Society.

Figure C1.5.17.(A) Enzymatic cycle of cholesterol oxidase, which catalyses tire oxidation of cholesterol by molecular oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate,...

Selected Examples of Organic Compounds of Biochemical, Pharmaceutical, and Environmental Significance That Show Natural Fluorescence or t Phosphorescence... 

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

Note The natural fluorescence colors of some flavonoids [7, 9] and anthracene derivatives [16] are altered by the ammonia treatment. This makes possible differentiation on the basis of color. Detection limits per chromatogram zone have been reported of 2 ng for morphine and heroin [2], 6 ng for ochratoxin A [5] and 1 pg for penicillic acid [13]. 

Thin-layer plates were made with silica gel-calcium sulfate and each contained a mixture of zinc silicate and zinc cadmium sulfide as phosphors. Separated components are generally visible under ultraviolet light by fluorescence quenching. This was true, in part, for the pyrethrins, except that some of the separated components possessed a natural fluorescence under the ultraviolet lamps. 

Riboflavin is heat-stable in the absence of light, but extremely photosensitive. It has a high degree of natural fluorescence when excited by UV light. This property can be used for detection and determination. Two coenzymes (Fig. 2), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are derived from riboflavin. 

In this chapter, we present the theory and results of measurements on humic acid fractions using fluorescence techniques. The fluorescence techniques are attractive for this application because of the natural fluorescence of humic materials, the hi sensitivity of fluorescence detection, and the ability to directly observe the morphology of the molecule in aqueous solutions without the need for drying or applying harsh chemical conditions. Several interesting types of information are obtained from fluorescence measurements ... 

K)°C, 45 min Induction of fluorescence in weakly pg] fluorescent or nonfluorescent pesticides and amplification of natural fluorescence. There are some differences between basic and acidic aluminium oxide layers. 

Pesticides, e.g. dursban, azinphos-methyl, menazon, imidan, phosalone, zinophos 200-225°C, 20-120 min Induced fluorescence or amplification of natural fluorescence detection limits 10-300 ng. [20]... 

Organophosphorus pesticides, e.g. coumaphos, menazon, maretin, dursban 200 °C, 45 min Induced fluorescence or amplification of natural fluorescence, detection limits 1-80 ng. [21]... 

Amplification of the natural fluorescence of some pesticides and bathochromic shift of the excitation and emission maxima detection limits 5-100 ng. 

Note A range of pesticides can be detected on cellulose layers using 3-hydroxyflavones without prior bromination. Thus, the natural fluorescence of robinetin or fisetin, which is weak in a non-polar environment, is significantly enhanced by the presence of polar pesticides [2, 5, 7, 8],... 

In Fig. 1 it can be seen that the reagent applied to the right-hand side of the chromatogram has, on the one hand, intensified the fluorescent zones of the cardenolides, but that there are, on the other hand, other substance zones whose fluorescence, compared with the natural fluorescence on the left-hand side of the chromatogram, has been weakened appreciably on treatment with the reagent. The reagent is not suitable for in situ quantitation. 

The natural fluorescence of ergot alkaloids is considerably augmented by the reagent [1]. Heating for longer than 3 min or to more than 80 °C leads to a reduction in fluorescence intensity and, hence, should be avoided [3]. 

Note Methanolic sodium hydroxide solution can replace potassium hydroxide solution [16] (q.v.). The production of color tones and fluorescence is very dependent on the duration and temperature of heating hence optimal conditions must be determined empirically [16]. Some thiophosphate insecticides do not form fluorescent derivatives [16]. The natural fluorescence of various cumarin derivatives is intensified [1]. 

Induced fluorescence or amplification [20] of natural fluorescence detection limits 10-300 ng. 

Induced fluorescence or amplification of natural fluorescence, detection limits 1-80 ng. 

Note The dipping solution can also be used as a spray solution [7]. Chromatogram of natural product extracts should always be examined under UV light before usin Benedict s reagent, since some natural fluorescences su e reduced to a greater or lesse degree by the reagent. 

Post-chromatographic detection in TLC usually proceeds according to one of three procedures (i) fluorescence detection (many organic substances exhibit natural fluorescence or can be derivatised to form fluorescent compounds) [399] (ii) UV absorption and (iii) visually (many chemical procedures - reagent spray, derivati-sation - render the spots on the TLC plate visible). There is no difficulty in detecting coloured substances... 

Fluorimetric methods of analysis make use of the natural fluorescence of the analyte, the formation of a fluorescent derivative or the quenching of the fluorescence of a suitable compound by the analyte. Fluorescence cannot occur unless there is light absorption, so that all fluorescent molecules absorb, but the reverse is not true only a small fraction of all absorbing compounds exhibits fluorescence. The types of molecule most likely to show useful fluorescence are those with delocalised ji-orbital systems. Often, the more rigid the molecule the stronger the fluorescence intensity. Naturally fluorescent compounds include Vitamin A, E (tocopherol). 

Mass spectrometry (MS) is also being used to add another dimension of analysis to achiral-chiral analysis. Recently, an achiral-chiral column-switching LC/LC-MS/MS method was reported for the pindolol enantiomers in human serum (Motoyama et al., 2002) and phenprocoumon metabolites (Kammerer et al., 1998). For analytes that have very poor chromophores or cannot naturally fluoresce, MS detection can be more sensitive for the underivatized form of the analyte. Also, MS detection can be particularly useful when very similar analytes that differ in mass (such as some amino acids and metabolites) cannot be satisfactorily separated chromatographically,... 

The natural fluorescence of CTC and its derivatives has been used extensively to determine small amounts of CTC in biological materials. Kohn (86) showed that the fluorescent complex formed by CTC with calcium ions and barbital could be extracted from animal tissues into an organic solvent and then measured spectrofluorometrically. The intense fluorescence of anhydro-CTC was used by Hayes and DuBuy (87) to determine CTC in animal tissues, tissue culture cells, and bacteria. Poiger and Schlatter (88) extracted CTC from biological material into ethyl acetate as the CTC-calcium trichloroacetate ion pair. The fluorescence of the antibiotic was then enhanced by the addition of magnesium ions and a base. 

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