Fluorescence molecules exhibiting

However, it should be noted that most fluorescent molecules exhibit broad and structureless absorption and emission bands, which means that each electronic state consists of an almost continuous manifold of vibrational levels. If the energy difference between the 0 and 1 vibrational levels of So (and Si) is, for instance, only about 500 cm4, the ratio Ah/No becomes about 0.09. Consequently, excitation can then occur from a vibrationally excited level of the S0 state. This explains why the absorption spectrum can partially overlap the fluorescence spectrum (see Section 3.1.2). 

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). 

Most dyes and pigments owe their colour to the selective absorption of incident light. In some compounds, colour can also be observed as a result of the emission of visible light of specific wavelengths. These compounds are referred to as luminescent. The most commonly encountered luminescent effects are fluorescence and phosphorescence. The transitions which can occur in a molecule exhibiting either fluorescence... 

Thus we see that in molecules possessing ->- 77 excited states inter-combinational transitions (intersystem crossing, phosphorescence, and non-radiative triplet decay) should be efficient compared to the same processes in aromatic hydrocarbons. This conclusion is consistent with the high phosphorescence efficiencies and low fluorescence efficiencies exhibited by most carbonyl and heterocyclic compounds. 

The analysis of the histograms of photon arrival times is equivalent in both cases and relies on fitting appropriate model functions to the measured decay. The selection of the fitting model depends on the investigated system and on practical considerations such as noise. For instance, when a cyan fluorescent protein (CFP) is used, a multi-exponential decay is expected furthermore, when CFP is used in FRET experiments more components should be considered for molecules exhibiting FRET. Several thousands of photons per pixel would be required to separate just two unknown fluorescent... 

The metaiioporphyrins form a diverse class of molecules exhibiting complex and varied photochemistries. Until recently time-resolved absorption and fluorescence spectroscopies were the only methods used to study metailoporphyrln excited state relaxation in a submicrosecond regime. In this paper we present the first picosecond time-resolved resonance Raman spectra of excited state metaiioporphyrins outside of a protein matrix. The inherent molecular specificity of resonance Raman scattering provides for a direct probe of bond strengths, geometries, and ligation states of photoexcited metaiioporphyrins. 

Fluorescence detectors can be more sensitive, but have a much narrower applicability. Only a small proportion of organic molecules exhibit natural fluorescence. One may choose to derivatise samples with a fluorescent or fluoro-genic agent, but this adds to the complexity of the analysis and the validation required. 

Fluorescein-5-thiosemicarbazide is a hydrazide derivative of fluorescein that can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage (Fig. 208) (Pierce). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits an excitation maximum at a wavelength of 492 nm and a maximal emission wavelength of 519 nm when dissolved in buffer at pH 8.6. In the same buffered environment, the compound has an extinction coefficient of approximately 78,000 M-1cm 1 at 492 nm. 

Fluorescent molecules span the entire region from the ultraviolet, through the visible, to the near infrared. Many dyes exhibit fluorescence, but to be of practical use, fluorescent dyes must satisfy certain requirements they must produce a pure color dictated by their absorption and emission spectra, they must have a high molar extinction, and most important, they must have a high quantum yield. These requirements are met by very few dyes. A disadvantage shared by many fluorescent dyes is their poor lightfastness, but there are some exceptions. 

Most fluorescent substances have rigid structures associated with fused aromatic rings and their fluorescence intensity is practically independent of the viscosity of the environment (4). On the other hand, their rotational movement as a whole will, of course, depend upon the local environment and since such rotations sweep out a larger volume than would be the case for auramine O, for example (i.e., larger V in eq. 2), so that larger domains in the polymeric system can be studied. Any fluorescent system will exhibit a polarization of fluorescence by virtue of the fact that the fluorescent molecules are anisotropic in regard both to emission and to absorption. This anisotropy can be described by fixed axes within the molecule, namely the dichroic axis of the molecule and the emission... 

In a study devoted to the fluoresceinamine fluorescence it was shown that this molecule exhibits no fluorescence until the amine s electron lone pair is made unavailable for electron transfer. Thus, protonation of the ammonium ion restores the fluorescence of the molecule in the deprotonated form, an intramolecular electron transfer fluorescence quenching occurs, the resorufin moiety of the molecule behaving here as an acceptor [157],... 

Fluorescence occurs when a compound absorbs radiation then emits it at a longer wavelength. It is highly selective. Fluorescence is exhibited by rigid molecules possessing a large number of delocalized n electrons. Electron-... 

Not all molecules that absorb light exhibit fluorescence. In non-fluorescent molecules the configurations of the ground and first excited states allow the excitation energy to be dispersed by non-radiative relaxation, or internal conversion, in which the energy is converted into heat. 

A collection of fluorescing molecules in a particular homogeneous environment has a characteristic lifetime. In a different environment, the same molecules may have a different lifetime. A collection of two species, either different molecules or similar molecules in different microenvironments, may exhibit a fluorescent decay with a time course that contains contributions fi-om both species. Thus we would observe a biexponential decay with two distinct lifetimes. 

Method B. Very few molecules exhibit dramatic fluoresence dynamics of the type observed for 3HF. In most cases the observed effects are more subtle, involving a slight time-dependent variation in the fluorescence band profile. Effects of this type can be due to vibrational relaxation, solvent relaxation and torsional relaxation. 

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