Fluorescence spectroscopies

Fluorescence spectroscopy is commonly used to characterize fluorescence effects in the UV and visual range of the electromagnetic spectrum. Such fluorescence is caused by the fact that the absorption of UV or visible light of specific wavelengths causes excitation of electrons within a molecule. If radiating relaxation occurs directly from the singlet Sj state, the process is called fluorescence. 

Readily measurable fluorescence intensities are found for molecules having aromatic and heteroaromatic rings, in particular when annulated rings are present, and in the case of conjugated 7x-electron systems. If the polymer molecules contain such fluorescence-active subunits they can be characterized by this technique, either directly via their fluorescence spectrum or via fluorescence quenching experiments (for polymers with appropriate quencher groups). It is 

Fundamentals. Fluorescence is the spontaneous emission of light after excitation of a species with light (see also p. 47). Fluorescence spectra of molecules provide valuable information about structural features [74]. 

In its neutral state the molecule shows strong fluorescence with bands at 425, 480 and 520 nm when excited with light of Aq = 480 nm. Cyclic voltammetry showed two reversible one-electron reduction processes (Fig. 5.14). 

The fluorescence of the molecule disappeared during the first reduction process this could not be evidenced at the low concentration of the reactand with UV-Vis absorption spectroscopy. The potential-dependent fluorescence of various 5-substituted indole trimer films has been investigated [77]. 

Potential-modulated fluorescence spectroscopy at liquid/liquid interfaces between immiscible liquids has been reported and a cell design has been provided [78], The dependence of the adsorption of the free, bare or the water-soluble porphyrins at the polarized water/l,2-dichloroethane interface has been studied [79], Observed spectral differences suggest a solvation structure at the interface that is different from that inside the bulk of the respective solution phases. For further studies with related porphyrins at the same interface, see [79]. Details of the transfer mechanism of the rose bengal dianion across the water/l,2-dichloroethane interface have been elucidated [80], 

Fluorescence Spectroscopy.—Fluorescence studies can be used to obtain information about proteins in a number of ways. Firstly there is the use of fluorescent labels or probes in order to obtain quantitative analytical results. For example, Lockridge and Le Du used the fluorescent probe -methyl 7-(dimethyl-carbamoxy)quinolinium iodide to establish that there were four active sites in the tetrameric cholinesterase molecule from human serum. Weiel et labelled the initiation factor protein (IF3) with fluorescein to study its binding to the 30 S ribosomal subunits and demonstrated 1 1 stoicheiometry. 

Secondly, fluorescence emission spectra of proteins usually allow conclusions to be drawn about the environment of the main fluorescent groups present (mainly the L-tryptophanyl residues). Fluorescence, in addition to circular dichroism, indicated that L-tryptophanyl residues in carboxypeptidase Y were buried in an apolar unsymmetrical environment (c.d. also indicated 40% / -conformation). Similarly in L-alanine aminopeptidase and dihydrofolate reductase fluorescence suggested L-tryptophanyl residues located in hydrophobic surroundings. By contrast, in bacteriorhodopsin one or more Trp residues appear to be exposed to a polar medium.  

A study of the fluorescence spectra of various lysozymes allowed Kuramitsu et al. to draw conclusions about the relationship between the Trp-108 residue and other groups. For example in hen egg-white lysozyme in which either Asp-52 or Glu-35 had been esterified and the turkey enzyme in which Asp-101 is replaced by Gly, the variation of fluorescence with pH showed that only the ionization of Asp-52 and Glu-35 affected the fluorescence of the Trp-108 residue. 

A fluorescence enhancement was demonstrated when kynuramine, a substrate for amine oxidase, became bound to the enzyme under anaerobic conditions. Lin has reported that when actin was labelled with dansyl-aziridine at Cys-373 the probe showed a blue shift in the emission maximum consistent with a hydro-phobic environment. Furthermore the excitation spectrum indicated that an L-tryptophanyl and an L-tyrosyl residue were in the near vicinity and that they transfer energy to the dansyl fluorophore. 

For cytochrome c oxidase, distances between subunit It, heme a and cytochrome c bound to subunit III could be estimated.Subunit II was labelled at a thiol group with A -(iodoacetamidoethyl)-l-aminonaphthalene-5-sulphonic acid and subunit III with either thionitrobenzoate-activatcd cytochrome c or a fluorescent porphyrin analogue. In this study factors allowing for the probable orientation of groups were included, being estimated from fluorescence depolarization measurements. The distances between subunit II and heme a, subunit II and cytochrome c (bound to subunit III) and between cytochrome c (bound to subunit III) and heme a were estimated to be 52, 35, and 25 A respectively. 

Fluorescence spectroscopy is a powerful technique to study the formation of hydrophobic aggregates in water solution. Pyrene is one of the most used fluorescent molecules to probe the formation of these kind of aggregates [18]. Pyrene has been extensively used to determine the micellar size [19] and the cmc [18] in urfactant solutions, but also to study the conformational transitions in amphiphilic polymers [20-21] and the cluster formation between surfactants and polymers [20-22]. 

The main feature making pyrene a useful probe for the study of hydrophobic microdomains is the sensitivity of the vibrational band structure of its fluorescence emission spectrum to the polarity of its environment [18]. The relative intensity of the peaks of the emission spectrum undergoes significant perturbation when the solvent polarity increases. In particular, the intensity of the first peak (7i) increases in polar solvents while that of the third peak (73) is essentially unaffected. So the ratio is sensitive to the solvent polarity and more 

In Fig. 4.3 two emission spectra of pyrene in a solution of a modified polymer (10 unit moll of 3-C12) are given. One of them (full line) is obtained in low ionic strength solution (0.1% NaCl) and the ratio /1//3 is found to be significantly the same as that in pure water, i.e. 1.85, indicating no formation of hydrophobic aggregates. When the ionic strength is very high (10% NaCl) the intensity of the third peak increases (dashed line in Fig. 4.3) and the value is 

In Fig. 4.4 the ratio is plotted as a function of NaCl concentration for the same modified polymers as those of Fig. 4.2. It is obvious that for three of the polymers (3-C12, 3-C14 and 3-C18) the pyrene experiences a transition from a high polarity environment to a lower polarity one 

Such a transition is not observed for the sample 3-C8, while a sample bearing 1 mol% of octadecyl groups (not shown) presents practically the same transition as the 3-C12. These results are in very good correlation with the viscosity results reported in Fig. 4.2. The Cl values determined by viscosity are indicated by arrows in Fig. 4.4. The onset of the decrease in the ratio fits well with the Cl values 

2 Fluorescence Spectroscopy. - A method for the spectrofluorimetric determination of six organophosphorus pesticides has been established. The fluorescence of the solutions was measured at excitation wavelength, 380 nm and emission wavelength, 488 nm. The optimum pH for measurement is 9.3 - 9.6 and transition metal ions interfere but can be removed by complexation. Detection limits varied between 10and 10 g/ml, depending on the substance. There are 

1 Tivo-coordinate Compounds. - The steric (and electronic) structures of a A, -aminoimino- phosphines (10) have been studied by X-ray crystallography, and by quantum-chemical calculations. Diphosphino-l,2,4-triphospholides (28) have also been examined by XRD.  

The dependence of the fluorescence intensity on the wavelength of the exciting light is known as the excitation spectrum, while the variation of the fluorescence intensity with the wavelength of the emitted light is referred to as the emission spectrum. 

The molecular group giving rise to fluorescence is termed fluorophore (fluorescence chromophore). The main fluorophores can be classified into natural fluorophores such as tryptophan residue in proteins, NAD(P)H, FMN/FAD and fluorescent indicators (probes) such as dansyl chloride, 8-anilino-l-naphthalene sulfonate (ANS), and ethidinm bromide. Nucleic adds do not have appreciable fluorescence, except for a minor base (Y-base) in tRNA (Table 7.4). 

The measurable parameters are the quantum yield ( )f), and the intensity and the position of peak emission (Xmax). The quantum yield or fluorescence efficiency is the fraction of molecnles that becomes de-excited by fluorescence and is defined as 

Natural fluorophore Trp Aqueous, pH 7 Tyr Aqueous, pH 7 Phe Aqueous, pH 7 Y-base Yeast tRNA  

Applications of fluorescence spectroscopy include ligand binding, probing of environment and measurement of distance between fluorophores. Fluorescence is very sensitive to the environment and the various parameters (e.g. ( )f and x) that are affected, 

The most important characteristic of fluorescence is that it can provide information on the nanometer length scale with very high sensitivity, allowing the system imder study to be characterized with high spatial resolution and with time resolutions of a few tens of picoseconds, or even lower Two-dimensional (2D) plots, in terms of fluorescence intensity dependence on time and wavelength, can be obtained, which can provide valuable information on the photophysical behavior of the system and its relation to the structural/morphological characteristics. 

Characterization of Polymer Blends Miscibility, Morphology, and Interfaces, First FditiozL Edited by S. Thomas, Y. Grohens, and P. Jyotishkumar. 

The combination of various fluorescence techniques, such as steady-state and time-resolved fluorescence spectroscopy and fluorescence microscopy, can provide extremely important information on the structure and dynamics of polymer blends. Many relevant examples will be presented in this chapter, with particular emphasis being placed on the most recent investigations. 

some fundamental aspects of fluorescence spectroscopy will be briefly presented and typical fluorescence experiments discussed. Their use in studies of intrinsically fluorescent polymers and in fluorescent labeled-polymer systems will then be illustrated. 

Under continuous-wave excitation of a fluorescent molecular system, typically in solution or in solid samples, it is possible to record absorption, fluorescence (both emission and excitation spectra) and phosphorescence spectra. 

In the last decades, fluorescence spectroscopy has been applied to a wide range of problems in the chemical, biological and material sciences. This tremendous success is strongly determined by the sensitivity of the process indeed many environmental factors can influence the photophysical behavior of the fluorescent state and hence they have an impact on the energy, efficiency or kinetics of the radiative process. The measurements can provide information on a wide range of molecular processes, including the interactions of medium with fluorophores, rotational diffusion 

Like Raman scattering, fluorescence spectroscopy involves a two-photon process so that it can be used to determine the second and the fourth rank order parameters. In this technique, a chromophore, either covalently linked to the polymer chain or a probe incorporated at small concentrations, absorbs incident light and emits fluorescence. If the incident electric field is linearly polarized in the e direction and the fluorescent light is collected through an analyzer in the es direction, the fluorescence intensity is given by 

In fluorescence spectroscopy, the orientation distribution of the guest probe is not necessarily identical to the actual orientation of the polymer chains, even if it is added at very small concentrations (i.e., a probe with high fluorescence efficiency). As a matter of fact, it is generally assumed that long linear probes are parallel to the polymer main chain, but this is not necessarily the case. Nevertheless, if the relation between the distribution of the probe axes and those of the polymer axes is known, the ODF of the structural units can be calculated from that of the probe thanks to the Legendre s addition theorem. Finally, the added probe seems to be mainly located in the amorphous domains of the polymer [69] so that fluorescence spectroscopy provides information relative to the noncrystalline regions of the polymeric samples. 

Problems related to the use of a guest dye can be reduced if the polymer contains a fluorescent chemical group. Gohil and Salem [70] took advantage of such intrinsic fluorescence to characterize the in-plane distribution of orientation in biaxially drawn PET films. In these experiments, the chain-intrinsic fluorescent label is due to the formation of dimers by two terephthalic moieties, exclusively within the noncrystalline regions. A comparison between sequential and simultaneous drawing along the MD and TD directions was undertaken for a fixed MD draw ratio of 3.5 and various TD draw ratios. The orientational order was characterized by two orientation ratios Rmd and RTD such that 

Recently, a formalism has been developed to determine the second and the fourth order parameters of films using polarized total internal reflection fluorescence (TIRF) [71]. Similarly to IR-ATR spectroscopy (Section 4), the experiment makes use of p- and s-polarized excitation, but the fluorescence emission (analyzed either in p- or s-direction) is detected normal to the substrate. Two approaches are developed based on the measurements of two intensity ratios. In the first one, the S angle has to be known experimentally or theoretically, and the order parameters (P2) and (P4) can be determined. In the second one, the order parameter (R ) is obtained by another technique, for instance IR-ATR spectroscopy, which allows deducing the order parameter (P4) and (cos2 5). 

NMR spectroscopy is a powerful technique to study molecular structure, order, and dynamics. Because of the anisotropy of the interactions of nuclear spins with each other and with their environment via dipolar, chemical shift, and quadrupolar interactions, the NMR frequencies depend on the orientation of a given molecular unit relative to the external magnetic field. NMR spectroscopy is thus quite valuable to characterize partially oriented systems. Solid-state NMR 

The second method uses pulsed lasers and the laser-induced fluorescence is detected by telescope. If the telescope and the laser source have a definite base distance, the crossing of laser beam and the acceptance angle of the telescope define the height of the atmospheric layer at which fluorescence is detected. There is also the technique of delayed coincidence, where the time interval between laser pulse and detected fluorescence pulse determines the distance of the observed molecules from the observer (Lidar) 

Many molecules which have absorption bands in the wavelength region of existing laser lines can be excited by absorption of laser photons into single isolated rotational-vibrational levels of the electronic ground state 1W -103) (jn the case of infrared laser lines) or of an excited electronic state (with visible or ultraviolet lines) 

This has been confirmed experimentally by several authors who investigated mainly diatomic molecules. 

Because of the large number of rotational levels in the upper and lower states, the overlap between the exciting laser line and the dopp-ler broadened absorption profile may be nonzero simultaneously for several transitions (u , / ) (v, f) with different vibrational quantum numbers v and rotational numbers J. This means, in other words, that the energy conservation law allows several upper levels to be populated by absorption of laser photons from different lower levels. 

There are, however, certain selection rules for electric dipole transitions which considerably reduce the number of possible transitions. They are extensively discussed and proved in reference and for diatomic molecules consist essentially of the following three rules  

After the appearance of the first book on fluorescence in 1951 [45], fluorescence spectroscopy became a widely used scientific tool in biochemistry, biophysics, and in material science. In the last few years, however, several new applications based on fluorescence have been developed, promoting fluorescence spectroscopy from a primarily scientific to a more routine method. The phenomena of fluorescence is for example exploited in simple analytical assays in environmental science and clinical chemistry, in cell identification and sorting in flow cytometry, and in imaging of single cells in medicine. The analyte, whose light emission is investi- 

Reference Data Table 4 Method Parameter Reference Sheet 

Required User Skills Unskilled for routine measurements 

Single/double beam Single and double beam 

Noise Signal/noise at high/low concentr. limit 10000 1 at high concentration 5 1 at low concentration 

The UV and IR spectra of a series of quaternary, dihydro-, tetrahydro-, and oxoprotoberberines have been recorded.The maxima for the fluorescence bands are between 540 and 570 nm for quaternary protoberberines, and between 320 and 330 nm for tetrahydroprotoberberines.  

A careful study of the Bohlmann bands of tetrahydroprotoberberines has confirmed that  

It can be used to measure the photoluminescence (PL) emission of rare earth ions, noble metal NPs, and semiconductor quantum dots. For quantum dots, based on their fluorescence spectra, the particle size can be evaluated, using the Brus effective mass model [21]. 

Vibrational relaxation involves transfer of the excess energy of a vibrationally excited species to molecules of the solvent. This process takes place in less than 10 s and leaves the molecules in the lowest vibrational state of an electronic excited state. 

Internal conversion is a type of relaxation that involves transfer of the excess energy of a species in the lowest vibrational level of an excited electronic state to solvent molecules and conversion of the excited species to a lower electronic state. 

Relationship between Excitation Spectra and Fluorescence Spectra 

Exploration of Luminescence Spectral Shape as a Function of Excited State Energy, Vibrational Modes, and Molecular Distortions. 

Quantum efficiency is described by the quantum yieid of fiuorescence, 

Absorption of light by a fluorescent molecule causes the excitation of an electron moving from a ground state to an excited state (Lakowicz, 1983). After the electron has been excited, it relaxes rapidly from the higher vibrational states to the lowest vibrational state of the excited electronic state after which, the excited state may decay to the ground state by the 

Fluorescence spectroscopy offers several inherent advantages for the characterization of molecular interactions and reactions. Firstly, it is 100-1000 times more sensitive than other spectrophotometric techniques. Secondly, fluorescent compounds are extremely sensitive to their environment. For example, vitamin A that is buried in the hydrophobic interior of a fat globule has fluorescent properties different from molecules that are in an aqueous solution. This environmental sensitivity enables characterization of viscosity changes such as those attributable to the thermal modifications of triglyceride structure, as well as the interactions of vitamin A with proteins. Third, most fluorescence methods are relatively rapid (less than 1 s with a Charge Coupled Device detector). One particularly advantageous property of fluorescence is that one can actually see it since it involves the emission of photons. The technique is suitable for at-line and on/in-line process control. 

If absorbance is less than 0.1, the intensity of the emitted light is proportional to the concentration of fluorophore and excitation and emission spectra are accurately recorded by a classical right-angle fluorescence device. When the absorbance of the sample exceeds 0.1, both emission and excitation spectra are reduced and excitation spectra are distorted. To avoid 

In the absorption process, the electrons are excited across the band gap, from the valence band to the conduction band, by providing an appropriate energy greater than the band gap of the material. These excited electrons must decay back to the valence band by radiative or non-radiative thermal processes. Non-radiative processes are the predominant routes to de-excitation when the electron-phonon coupling is strong. Phonon coupling provides quasi-continuous states which the 

It has been found that the emission due to Mn doping can be further tuned by changing the crystal field around the Mn + ions. Instead of the S ions, as in the case of ZnS, an oxide host can be used to induce a much larger crystal field splitting of the t2g-eg levels in Mn + ions, for example by doping Mn + ions in ZnO nanocrystals [20], In this case, the Mn d-d emission is observed at 470 nm (2.6 eV), representing a blue shift of 0.5 eV compared to the case of Mn emission in Mn-doped ZnS. 

Generally, the lifetime of an excited species is short because there are several ways an excited molecule can give up the excess energy. Apart from 

A substance must absorb light in order to fluoresce as a result, any substance that can be measured fluorimetrically can also be determined speetropho-tometrically. As fluorescence is one of several mechanisms by which a molecule returns to the ground 

In conventional fluorescence analysis the majority of quantitative measurements is made using fixed wavelengths for excitation and emission. At low concentrations, a plot of the fluorescent power of a solution vi. the concentration of the emitting species ordinarily is linear. As to the sensitivity of fluorescence procedures in analysis, it is convenient to separate the contributions from the properties of the fluorescent molecule itself absolute sensitivity), the performance of the instrument instrumental sensitivity) and the chemistry involved in the preparation of the sample method sensitivity). The absolute sensitivity is determined chiefly by the molar absorptivity and the fluorescence efficiency of the analyte molecule itself. High fluorescence efficiency is usually associated with some rigidity. The method sensitivity takes account of pre-concentration steps in the preparation of the sample on the one hand and the limitations imposed by the fluorescence of the blank on the other. The sensitivities and selectivi-ties attained by fluorescence, phosphorescence and chemiluminescence are hardly paralleled by other techniques. In many cases it is not required that the analyte be isolated from the matrix. 

Wavelength calibration of fluorimeters is highly important a calibration sample is to be used. Fluorescence intensity samples such as Rhodamine B are routinely measured to calibrate and monitor the performance of fluorescence spectrophotometers [507], Also PMMA/fluorescent materials are used to check instrumental stability resolution and wavelength precision (Anadis Instruments/Malden). 

Huorescence spectroscopy has been reviewed [508]. Various monographs deal with fluorescence [509,510], cfr. also Bibliography for fluorescent probes, cfr. ref. [511], and for standards in fluorescence spectroscopy ref. [512]. 

2) This is very much a realization of the basic idea of Synge, from 1928, for ultramicroscopy. 

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The attachment of pyrene or another fluorescent marker to a phospholipid or its addition to an insoluble monolayer facilitates their study via fluorescence spectroscopy [163]. Pyrene is often chosen due to its high quantum yield and spectroscopic sensitivity to the polarity of the local environment. In addition, one of several amphiphilic quenching molecules allows measurement of the pyrene lateral diffusion in the mono-layer via the change in the fluorescence decay due to the bimolecular quenching reaction [164,165]. 

Lee D and Albrecht A C 1985 A unified view of Raman, resonance Raman, and fluorescence spectroscopy (and their analogues in two-photon absorption) Advances in Infrared and Raman Spectroscopy vo 12, ed R J H Clark and R E Hester (New York Wiley) pp 179-213... 

Lakowioz J R 1983 Principles of Fluorescence Spectroscopy (New York Plenum) oh 3... 

Fries J R, Brand L, Eggeling C, Kdllner M and Seidel CAM 1998 Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy J. Phys. Chem. A 102 6602-13... 

Ambrose W P and Moerner W E 1991 Fluorescence spectroscopy and spectral diffusion of single impurity molecules in a crystal Nature 349 225-7... 

Tittel J, Gdhde W, Koberling F, Basche T, Kornowski A, Weller H and Eychmuller A 1997 Fluorescence spectroscopy on single CdS nanocrystals J. Chem. Phys. B 101 3013-16... 

Weiss S 1999 Fluorescence spectroscopy of single biomolecules Science 283 1676-83... 

Eggeling C, Fries J R, Brand L, Gunther R and Seidel CAM 1998 Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy Proc. Natl Acad. Sc/. USA 95 1556-61... 

Ha T, Ting A Y, Liang J, Caldwell W B, Deniz A A, Chemla D S, Schultz P G and Weiss S 1999 Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism Proc. Natl Acad. Sc/. USA 96 893-8... 

Comprehensiveiy cataiogues phenomena and techniques encountered in flash photoiysis and fluorescence spectroscopies. 

Table 7.16 Fluorescence Spectroscopy of Some Organic Compounds Table 7.17 Fluorescence Quantum Yield Values...

Single vibronic level, or dispersed, fluorescence spectroscopy... 

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