Fluorescence-quenching

Fluorescence is characterized by parameters such as intensity, quantum yield, and lifetime. The fluorescence intensity at a given wavelength is equal to the number of photons emitted by fluorescence multiplied by the photon energy  

The fluorescence quantum yield 4 f is equal to the number of photons emitted by fluorescence divided by the number of absorbed photons. In general, Ip and bp are proportional to each other. The fluorescence lifetime to is the time spent by the fluorophore 

Forster energy transfer or energy transfer at a distance occurs between two molecules, a donor (the excited fluorophore), and an acceptor (a chromophore or fluorophore). Energy is transferred by resonance, i.e., the electron of the excited molecule induces an oscillating electric field that excites the acceptor electrons. As a result of this energy transfer, the fluorescence intensity and quantum yield of the emitter will decrease. Energy transfer is described in Chapter 14. 

The fluorescence quenching of an excited atom or molecule may occur in two ways either by the physical processes of energy transfer (see Section IV. 16) or by a chemical reaction. The physical processes are those resulting in energy exchange whereas the chemical processes are accompanied by rearrangement of particles. 

Since mercury atoms play an important part in photosensitized reactions (see Section VIII.26), certain data referring to quenching of Hg (6 Pi) fluorescence will be given. The physical processes possible here are 

Since the quenching activity of atoms (He and Ar) is very weak compared to that of diatomic and polyatomic gases, for molecules the transition Hg ( Pi) Hg ( Po) would be associated with process Hg + M - Hg + M (the asterisk denotes vibrational excitation (see [251]). 

The rate constants of process Hg ( P Hg q Po) [505] have been measured for a large number of molecules and appear to be 10 —10 cm s, indicating that the probability of this process is great. 

Process Hg ( Pi) - Hg( So), in which the P level energy converts to vibrational energy of the molecular collision partner, has not been sufficiently studied and its probability can be estimate from sparse information only. 

Nemzek and Ware [7] have studied the fluorescence decay of 1,2-benzanthracene (and naphthalene) in 1,2-propanediol or purified mineral oil by the single photon counting technique over the temperature range 10—45°C. The fluorescence lifetimes, t0, were measured. In further experiments, which included a heavy atom fluorescence quencher, carbon tetrabromide in concentration [Q] 0.05—0.29 mol dm-3, no longer could the decay be characterised by an exponential with a constant lifetime. However, the decay of fluorescence was well described by an expression of the form 

Nemzek and Ware [7] evaluated the mutual diffusion coefficient, D, and encounter distance, J2eff. D was found to be in close agreement with values of molecular diffusion coefficients from independent measurements. The encounter distance was 0.90nm in 1,2-propanediol. There were small 

Assuming that the true encounter distance is about 1.20 nm (the exact value is not very important), feact 4 x 109 dm3 mol-1 s-1. The quenching of 1,2-benzanthracene and naphthalene fluorescence by carbon tetrabromide can also be followed by illuminating a solution with light of constant intensity and measuring the fluorescence intensity, /, for a number-of solutions containing different quencher concentrations. These steady-state experiments of Nemzek and Ware [7] are discussed in Sect. 5.5. 

64 to 0.48 nm as the nitrobenzene concentration increased from 0.001 to 0.78 mol dm . The decrease of R as [Q] increases is of similar trend and magnitude to that noted by Nemzek and Ware [7]. Beddard et al. [8] suggest that the rate coefficient for quenching of the encounter pair is 

By using this expression and eqn 12.18, the quantum yield of fluorescence is written as 

The observed fluorescence lifetime can be measured with a pulsed laser technique. First, the sample is excited with a short Hght pulse from a laser using a wavelength at which S absorbs strongly. Then, the exponential decay of the fluorescence intensity after the pulse is monitored. From eqns 12.21 and 12.22, it follows that 

In water, the fluorescence quantum yield and observed fluorescence lifetime of tryptophan are p = 0.20 and to = 2.6 ns, respectively. It follows from eqn 12.23 that the fluorescence rate constant /cp is 

The dependence of the fluorescence intensity on the presence of other species gives valuable information about photobiological processes and can also be used to measure molecular distances in biological systems. 

Now we consider the kinetic information about photochemical processes that can be obtained by quenching studies. Fluorescence quenching is the nonradiative removal of the excitation energy from a fluorescent molecule and the elimination of its fluorescence. Quenching maybe either a desired process, such as in energy or electron transfer, or an undesired side reaction that can decrease the quantum yield of a desired photochemical process. Quenching effects may be studied by monitoring the fluorescence of a species involved in the photochemical reaction. 

In proteins the Trp fluorescence is often quenched by intramolecular interactions with different amino acids. These quenching processes contribute to the magnitude of the non-radiative relaxation rate constant, kuR, of the excited state, cysteine and especially disulfide bridges will quench Trp fluorescence 

Histidine may quench Trp fluorescence through a proton transfer mechanism. There is evidence (17) that position 4 of the indole ring acquires more electron density in the excited singlet state and can accept a proton from a proton donor such as histidine. 

The neutral amide function of glutamine or asparagine, as well as the amide backbone of the protein, can quench Trp fluorescence. This is another short-range electron transfer process. The short lifetime of one of the Trp fluorescence decay components may be due to the dose proximity of the indole ring to the amide backbone in that particular rotamer state. 

When the Trp residue is located in a hydrophobic environment of the protein its 1 f is enhanced and importantly the fluorescence spectral maximum is shifted to a much higher energy than that normally seen for a solvent exposed residue. 

In a protein containing more than one Trp residue the Stern Volmer quenching curve often shows downward curvature (Pigure hi). This would result because the less accessible Trp residues will be quenched after the more exposed residue, and will have a lower value of k. However, the direction of curvature will depend on the relative values of and tj, especially in the case of a single chromophore when upward curvafttre is observed. This is due to the fact that at a high quencher concentration diffusion controlled kinetics are not observed, and there will be an appreciable fraction of the chromophore that is surrounded by the quencher at the instant of excitation. That molecule will be immediately quenched and F will be less than that expected for a diffusion controlled process. 

The intensity of fluorescence can be decreased (quenched) by several processes, such as collisions (in solution), excimer or exdplex formation, and energy, electron or proton transfer [5]. In the context of polymer blends studies, the process of collisional fluorescence quenching, either static or dynamic (as described by the Stern-Volmer quenching), is not particularly relevant. 

In order for FRET to occur, there must be a spectral overlap between the donor emission and the acceptor absorption. In its simplest form, the rate constant for transfer between a donor and an acceptor at a distance r is  

F re 25.4 (a) Forster dipole-coupling and (b) Dexter electron-exchange models for energy transfer (from D to A). 

E)ue to its dependence on r the Forster transfer rate (Eq. (25.14)) depends heavily on the separation between the donor and acceptor fluorophores/mole-cules, and efficient transfer only occurs if this separation is less than the Forster transfer radius (Rq). Typical values of Rq are on the order of only a few nanometers, and therefore FRET is very sensitive to distances of this magnitude. 

Polymer interfaces are usually much broader than inorganic interfaces because, even in the case of immiscible polymers, some segment interpenetration occurs at the interface between the individual domains, being the interfacial width (iv) proportional to where x is the Flory-Huggins chi-parameter [13]. Therefore, 

This is a steady-state competitive method, applicable when a solute is capable of fluorescing. We consider the simplest case. The solute A undergoes excitation to the excited singlet state A upon absorption of radiation of frequency 

The excitation process may generate an excited molecule in any allowed vibrational state, but tbe excess vibrational energy is rapidly lost, and the excited state species may then emit a photon of frequency Vem, this singlet-singlet transition from the excited to ground state being fluorescence. 

A may also return to the ground state via a radiationless transition, most commonly by collisional transfer of energy to a solvent molecule. 

In the presence of exciting radiation of constant energy, a steady state is established between the excitation and deexcitation processes. 

If a second solute Q is added that is able, in a second-order reaction, to make available an additional route for return to the ground state, we can write 

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Photodegradation as well as fluorescence quenching has been observed in chlorophyll monolayers [302,316]. Whitten [317] observed a substantial decrease in the area of mixed films of tripalmitin and a ci5-thioindigo dye as isomerization to the trti 5-thioindigo dye occurred on irradiation with UV light. 

There are two approaches to estimation of AG fThe first is an empirical approach (36) based on dynamics of fluorescence quenching of aromatic hydrocarbons ia acetonitrile solution. Accordingly,... 

NEW FLUORESCENCE QUENCHING METHOD FOR DETERMINATION OF COPPER (II) IN WATER... 

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

Physical methods Physical methods include photometric absorption and fluorescence and phosphorescence inhibition, which is wrongly referred to as fluorescence quenching [1], and the detection of radioactively labelled substances by means of autoradiographic techniques, scintillation procedures or other radiometric methods. These methods are nondestructive (Chapt. 2). 

Fig. 4 Explanation of the fluorescence-quenching effect [2]. — (A) chromatograms of the same quantities of saccharin and dulcin observed under UV 254 light, (B) schematic representation of fluorescence quenching, (C) spectral reflectance curves of saccharin and dulcin.

Short-wavelength UV radiation (A = 254 nm) is employed for excitation. This allows aromatic organic compounds, in particular, to be detected by fluorescence quenching. Uranylacetate may also be excited at A = 366 nm. 

Fig. 22 Optical trains of the commercially available scanners. (A) absorption, (B) fluorescence quenching and true fluorescence.

However, the optical train illustrated in Figure 22B allows the determination of fluorescence quenching. The interfering effect described above now becomes the major effect and determines the result obtained. For this purpose the deuterium lamp is replaced by a mercury vapor lamp, whose short-wavelength emission line (2 = 254 nm) excites the luminescence indicator in the layer. Since the radiation intensity is now much greater than was the case for the deuterium lamp, the fluorescence emitted by the indicator is also much more intense and is, thus, readily measured. 

Fig. 25 Calibration curve for the determination of dulcin by fluorescence quenching and absorption [2],...

Color reproduction of the chromatograms can be achieved by color photography — the best, but also the most expensive method of documenting thin-layer chromatograms. It can be used not only to produce true-color reproductions of colored zones but also — with the aid of a Reprostar (Fig. 64) or a UVIS analysis lamp (Fig. 6) — of fluorescent or fluorescence-quenched zones. When photograph-... 

The required exposure times are difficult to estimate. They are best found by trial and error. Documentation of fluorescence quenching at A = 254 nm usually only requires one trial. The exposure time found to be adequate here is normally suitable for all following exposures of fluorescence quenching if the exposure conditions are maintained constant (camera type, film type, distance of objective and lamp, aperture etc.). The exposure time required for fluorescent chromatograms is primarily dependent on the intensity of the fluorescence and, therefore, has to be optimized for each chromatogram. It is best to operate with a range of exposure times, e.g. aperture 8 with exposures of 15,30,60,120 and 240 seconds. Experience has shown that one exposure is always optimal. 

Note In the case of HPTLC plates the detection limit for the visual recognition of the violet = 530 nm) colored chromatogram zones was 20 ng per chromatogram zone. With the exception of the two tetrahydrosteroids the cor-ticosteriods could be detected on TLC plates with fluorescent indicators by reason of fluorescence quenching (Fig. 1 A). Figure 2 illustrates the absorption scans of the separations illustrated in Figures 1A and 1B. 

Acid Color Fluorescence quenching (>l = 254 nm) Sensitivity (pg/zone) TLC HPTLC ... 

Note The alternative fast blue salt BB produced the most intensely colored chromatogram zones for visual analysis in daylight, while fluorescence quenching in UV light (A = 254 nm) was greater with fast blue salt B and fast blue salt RR (Figs. 1 and 2). 

The intensity and colour of the fluorescence of many substances depend upon the pH of the solution indeed, some substances are so sensitive to pH that they can be used as pH indicators. These are termed fluorescent or luminescent indicators. Those substances which fluoresce in ultraviolet light and change in colour or have their fluorescence quenched with change in pH can be used as fluorescent indicators in acid-base titrations. The merit of such indicators is that they can be employed in the titration of coloured (and sometimes of intensely coloured) solutions in which the colour changes of the usual indicators would... 

Forward Electron Transfer as Studied by Fluorescence Quenching 69... 

Figure 10 illustrates Stern-Volmer plots for the fluorescence quenching of APh-x by MV2+ and SPV in aqueous solution [74]. With MV2+, the quenching is so effective that it occurs at very low quencher concentrations (in the range of 10 6 M), whereas with SPV, it proceeds to about the same extent at two-orders of magnitude higher quencher concentration (in the range of 10 4 M). 

Table 2. Rate constants for the fluorescence quenching of APh-8 (8) by SPV (14) and MV2 + (13) in aqueous solution [74]...

The fluorescence quenching depends on the content of the Phen units (the x values) in APh-x. An aqueous solution of APh-9 contained as many charged groups (SOJ) as about 10 times that of APh-50, when compared at the same molar concentration of the Phen residues. When AMPS homopolymer (PAMPS) was added to a solution of APh-50 so that the SOJ residue concentration was equal to that for APh-9, the kq value for the APh-50 quenching by MV2 + decreased from 2.1 x 1012 to 4.2 x 1011 M-1 s 1, which is close to the kq value for APh-9 (Table 2). From these facts the lower kq values for APh-x with lower x (higher... 

Figure 11 shows Stern-Volmer plots for fluorescence quenching of the amphiphilic cationic copolymer QPh-x [74]. The quenching of QPh-x with MV2+ is expected to be much less effective than that of APh-x. The quenching data for the QPh-x system are presented in Table 3. For comparison, the data for a related... 

More recently, several groups have investigated electrostatic effects on the fluorescence quenching of hydrophobic chromophores covalently attached to various polyanions. The photophysics of the chromophores incorporated in the polyeletrolytes at small mole fractions is relatively simple, because no interaction is expected to occur between the incorporated chromophores. For this reason, most of the studies have focused on amphiphilic polyeletrolytes loaded with a low amount of hydrophobic chromophores. 

Similar data were reported by Turro et al., [62,63] who synthesized a copolymer of AA with 1.5 mol% of 2-[4-(l-pyrene)butanoyl]aminopropenoic acid, 19 and studied the fluorescence quenching with Tl +, Cu2+, and 1 ions in aqueous solution. 

Webber et al. [60, 78] also studied the fluorescence quenching of diphenylan-thracene (DPA) covalently bound to poly(methacrylic acid), PMAvDPA (23) [60], and to sodium poly(styrenesulfonate), PSSvDPA (24 )[78]. The fluorescence quenching of the excited DPA moiety by MV2+ and Cu2+ was also highly efficient. For example, with PMAvDPA of 0.073 mol% DPA content, the kq values at pH... 

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