Dynamic quenching of the multiband fluorescence of 3-hydroxyflavone

The properties of emission, absorption, and dual fluorescence excitation of 3-hydroxyflavone in acetonitrile are studied under the conditions of dynamic quenching by potassium iodide with concentrations up to 4 × 10^?2 M. The normal and tautomeric forms undergo quenching, which is more efficient for the tautomeric form. An interesting circumstance is that the absorption in the S ₀ → S ₁ and S ₀ → S ₂ singlet bands of the solution increases with increasing quencher concentration in the whole region of concentrations used, the steepest rise being recorded in the concentration region from 0 to 5 × 10^?3 M. The intensities and quantum yields of the two fluorescence bands show rather complicated nonlinear dependences on the quencher concentration. The long-wavelength fluorescence band, which belongs to the tautomeric form of 3-hydroxyflavone, is quenched considerably stronger. The experimental results reveal the kinetic character of the excited-state proton transfer in molecules of 3-hydroxyflavone in acetonitrile.     

Determination of the stern-volmer constant in quantum systems with dual fluorescence

The mathematical relations describing the properties of the steady-state spontaneous emission of quantum systems with dual fluorescence under conditions of dynamic quenching of excited states by foreign impurities are analyzed. The direct dependence of the intensity and yield of the photoproduct fluorescence on the quencher concentration is not simple and cannot serve as a convenient base for determining the Stern-Volmer constant. It is shown that, in the case of a kinetic character of product formation, the fluorescence intensity ratio of the initial dye and its photoproduct linearly increases with the quencher concentration. The relation obtained can be used to determine the constant of bimolecular quenching of the excited states of reaction products. This conclusion is based on the analysis of the experimental fluorescence spectra of 3-hydroxyflavone, obtained upon excitation in the region of the S ₁ absorption band under conditions of dynamic quenching by potassium iodide. This analysis can be applied to a wide range of luminophores with photoreactions accompanied by dual fluorescence (charge transfer, proton transfer, phosphorescence, complexation, etc.).     

Effect of temperature on the dynamic quenching of the dual fluorescence of molecules

The properties of the dual fluorescence of 3-hydroxyflavone in acetonitrile are studied under conditions of dynamic quenching by the spin quencher TEMPO in the temperature range from 20 to 80°C. 3-Hydroxyflavone is characterized by the intramolecular excited state proton transfer and its spectrum consists of two well-spaced fluorescence bands belonging to the normal and tautomeric forms. The fluorescence was selectively excited at wavelengths of 290, 304, and 340 nm, belonging to different absorption bands of the luminophore. The character and degree of the temperature quenching of the fluorescence depend on the excitation wavelength and are considerably different for normal and tautomeric fluorescence bands. The Stern-Volmer constants for both forms, calculated from the experimental data on the assumption of the diffusion mechanism of quenching of the excited states, increase with temperature. Both in the pure solutions and in the solutions with the quencher, the intensity ratio of the fluorescence of the initial form and the product increases with heating in the entire temperature range from 20 to 80°C for all the above excitation wavelengths. Original Russian Text ? V.I. Tomin, 2008, published in Optika i Spektroskopiya, 2008, Vol. 104, No. 6, pp. 926–933.     

Temperature dependences of dual fluorescence as criterion for determining character of photoreactions

We performed comparative studies of the temperature quenching of dual fluorescence of acetonitrile solutions of several molecular probes with proton transfer reaction in an excited singlet state of 4′-(dieth-ylamino)-3-hydroxyflavone (FET), 1-methyl-2-(4-methoxy)phenyl-3-hydroxy-4(1H)-quinolone (QMOM), and 3-hydroxyflavone (3HF) parent molecule at different energies of excitation quanta. In accordance with expressions obtained from balance equations for photoreactions of the kinetic and thermodynamic character, the intensity ratios of fluorescence bands as functions of the degree of quenching behave differently. Namely, the quenching increases the relative intensity of bands normal form/tautomer for reactions of the kinetic type, retaining this ratio unchanged for reactions of the thermodynamic character. Our experimental studies showed that, for fluorescent probes with the kinetic reaction (3HF and QMOM), the intensity ratio fluorescence bands increases almost linearly with the degree of quenching, whereas, in the thermodynamic case (FET), this ratio is independent of this parameter. Conclusions about the character of reactions that we obtained in this work agree well with data of independent investigations of these molecules by laser spectroscopy with high time resolution, and the obtained relations allow us also to judge the mechanism of temperature quenching in the case of the reaction of the kinetic type. The method can be used for comparatively simple express selection of molecular probes, candidate for new applications.     

Separation of a weak fluorescence signal of 3-hydroxyflavone using dynamic quenching

The spectral and time characteristics of the dual fluorescence of a 3-hydroxyflavone probe in a solution with a fluorescence quencher are studied in the temperature range of 20–80°C. At room temperature, the fluorescence consists of two bands that belong to the normal and tautomeric forms of the luminophore, while heating of the solution results in the appearance of an additional fluorescence band belonging to the anionic form of the luminophore. The additional band is strongly overlapped with the band of the normal form, and its intensity rapidly increases with temperature to exceed the intensity of the normal band. The introduction of the TEMPO spin quencher of excited states into the solution completely quenches the fluorescence of the anionic form, which allows one to record the pure dual fluorescence of 3-hydroxyflavone in the entire temperature range studied. The detection of probe signals in the pure form is important for applications of proton transfer molecular probes using the intensity ratio of the fluorescence bands as the main sensitive parameter.     

Fluorescent probe based on proton transfer in 4′-(diethylamino)-3-hydroxyflavone: Spectral and luminescent characteristics upon selective excitation

The spectral characteristics of acetonitrile solutions of 4′-(diethylamino)-3-hydroxyflavone dye with dual fluorescence are studied under selective excitation. This dye is a structural analog of 3-hydroxyflavone and exhibits excited-state proton transfer, which, in contrast to 3-hydroxyflavone, has a thermodynamic rather than a kinetic character. The fluorescence spectra at different excitation photon energies and the excitation spectra of different fluorescence bands are studied. It is found that the intensity ratio of the normal and tautomeric fluorescence bands lying near 507 and 570 nm, respectively, depends on the excitation wavelength, namely, this ratio is 1.45 and almost does not change in the region of the main absorption band (370–420 nm), while, in the region of the second singlet band (near 280 nm), it decreases to 1.15. This can be explained by an increase in the probability of proton transfer with formation of a tautomeric form in the case of excitation into the second band. Another interesting feature is the existence of a latent third emission band peaked at 535 nm, which was found and reliably recorded upon excitation at wavelengths of 470–500 nm. Addition of water quenches this emission, which indicates that it belongs to the anionic form of the dye.     

Studying reactions that proceed from highest excited states of molecules using fluorescence quenching

The steady-state monochromatic excitation of a luminophore that has fluorescing products is considered. The effect of dynamic quenching of highest excited states on the fluorescence of singlet states under its excitation via singlet S ₁ and S _n (n ≥ 2) states is discussed. It is shown that the use of the method of fluorescence dynamic quenching by foreign impurities opens new possibilities for studying photoreactions that proceed via S _n singlet states. A large number of primary photoprocesses are considered which include the electron density redistribution (the internal electron transfer) in the excited state, protolytic reactions, intramolecular proton transfer (phototautomerization), hydrogen bonding, and formation of excimers and exciplexes. It is shown that, upon dynamic quenching, the bimolecular quenching constant of an excited level depends on the amount of thermal energy released in the luminophore before the occurrence of the light emission event. Based on the experimental measurements of the fluorescence spectra at different quencher contents, the calculation of the Stern-Volmer constant for reaction products is considered in detail. It is shown that this constant can be most reliably determined from the dependence of the fluorescence intensity ratio of the initial reagents and the quencher product rather than from the dependence of the fluorescence intensity of the products on the concentration of the quencher. The relations determined are used in analysis of the experimental fluorescence spectra of solutions of 3-hydroxyflavone excited by radiation with different wavelengths lying in the range of the S ₁ and S ₂ absorption bands. The temperature behavior of the Stern-Volmer constant for different fluorescence bands of 3-hydroxyflavone is considered. It is shown that, if these constants for the normal and tautomeric forms are correctly determined, their temperature dependences are similar.     

Effect of temperature and dynamic fluorescence quenching on proton transfer in 3-hydroxyflavone

The effect of temperature and a fluorescence quencher on the properties of the excited states of 3-hydroxyflavone is considered. The absorption spectra and the spectra of dual fluorescence exited by electromagnetic radiation in the region of the S ₁ absorption band of 3-hydroxyflavone in acetonitrile are measured and analyzed in the temperature range of 20–80°C. The fluorescence lifetimes are also measured at different temperatures. As a quencher of excited states we used the TEMPO spin quencher. The analysis of the fluorescence parameters shows that the heating of the solution to 60°C leads to a considerable (by a factor of 1.24) increase in the proton-transfer rate for the first absorption band. The introduction of a quencher decreases the yield of the two fluorescence bands by the diffusion mechanism and increases the proton-transfer rate with respect to the rate in the pure solvent by a factor of 1.16 at room temperature and 1.65 at T = 80°C.     

Investigation of excited-state proton transfer in 3-hydroxyflavone molecules

The dual fluorescence spectra of 3-hydroxyflavone molecules excited by electromagnetic radiation in the region of the S ₁ and S ₂ absorption bands in the temperature region of 20–80°C are studied using the dynamic quenching of the excited state. An analysis of the fluorescence parameters shows that heating the solution from room temperature to 60°C increases the proton transfer rate by a factor of 1.24 in the case of standard excitation into the main absorption band and even stronger (by a factor of 6.9) in the case of excitation into the second absorption band. The presence of a quencher reduces the yield of the two emission bands and noticeably increases the proton transfer rate, by a factor of 1.16 at room temperature and by a factor of 1.25 at 80°C. Upon excitation into the second singlet band, the transfer rate increases even more (especially at higher temperatures), by a factors of 1.24 and 3.5 for the same temperatures. The temperature dependences of the transfer rate constant allowed us to estimate the activation energies of the proton transfer reaction under different physical conditions and reach conclusions about the mechanism by which this reaction proceeds. It is found that the proton transfer activation energy decreases from 500 to 360 cm⁻¹ when measured in temperature ranges of 20–40 and 20–60°C. The introduction of a quencher with a concentration of 5 × 10⁻³ M increases the activation barrier to 534 and 471 cm⁻¹ in the same temperature ranges.     

Spectral properties of molecular charge-transfer probe QMOM

The spectral characteristics of solutions of a dye with dual fluorescence, 1-methyl-2-(4-methoxy)phenyl-3-hydroxy-4(1H)-quinolone, in acetonitrile are studied upon selective excitation. This dye is a structural analogue of 3-hydroxyflavone and also exhibits excited-state proton transfer, which, as well as in the case of 3-hydroxyflavone, has a kinetic nature. The fluorescence spectra are studied upon excitation by photons of various energies, and the excitation spectra are recorded at wavelengths of different fluorescence bands. It is found that the intensity ratio of the emission of the normal and tautomeric forms (at wavelength of 415 and 518 nm, respectively) is almost the same (0.23–0.25) for excitation in the regions of the main and the second absorption bands. At the same time, in the case of excitation between these bands, this ratio decreases to 0.19. The second interesting feature is the existence of a third latent emission band peaked at about 480 nm, which is reliably detected upon excitation at wavelengths in the region of 400–450 nm. This study shows that this emission belongs to the anionic form of the dye. This form is also responsible for a decrease in the intensity ratio of the emission of the two main forms in the case of excitation between the first and second absorption bands.     

The influence of temperature and dynamic quenching on the properties of 3-hydroxyflavone excited states

The influence of temperature and dynamic quenching on the properties of excited states of the normal and tautomeric 3-hydoxyflavone forms was studied. The stationary two-band fluorescence spectra of this luminophore in acetonitrile were recorded and analyzed. The spectra were observed under excitation by electromagnetic radiation in the region of the S ₁ absorption band over the temperature range 20–80°C. TEMPO was used as a quencher of the excited state. Heating caused temperature quenching of luminescence, and the tautomer formed via the excited state of the normal form of the luminophore was quenched more strongly both in pure solvent and in the presence of the quencher. An analysis of two-band fluorescence parameters led us to conclude that solution heating over the temperature range studied increased the rate of proton transfer by 1.25 times. The introduction of the quencher also accelerated proton transfer by 1.16–1.25 times as the temperature increased from room temperature to 80°C.     

Influence of deformations of a polymer matrix on the characteristics of dual fluorescence of 3-hydroxyflavone

The possibility of considerably changing the conditions for the proton transfer reaction in 3-hydroxyflavone molecules in polyvinyl alcohol (PVA) polymer matrices by stretching deformations is demonstrated. Samples of this kind are traditionally used to obtain ensembles of fluorophore molecules oriented along a chosen axis and for polarization measurements. The fluorescence spectrum of 3-hydroxyflavone in PVA has two characteristic bands in the violet and green spectral regions, which indicates excited-state proton transfer. Stretching leads to a strong reduction in the violet band, whose contribution in undeformed samples is comparable to the contribution of green fluorescence. Even twofold stretching of PVA films strongly decreases the violet band intensity, which is more pronounced in the case of sixfold stretching. In the latter case, the fluorescence spectrum behavior is very close to the pattern observed in nonpolar and aprotic solvents, in which the violet fluorescence intensity is very low. The data obtained indicate that mechanical stretching allows one to eliminate the main intermolecular factors that slow down the proton transfer between the active groups in PVA.     

Intramolecular proton transfer from the highest singlet states in 3-hydroxyflavone

It is found that the excitation spectra of the dual fluorescence of 3-hydroxyflavone are different for different recording wavelengths and that the intensity ratio of the emission of the normal and tautomeric (with intramolecular proton transfer) forms upon selective UV excitation in the regions of the S ₁, S ₂, and S ₃ singlet absorption bands strongly depends on the excitation wavelength. The results obtained directly point to the existence of an additional channel of population of the excited state of the tautomeric form and are explained by the intramolecular proton transfer through the S ₂ and S ₃ excited singlet states of fluorophore molecules. The constants of this transfer are estimated using analytical relations for the steady-state fluorescence excitation.     

On estimating probability of excited-state intramolecular proton transfer

Higher singlet states can play an important role in various intramolecular processes. Recent investigations of the time-resolved (with a picosecond resolution) spectra of the dual fluorescence of 3-hydroxyflavone molecules excited in the region of the S ₁ and S ₂ absorption bands by pulses with a duration of ∼44 ps have directly shown the occurrence of the proton transfer from the carboxyl to the carbonyl group of the molecule upon excitation into the second singlet absorption band. The reaction times estimated from the emission characteristics are comparable with the electronic level lifetime (several picoseconds), as a result of which the direct measurements are rather difficult. The proton transfer through the S ₂ state is also recorded in the steady-state fluorescence excitation spectra. In this study, it is shown how the reaction rate can be estimated from these data.     

Investigation of reactions from the highest excited states of molecules by fluorescent spectroscopy methods

The effect of the highest excited states on the yield of photoproducts that are usually formed upon excitation of the first singlet electronic state of polyatomic molecules is discussed. It is shown that the excitation of molecular objects through the highest singlet states can, in some cases, increase the yield of reaction products. This allows one to estimate the probabilities of reactions from the corresponding states. The consideration concerns a wide range of primary photoreactions, including the electronic density redistribution (the intramolecular electron transfer) in the excited state, the protolytic reactions, the intramolecular proton transfer (the phototautomerization), the hydrogen bond formation, and the formation of excimers and exciplexes. The relations obtained are used to analyze the experimental fluorescence spectra of 3-hydroxyflavone solutions, excited by electromagnetic radiation with different wavelengths in the region of the S ₁, S ₂, and S ₃ absorption bands. The analysis fulfilled shows that the highest singlet states play an important role in the formation of tautomers in 3-hydroxyflavone due to the intramolecular proton transfer.     

Energy and Electron Transfer in the Field of Transition Metal Complexes

The bimolecular quenching of an excited state molecule in fluid solution can occur in three fundamental ways:^1–3 (i) chemical reaction between the excited state and the quencher, (ii) electronic energy transfer from the excited state to the quencher, or (iii) deactivation of the excited state by some catalytic action of the quencher. The intimate nature of the quenching mechanism is often difficult to elucidate for a single excited state-quencher couple. More useful information can generally be obtained from correlations of data concerning homogeneous families of excited states and/or quenchers.     

Determination of the diffusion and viscosity coefficients from the properties of dual fluorescence of molecules in solutions

We have proposed and substantiated an approach that makes it possible to determine the diffusion and microviscosity coefficients in solutions from characteristics of the dual fluorescence of molecular probes. This approach uses the Stern-Volmer constants obtained upon fluorescence dynamic quenching in solutions. The relations that follow from the balance equations in terms of the formalism of two-level reactions in the excited state for the case of photoreactions of the kinetic character yield the dependences of the intensity ratio of the fluorescence bands of the normal form and tautomer on the degree of quenching. In accordance with these dependences, the dynamic quenching of the diffusion character (including the temperature quenching) changes the intensity distribution, and, based on these dependences, the Stern-Volmer constants and the bimolecular quenching constants can be determined, from which, using appropriate models, the diffusion and viscosity coefficients can be found. The merit of the method is its simplicity and availability, since it is based on the use of the data of steady-state measurements of fluorescence spectra with widespread standard instruments.     

Direct observation of proton transfer from the excited <Emphasis Type="Italic">S</Emphasis><Subscript>2</Subscript> state in the 3-hydroxyflavone molecule

The spectra of dual fluorescence of 3-hydroxyflavone molecules excited by 44-ps pulses in the region of the S ₁ and S ₂ absorption bands are measured with a picosecond resolution. The dynamics of the spectra directly demonstrates the time development of the proton transfer from the carboxyl to the carbonyl group of the molecule. Upon excitation into the main absorption band, the transfer process occurs for about 210 ps. The excitation into the region of the S ₂ band results in a faster (～170 ps) process, and the relative contribution made to the total spectrum by the long-wavelength band, which belongs to the proton-transfer state, is higher in this case for all the time ranges of luminescence recording. The data obtained directly point to an additional channel of proton transfer via the S ₂ state. The probability of this process is estimated to be 0.84 × 10¹² s^?1.     

Effect of temperature on the fluorescence emission of 2H-chromen-2-one derivative in non-polar and polar solvents

The effect of temperature (293-333 K) on the fluorescence emission of 4-(5-methyl-3-phenyl-benzofuran-2-yl)-6-chloro-2H-chromen-2-one (MPBClC) is recorded in non-polar (1,4-dioxane, and toluene), and polar (butanol and DMSO) solvents. It is found that there is no shift in the position of fluorescence maxima, but the intensity decreases with increase in temperature, which depends on the polarity of the solvent. A mechanism of fluorescence quenching with rise in temperature is discussed in terms of the relative location of lowest ¹(ππ^?) and ³(nπ^?) states, and the energy difference between them. The change in temperature brings about a change in the probabilities of radiative and non-radiative transition. The radiationless deactivation of excited-state in the absence of quencher is temperature-dependent and its thermal activation energy has been determined.     

A Comparative Study into Two Dual Fluorescent Mechanisms via Positional Isomers of N-hydroxyarene-1,8-naphthalimides

Three isomers of hydroxy substituted N-aryl-1, 8-naphthalimides based on N-aryl naphthalic anhydride fluorophore have been synthesized. The decrease in fluorescence intensity from ortho to para substitution of hydroxy group on N-aryl reveals that para substituted isomer undergoes ESEC (Excited State with Extended Conjugation) mechanism which is proved by low quantum yield and appearance of dual emission. The ortho isomer, however, has high quantum yield and no tautomer emission, indicating ESIPT (Excited State Intramolecular Proton Transfer) mechanism is not operating. Similarly, all these isomers show strong fluorescence quenching in presence of strong H-bonding solvents like DMSO and pyridine, but there was neither the shift of emission bands nor the appearance of new bands for proton transfer to these solvents. Thus, it also indicates the absence of excited state proton transfer mechanism. Both the ortho isomer, and to a greater degree the meta isomer, showed larger quenching constants (Kapp) with pyridine than DMSO. This trend opposes the hydrogen-bond affinity for these solvents with phenol and points to a 2-point recognition interaction. In addition, a naphthalimide derivative using 2-aminoimidazole was prepared and examined for optimal positioning of a six-membered ring hydrogen bond pattern. No dual fluorescence was observed for this compound either. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.