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.     

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.     

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.     

Dynamic quenching of the dual fluorescence of molecules

Mathematical relations describing the properties of spontaneous steady-state dual fluorescence under conditions of dynamic quenching of excited states by foreign impurities are derived. It is shown that, in the case of a kinetic character of the reaction, the initial form of the dye and its photoproduct are quenched, the intensity ratio of the fluorescence bands of the initial form and the product linearly increasing with the quencher concentration. Analysis performed is applicable to a wide range of photoreactions accompanied by the dual fluorescence (charge transfer, proton transfer, complexation, etc.). The properties of the fluorescence, absorption, and dual fluorescence excitation for 3-hydroxyflavone in acetonitrile under conditions of dynamic quenching by the TEMPO spin quencher with a concentration below 1.25 × 10^?2 M are studied. 3-Hydroxyflavone is characterized by the excited-state intramolecular proton transfer and by the fluorescence spectrum consisting of two well-spaced bands. The observed dependences of the intensity of both fluorescence bands on the quencher concentration correspond to the theoretical conclusions. The Stern-Volmer constants calculated from the experimental data on the assumption of diffusion quenching of the excited states are 858 and 1141 M^?1 for the normal and tautomeric fluorescence bands, respectively. The experimental results reveal the kinetic character of the excited-state proton transfer in 3-hydroxyflavone in acetonitrile.     

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.     

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.     

Dependence of the properties of the dual luminescence of 3-hydroxyflavone on the excitation wavelength

The luminescence and luminescence excitation spectra of 3-hydroxyflavone in acetonitrile obtained at different excitation/recording wavelengths are studied. The dependences of the position of the normal luminescence band maximum and of the intensity ratio of the normal and proton-transfer bands, on the excitation wavelength are found and studied for the first time. It is found that the spectral contour of dual luminescence also depends on the excitation wavelength and the blue emission band is cut off at a sufficiently long-wavelength excitation in the region of 390 nm. In the luminescence excitation spectrum, an additional wide band is observed in the proton-transfer region at 200–260 nm. Excitation in the region of 380–440 nm allowed us to reveal a wide structureless band near 470 nm (with the maximum of its excitation near 420 nm) belonging to the anionic form of 3-hydroxyflavone. Addition of water at a concentration of ～2.2 M quenches this band almost completely.     

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.     

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.     

On the possibility of measuring the temperature by the luminescence of a probe with intramolecular proton transfer

The temperature dependences of the fluorescence characteristics of three forms (normal, tautomeric, and anionic) of 3-hydroxyflavone in methanol upon selective excitation in different UV ranges are studied. The fluorescence of all the forms undergoes temperature quenching, whose efficiency depends on the excitation energy. It is found that the intensity ratio of the fluorescence of different forms also varies with temperature; the excitation regions in which these variations are most pronounced are determined. The highest temperature sensitivity is observed for the luminescence intensity ratio of the normal and tautomeric forms upon excitation in the region of 280 nm, this ratio continuously increasing from 0.5 to 1.0 in the temperature range of 20–70°C. The dependence found allows one to measure the temperature of the solution in some temperature regions with an error of ～1 ± 0.5% with the help of a standard Hitachi F-2500 spectrofluorimeter.     

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.).     

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 anionic form of 3-hydroxyflavone

The spectral and temporal characteristics of the fluorescence of the anionic form of some 3-hydroxiflavones in different solvents are studied. This form is observed under selective excitation in the region of the long-wavelength slope of the main absorption band, and its spectrum consists of a wide structureless band lying between the short- and long-wavelength fluorescence bands of these molecular probes. The fluorescence excitation spectra of the anionic form differ from the corresponding spectra of the normal and tautomeric forms. The addition of water to the solution leads to a gradual fluorescence quenching which is static or belongs to the second kind according to Vavilov’s classification, i.e., occurs in the ground state.     

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.     

Temporal characteristics of the fluorescence of the anionic form of 3-hydroxyflavone

The spectral and temporal characteristics of the fluorescence of the anionic form of 3-hydroxyflavone in acetonitrile are studied. This form can be selected upon excitation in the region from 380 to 440 nm with the maximum near 420 nm. The fluorescence spectrum of this form has the shape of a wide structureless band peaked at about 470 nm. The lifetimes of the fluorescence of the anionic form in the region from 460 to 530 nm are measured; the average lifetimes do not depend on the recording wavelength in the entire region and are equal to 3.7 ± 0.2 ns. Addition of water to the solution leads to a gradual quenching of the fluorescence and its complete vanishing at a concentration of 10 M. This is a static quenching or quenching of the first kind according to Vavilov’s classification; i.e., it occurs in the ground state.     

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.     

Modulation of charge transfer rate in (N,N′-dimethylamino)benzonitrile liquid solutions

We studied the properties of the emission, absorption and excitation of dual fluorescence of (N,N′?dimethylamino)benzonitrile in a polar aprotic solvent acetonitrile under selective irradiation of solutions by light with different energies of quanta to elucidate mechanisms of dual fluorescence arising in this solvent at different temperatures in the range 274–313 K. In all cases, dual fluorescence of the solute in acetonitrile was observed, which is caused by emission from locally excited Franck-Condon and charge-transfer states. A change in the energy of excitation quanta has a weak effect on the position of the fluorescence bands; however, the intensity ratio between the bands noticeably changes in favour of the intensity of the long-wavelength band at excitation in the range of the long-wavelength absorption band. An interesting and unusual fact is that solution heating is accompanied by essential growth of quantum yield of dual fluorescence at all wavelengths of the excitation. To explain the observed effects, the same dependences were measured and analysed for DMABN in neutral solvent n-hexane in the same conditions. We involve also the data of quantum-mechanical calculations, which show that there is a considerable probability of occurrence in solutions of DMABN rotational isomers with differing orientation of the dimethylamino group with respect to the benzonitrile. In the excited state, these have different charge-transfer rates, resulting in a modulation in the intensity ratio of the observed fluorescence bands with change excitation energy quanta on the red wing of the absorption band, doi: 10.1134/S0030400X12050219.     

Modulation of the proton transfer rate by excitation photons

The effect of the exciting photon energy on the excited state proton transfer in a dye with dual fluorescence—FET (4′-diethylamino)-3-hydroxyflavone)—is studied. The steady-state fluorescence spectra are studied upon selective excitation by photons with different energies in the region of the main absorption band, as well as at its long-wavelength wing, in the temperature range of 2–30°C. It is found that, at all temperatures, the ratio of the integral emission of the normal and tautomeric forms, which are observed at 480 and 570 nm, respectively, depends on the excitation wavelength; namely, this ratio noticeably decreases with increasing excitation wavelength in the region of the main absorption band and its long-wavelength wing at 390–440 nm, and the rate of this decrease depends on temperature. In the same region, the long-wavelength excitation effect, which is atypical for inviscid solvents at room temperature, is observed; i.e., a short-wavelength emission band is bathochromically shifted by 6–15 nm depending on temperature. This spectral shift is directly related to the inhomogeneous broadening of the electronic spectra of the normal FET form, which is very large due to a considerable (>10 D) difference in the dye dipole moments. Most probably, the excitation creates the possibility of emission from nonrelaxed nonequilibrium orientational sublevels because their lifetime becomes shorter due to the proton transfer reaction, the rate of which in acetonitrile is comparable with the rate of intermolecular orientational relaxation. It is proposed to explain these dependences using energy diagram taking into account the dependence of the free energy on the orientational polarization of the solvent.     

Molecular probe FET: Long-wavelength fluorescence excitation effect and proton transfer reaction

We studied the steady-state fluorescence spectra of solutions of FET (4′-(diethylamino)-3-hydroxyflavone) in acetonitrile that were excited at different temperatures by quanta with different energies located in the range of the main absorption band and in its long-wavelength wing. We found that, at room temperature, the emission intensity ratio of the bands of the normal and tautomeric forms, which are located at 505 and 570 nm, respectively, depends on the excitation wavelength. In the range of the main absorption band 300–360 nm, this ratio remains nearly the same, i.e., 1.45, while, upon excitation in the range of the long-wavelength wing 360–380 nm of the main band, it decreases to 1.33 at a wavelength of 460 nm. In this same range, a long-wavelength excitation effect that is unusual for liquid inviscid solvents at room temperature, i.e., a bathochromic shift of the entire short-wavelength emission band by 11 nm, manifests itself. We propose to explain these dependences using energy diagrams, which take into account the dependence of free energy on the orientational polarization of the polar solvent. The observed effect of the long-wavelength shift of the fluorescence spectrum with increasing excitation wavelength is explained in terms of the inhomogeneous broadening of electronic spectra of polar solutions, and it should be described using the scheme of energy states that takes into account sublevels of orientational broadening due to orientational dipole-dipole interactions of the fluorophore with nearest molecules of the polar solvent, as well as the relation between the fluorophore lifetime in the excited state and the dielectric relaxation time of solvent molecules in the field of the fluorophore dipole.     

Effects of long-wavelength fluorescence excitation in N, N′-dimethylaminobenzonitrile liquid solutions

We have studied the properties of the emission, absorption, and excitation of dual fluorescence of N,N??-dimethylaminobenzonitrile in a set of solvents of different polarity under selective irradiation of solutions by light with different energies of quanta in the range of the long-wavelength absorption band. In all cases, dual fluorescence is observed, which is caused by emission from locally excited Franck-Condon and charge-transfer states. A change in the energy of excitation quanta has no effect on the position of the fluorescence bands; however, the intensity ratio between the bands noticeably changes in favor of the intensity of the long-wavelength band, which belongs to the charge-transfer state. To explain the observed effects, we involve data of quantum-mechanical calculations, which show that there is a considerable probability of occurrence in solutions of these systems of rotational isomers that differ in the orientation of the dimethylamino group with respect to benzonitrile. In the excited state, these rotamers have different charge-transfer reaction rates, which leads to a change in the intensity ratio of the observed fluorescence bands upon using the selective excitation.