ISSN 0030 400X, Optics and Spectroscopy, 2012, Vol. 113, No. 1, pp. 4152. Pleiades Publishing, Ltd.

., 2012. Original Russian Text V.I. Tomin, 2012, published in Optika i Spektroskopiya, 2012, Vol. 113, No. 1, pp. 4455.

CONDENSED MATTER SPECTROSCOPY

Fluorescence Quenching as a Method of Identification of the Character of Photoreactions in Excited States of Molecules
V. I. Tomin
Institute of Physics, Pomeranian University, S l upsk, 76 200 Poland e mail: tomin@apsl.edu.pl
Received November 15, 2011

AbstractWe consider the steady state monochromatic excitation of a luminophore that has fluorescent products. We consider how the fluorescence of singlet states is affected by different means of quenching high lying excited states such as quenching with impurities, temperature quenching, and shortening the lifetime with induced transitions in the light field. We show that the use of different methods of fluorescence quench ing opens new possibilities for studying photoreactions that proceed via excited singlet states. Our consider ation concerns a wide range of primary photoprocesses, such as electron density redistribution (internal elec tron transfer) in the excited state, protolytic reactions, intramolecular proton transfer (phototautomeriza tion), hydrogen bond formation, intermolecular relaxation of molecules in solutions, and formation of excimers and exciplexes. The obtained relations have been used to analyze experimental fluorescence spectra of solutions of some organic compounds, including derivatives of 3 hydroxyflavone and 3 aminophthalim ide, subjected to quenching by different methods. DOI: 10.1134/S0030400X12060215

INTRODUCTION In recent years, various applications of photolumi nescence methods have been studied intensely, not only in traditional fields of physics and chemistry, but also in the life sciences, such as biology, medicine, bio medical optics, and photonics [15]. Absorption and fluorescence processes in complex molecules are com monly observed between the ground S0 and first excited S1 singlet states. The first singlet state is final in the process of light absorption and, simultaneously, serves as an initial level in the formation of all charac teristics of fluorescence of molecules that are either in the vapor phase or in the condensed state. Various photophysical and photochemical processes in organic molecules, such as, e.g., formation of exci mers and exciplexes, electron density redistribution, transition to triplet metastable states, phototautomer ization, various configurational changes of molecules in solvates, etc., are directly related to the S1 state. There are also systems the photoreactions in excited states of which give rise to dual fluorescence. A valuable property of molecules with dual fluores cence is that the mutual change in the intensity of their fluorescence bands depends on intermolecular inter actions. This property is applied in the self calibration procedure of the response reaction to the properties of the environment, which is widely used in studies of
41

various physicochemical objects using probes with internal proton transfer in the excited state [48]. The relative (ratiometric) signal of the two luminescence bands of a molecular probe allows one to obviate (i) procedures of determining the absolute sensitivity of fluorescence measurements at wavelengths in the ranges of luminescence to be recorded, (ii) taking into account concentration changes of the fluorescent probe in the sample, and (iii) uncontrollable fluores cence quenching by impurities in the course of the measurement procedure. In this work, we consider how various types of quenching of excited states, such as quenching by impurities, temperature quenching, and induced tran sitions in the field of light radiation, affect the proper ties of luminophores with dual fluorescence and fluo rescence that proceeds simultaneously with a strong relaxation shift of spectra. We consider the influence of different kinds of quenching on the characteristics of dual fluorescence of molecules and show how one can distinguish between the kinetic and thermodynamic photoreactions that proceed in excited states of mole cules. Experimental data that support the obtained conclusions are presented. Some aspects concerning various quenching mechanisms have been previously presented in our previous works, while the present paper is of a generalizing character.

42 ()
N* S1
N

TOMIN (b)
k+ k k nR kR
N P S1

N* P
*

S1

k+ k k nR kR
N P S1

P*

ex

k nR

ex

N kR
P P

N P k nR S0 k0
P

kR

P
N S0

S0 k0

P N

N S0

Fig. 1. Scheme of energy levels for the description of the reaction of formation of a fluorescent product (P) from the normal form (N) of a fluorophore: (a) reaction proceeds with an energy barrier; (b) reaction proceeds without any energy barrier.

MODEL AND THEORETICAL DESCRIPTION OF FLUORESCENCE TAKING INTO ACCOUNT QUENCHING Figure 1 presents the scheme of levels for the description of an arbitrary photoreaction that starts from the first singlet state. It reflects the occurrence of the following main energy levels: the ground S 0N and excited S1N levels of the normal (N) form and the ground S1P and excited S 0P levels of the photoreaction product (). We will also consider that the first excited states of the molecule and product possess measurable fluorescence. We will not specify the particular type of photoreactionthis, e.g., may be a photoattachment reaction, such as the formation of an excimer (a com plex with an unexcited fluorophore molecule) or exci plex (a complex with a solvent molecule), or the reac tion of formation of a molecular conformer, or the reaction of tautomerization. It is only important that the system of energy levels of the photoproduct will differ from that of the initial molecule, as is shown in Fig. 1a. In the majority of cases, the formation of the photoproduct is energetically a more favorable pro cess; therefore, its singlet level S1p usually lies some what lower than the level S1N and the reaction proceeds with overcoming of a certain energy barrier. Let us consider basic transitions in the scheme in Fig. 1 that occur as a result of the light absorption and relaxation of the excitation energy. A molecule that resides in the ground state S 0N absorbs light with the frequency B in the range of its fundamental absorp tion band and transits to the first excited level S1N , which is responsible for the short wavelength fluores cence band (the transition S1N S 0N with the fre N quency N and probability k R ). From this state, non N radiative transitions k nR and the reaction of formation

of the photoproduct in the excited state S1p (with the rate constant k+) also occur. After the lifetime , the state S1p spontaneously decays as a result of the fluores P cence transition with the probability kR , as well as due P to the nonradiative transition with the probability k nR and the reverse transition with the probability k to the level S1N . Therefore, the second spin emission band is related to transitions from the excited state S1P of the photoproduct to the ground state S 0P . For some photo reactions, the intensity of this transition can often even exceed the intensity of the short wavelength band; in some cases, the transition frequency is shifted with respect to the blue band by up to ~100 120 nm, so that the bands overlap each other very weakly. The latter circumstance makes it possible to easily determine the parameters of their contours with high accuracy. Consequently, with this design of experiment, two fluorescence bands can usually be easily observed, with the long wavelength band being related with the scheme of levels of the photoproduct. The investigation of this dual fluorescence is an effi cient tool for studying not only these N and P systems themselves, but also how they are affected by the fields of the environment and introduction. The fluores cence of systems of this kind, e.g., excimers, exci plexes [2, 3, 9], charge transfer molecules [2, 6, 10, 11], and ESIPT (excited state internal proton transfer) molecules [24, 1214]), has been studied intensely. In the simplest case of the standard steady state excitation regime of a fluorophore into its first absorp tion band by light with the radiation density uex, the following equation can be written for the S1P level:
P P k+ N * = {kR + knR + k} P *.

(1)

This expression reflects the balance between parti cles in the state S1P due to their transition from the
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FLUORESCENCE QUENCHING AS A METHOD

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state S1N of the fluorophore (left hand side of the equality) and their decay by spontaneous emission, nonradiative deactivation, and reverse reaction of for mation of the photoproduct (right hand side of the equation). From (1), we immediately obtain the con centration ratio between the excited particles of the fluorophore and its photoproduct,
P P N * = k R + k nR + k . P* k+

(2)

samples. The indices N and P in (5) refer to the initial form and its photoproduct. We will follow the scheme that we used in [15] to study how the dynamic question affects the dual fluo rescence of a complex molecule. We restrict our con sideration to the frequently occurring case of an effi cient photoreaction, in which the formation rate of the photoproduct is very high compared to the sum of constants that determine the fluorescence decay of the normal form,
N N N . (6) k+ k R + knR + kT In this case, the quenching will mainly cause only a decrease in the lifetime of the photoproduct and, then, in relations (2) and (3), the relative population will depend on the quenching probability,
P P P N * = k R + k nR + k + kT , P* k+

The numerator of the right hand side of this expression can be considered to be the inverse (exper imentally determined) radiation decay constant (life time) of the level S1P if it is excited by a pulse,

P =

1 . P P kR + knR + k

(3)

Expression (3) is convenient to use, since can be determined experimentally. Then, expression (2), sub ject to (3), takes the form

(7)

N* = 1 , (4) P* k+ P and, consequently, the relative population of fluores cent states is inversely proportional to the formation rate of the photoproduct and its fluorescence lifetime . In principle, from the known relative populations N * (which are proportional to the intensities of the P* corresponding fluorescence bands) and the lifetime of the photoproduct, one can determine the averaged rate constant k+ of its formation. Consider a more complicated problem in which the probability of quenching of spontaneous transi tions from the states S1N and S1P by several known mechanisms is taken into account in the general form,
N ,P N ,P kT = kQ Q + ktN ,P (t 0 ) + BN ,PU N ,P .

1 . (8) P + + k + kT It can be seen that the quenching will increase the relative population N * and decrease the lifetime P* provided that the contribution from the quenching probability is significant compared to the sum of other depopulation probabilities of the state,
P =
P kR P knR P P P . (9) kR + knR + k kT Clearly, at low values of the inverse photoreaction rate k,

(5)

In the general case, this problem takes into account N ,P the probability kQ Q of dynamic quenching of the N ,P excited level by impurities (where kQ = P kqN ,P is the quenching probability per unit concentration, kqN ,P is the diffusion bimolecular constant of quenching by an impurity quencher, and Q is the quencher concentra tion), ktN ,P (t 0) is the temperature quenching probabil ity, and BN ,PU N ,P is the probability of induced emission in the laser radiation field that reduces the lifetime of the level (where U N,P is the radiation field energy den sity and BN ,P is the Einstein coefficient for induced emission transitions). The latter mechanism should be taken into account increasingly frequently when using laser spectroscopy methods with powerful excitation, especially, upon two and three photon irradiation of
OPTICS AND SPECTROSCOPY Vol. 113 No. 1 2012

(10) k + k , i.e., when there is no fast energy exchange between the states of the normal form and the reaction product, this inequality will be implemented more easily. As can be seen from expression (7), the ratio N * directly P* N ,P depends on all quenching processes kT . Diffusion quenchers will interact not only with levels of the tau tomer, but also with excited states of the normal form. However, this will not affect the intensity ratio N * , P* since the sum of the probabilities of all processes that deplete the level S1N will be smaller than the reaction rate k+ and can be represented by the relation
N N N (11) kR + knR + k+ + kT k+ , which contradicts (6). Let us consider in more detail the general case of the influence of quenching on the relative populations N * taking into account the quenching terms in for P* mula (5). Clearly, if other conditions are the same, an increase in the concentration of the quencher leads to

44

TOMIN

a linear dependence of N * on the content of the P* P quencher with the proportionality coefficient kQ , which makes it possible to determine the bimolecular quenching constants from the appropriate experimen tal data. A similar linear dependence should manifest itself with increasing laser irradiation intensity due to induced transitions, provided that other quenching factors remain the same. This dependence will give the possibility of determining the Einstein coefficient for induced radiative transitions. However, heating the solution will act differently and, here, one should not expect an ordinary linear dependence on temperature. This quenching is also bimolecular, and, commonly, there are two mechanisms by which it occurs: quench ing by the solvent itself and quenching by molecular oxygen, which is always contained in solutions at atmospheric pressure. The bimolecular quenching constant is directly proportional to the diffusion coef ficient D

acteristic dependence of N * on the quencher concen P* tration is linear with respect to the intensity of the exciting field but is nonlinear with respect to tempera ture in the range in which the viscosity of the solution depends on temperature. It is also interesting to consider photoreactions with other limiting case in which the role played by the rate constant of the reverse reaction dominates over other deactivation rates of the singlet S1P of the prod uct,
P P P . k k R + knR + kT

(14)

Then, we can assume that the deactivation of the state is determined by the reverse reaction
P P P {kR + knR + k + kT } = k .

(15)

With allowance for this equality, it follows from (4) that

kQ = 4NRc D .

(12)

Here, N is the concentration density of molecules and Rc is the closest approach distance between molecules, which is usually taken as a sum of the radii of the fluo rophore and quencher molecules, RM and RQ, respec tively. The total diffusion coefficient D is a sum of the translational diffusion coefficients of the fluorophore and solvent molecules, DM and DQ, respectively, and can be represented [3] by the StokesEinstein relation

N * = k . P* k+

(16)

(13) D = Dm + DQ = kT 1 + 1 , f Rm RQ where k is the Boltzmann constant, is the viscosity of the medium, and f is the coefficient that depends on the boundary conditions for the rotation of the fluoro phore molecule (it is 6 if solvent molecules attach themselves to the fluorophore molecule, and they rotate as a whole, and it is 4 for a free rotation of the fluorophore molecule in the solvent). It follows from relations (12) and (13) that the bimolecular quenching coefficient linearly depends on temperature only if the viscosity remains to be a constant and temperature independent parameter. However, as a rule, in liquid solutions, the viscosity also depends on temperature, especially near freezing points, and, therefore, the dif fusion and the probability of bimolecular quenching nonlinearly depend on . In principle, this depen dence may prove to be useful in determining the microviscosity of the solution at different tempera tures from the temperature quenching of fluorescence. We note that, although the quenching mechanisms differ from each other, their common feature is that the relative concentration N * substantially depends P* on the degree of quenching. Therefore, if relation (9) between the rate constants is implemented, the char

Clearly, in this case, the relative populations N * P* depend now only on the probability ratio between the direct and reverse reactions and, consequently, all dependences of N * on the quenching probability van P* ish. Nevertheless, this does not mean that there is no quenchingquenching takes place, but manifests equally for the two bands. Furthermore, if there was a quencher that would act only on one of the fluores cence bands, irrespective of the mechanism of action of this quencher, both bands should be quenched because they strongly interact with each other during the lifetime of the excited state. Recall that, as is known [4, 68, 16], primary pho toreactions can be divided into kinetic and thermody namic reactions. The first, kinetic, type includes reac tions with weak interaction (energy exchange) of the fluorescent states of the normal form and product. This is related to a considerable energy gap between the excited states of the fluorophore and product and an energy barrier that separates these states. These reactions are characterized by equilibrium that is shifted toward the product, as a result of which they are almost irreversible. In connection with this, the probability k of the reverse transition for these reac tions will be considerably smaller than the rate con stant k+ of the direct reaction and, in some cases, k can be even smaller than the sum of rate constants of the radiative and nonradiative electronic transitions S1P S 0P (which is rather large for polyatomic mole cules, 1091010 s1); i.e.,
k
P P P kR + knR + kQ Q.

(17)
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FLUORESCENCE QUENCHING AS A METHOD

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In this case, the quenching affects the relative popula tion N * of the levels; i.e., it is possible to use expres P* sion (7). The thermodynamic schemes are characterized by an intense energy exchange between the excited states of the normal form and its photoproduct, and this, obviously, should occur at a close location of singlet levels, i.e., at a small energy gap. Fast energy exchange occurs at a rather high probability of the reverse tran sition; i.e., inequality (14) can be considered to be a good condition for this exchange. In this case, because of the strong energy exchange, both fluorescent states decay in a similar way during the measurement pro cess, and this is commonly considered to be the main experimental fact justifying the statement that the reaction is of thermodynamic character on the basis of kinetic measurements of the character of the fluores cence decay of the two forms. An efficient overlap of the energetic vibrational systems and a small energy gap are necessary conditions for the occurrence of these reactions. This feature fundamentally distin guishes this case from the kinetic one. In [16], the behavior of the dual fluorescence of some derivatives of flavones upon quenching of the excited state by a TEMPO quencher was studied and particular types of reactions were determined for each compound. We note another interesting possibility of changing the character of the reaction by means of quenching. Indeed, inequality (14) shows that rather strong quenching can violate its feasibility and, consequently, cause the reaction to proceed according to the kinetic scheme without changing the arrangement of the excited levels of the luminophore and product. This will directly affect the recorded fluorescence decay kinetics of different bands. Let us now clarify how the obtained relations can be used to process experimental data and to extract useful information on reactions. In experiment, we cannot directly measure the populations of energy lev els, since, in luminescence experiments, we measure the intensities of corresponding signals, which are directly proportional to the populations. Let us take into consideration that, for the ith form, the fluores cence intensity is directly proportional to the concen tration of molecules in the corresponding state Ni, the probability kR of the radiative transition, the average energy of the quantum hi, and the recording sensitiv ity ci,

The obtained relation contains the same two char acteristic dependences that are indicated in expres sions (7) for the populations of the levels, which makes it possible to determine useful properties of excited states. Formulas (19) and (5) yield a linear depen dence of the relative intensity on the quencher con centration, and it allows us to determine from the experimental data the bimolecular quenching con P stant kQ for the excited state of the reaction product provided that other constants that determine the slope of the curve IN/IT on the quencher concentration, N P namely, cN, cP, N, P, k R , kR , and k+, are known. It is most convenient to rule out the necessity of using the constants that appear in (19) by applying rel ative values for IN/IT at the zero quenching probability (e.g., upon dynamic quenching at a quencher concen tration of Q = 0) and quenching conditions. Let the signal in the pure solvent without quenching be denoted as
P N P I c k k R + k nR + k . 0 (20) I NP = N = N N R T k+ I P 0 cP P kR Then, in view of (5), expression (20) can be rewrit ten as

I NP P = 1 + kQ Q + ktP (t 0 ) + BPU P , 0 I NP

(21)

I i = ci N i kR h i .

(18)

Then, for the relative fluorescence intensity of the forms N and , taking into account (7), we obtain
N k R + knR + k + kQ I N cN N kR . = T k+ I P cP P kR P P P

P P where kQ , and the fluorescence lifetime P of = P kQ the product in the solution without the quencher is defined by expression (3). As can be seen from expressions (19) and (21), quenching directly affects the yield of the photoprod uct; namely, addition of a quenching substance increases the parameter IN/I, with other conditions remain unchanged. (In principle, the quencher can also affect the probability of the product formation.) Ratio (19) can be used to determine parameters that characterize quenching mechanisms reflected in for mula (5), e.g., constants of bimolecular quenching by impurities, if one measures dependences of relative intensities on the quencher concentration provided that other quenching parameters remain the same. The next conclusion refers to the possibility of finding temperature quenching constants from changes in the relative intensities of bands with heating the sample. Finally, if light quenching is used and the density of the radiation that acts on the sample is mon itored, it becomes possible to determine the probabil ity of induced transitions. Clearly, for the intensity ratio in the thermody namic case, in which expression (16) is valid, there is no dependence on the degree of quenching,
N I N cN N kR k . = T I P cP P kR k+

(19)
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46 Ifl 105, pulses () 10

TOMIN

IN/IP 0.029

IN/IP 0.03

0.026

0.02 0.01

8
0 4 8 Q, mM 12 0.023

(b)
1 2 3 12 Q 102, mM

4 P N

400

500

600

700 , nm

Fig. 2. Fluorescence spectra of solutions of 3 hydroxyflavone in acetonitrile with different concentrations of the TEMPO quencher (16). The concentration of TEMPO was in the range 05 103 M and was varied with a step of 103 M. The short wavelength fluorescence band is also shown on an enlarged scale (24). The excitation wavelength is 340 nm. The insets on the top show intensity ratios IN/I for quenching with (a) TEMPO and (b) potassium iodide.

We can assume that the proposed general descrip tion for taking into account the formation of the pho toreaction product refers to a wide circle of physico chemical processes that proceed after the singlet exci tation. It is likely that among them are the formation of excimers and exciplexes (classical examples are rep resented by pyrene in nonpolar solvents and anthracene with dimethylaniline [2, 3, 9]), electron transfer (strong electron density redistribution) in the first excited singlet state of complex molecules (N,N' dimethylaminobenzonitrile and its derivatives [10, 11]), intramolecular proton transfer (molecules from the families of 3 hydroxyflavone and 3 hydroxy chromone, 3 hydroxyquinolone, etc.), and hydrogen bond formation. Seemingly, these reactions also include the formation of a triplet metastable state of complex molecules and a number of other classical reactions such as, e.g., cistrans isomerization. The obtained conclusions were used to process experimental data for solutions of various organic compounds, including molecules of the 3 hydroxyfla vone family, in the excited state of which internal pro ton transfer reactions are observed upon excitation of fluorescence via different singlet states. The structural formula of this organic compound is presented in Fig. 2. EXPERIMENTAL DATA AND DISCUSSION We used solutions of 3 hydroxyflavone (Indofile Chemical Co., additionally doubly purified by recrys

tallization) in acetonitrile (Scharlau, pure grade, for UVVIS spectroscopy) with a concentration of 10 5 M. As a quencher, we used potassium iodide and TEMPO (2,2,6,6 tetramethylpiperidinyl 1 oxy, reagent grade, Aldrich; spin quencher) without addi tional purification. The absorption and emission spec tra of quencher solutions were monitored in pure sol vents to take into account possible absorption and emission in fluorescence ranges of dyes under study. Fluorescence and fluorescence excitation spectra were recorded on a Hitachi F 2500 spectrofluorimeter, and absorption spectra were measured using a Hitachi U 2810 spectrophotometer. Usually, a cell with a sample was thermostatted and luminescence was registered with a standard 90 scheme with respect to the excita tion beam. Figure 2 shows the fluorescence spectra of 3 hydroxyflavone in acetonitrile obtained upon excita tion by ultraviolet radiation at a wavelength of = 340 nm. The first band with a maximum near 385 nm belongs to the N form, and the long wavelength (green) near 525 nm is attributed to the state S1P of a tautomeric form, which is a photoproduct of the nor mal form, which arises upon photoexcitation. The tautomer is formed from the initial molecule as a result of the passage of a proton of the carboxyl group to the oxygen atom of this group, because this configuration of 3 hydroxyflavone is energetically more favorable. This description of the dual fluorescence of 3 hydrox yflavone and its mechanism were given for the first
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time in [17, 18] and have been confirmed in a number of works [13, 14]. The reaction was termed the reac tion of internal proton transfer. The action of the quencher leads to considerable quenching of the emission intensity of the two bands, with neither the fluorescence contour nor its position experiencing noticeable changes. The quenching effect is more pronounced for the green band; there fore, the ratio IN/I continuously increases at all con centrations of the quencher (see Fig. 2, insets (a) for TEMPO and (b) for KJ.). For the TEMPO quencher, the ratio IN/I linearly increases with the quencher concentration in the range 01.2 102 M and, for the KJ quencher, this ratio increases almost linearly in the range 03.0 102 M. In accordance with the theory, these data indicate that, in the excited state of 3 hydroxyflavone, a kinetic internal proton transfer reaction proceeds. This conclusion is also consistent (i) with the data of kinetic measurements of the fluo rescence decay of the two bands; (ii) with a large spec tral spacing between the two fluorescence bands; and (iii) with a small ratio of their intensities, which points to a high reaction rate of the internal proton transfer [13, 14]. Processing of the experimental data presented in Fig. 2 makes it possible to calculate the SternVolmer constant. An increase in temperature to 80 is accompanied by a considerable increase in the Stern Volmer constant, from 13 to 87.6 1 for the short wavelength fluorescence band [15]. To explain this dependence, let us consider the physical meaning of the SternVolmer constant and recall how it is defined and with which it is related. According to the classical notions, this constant characterizes the number of contacts of an excited molecule with molecules of an impurity (quencher) per unit time. During the con tact, the luminophore molecule loses its excitation energy and returns to the ground state. The energy transferred to the quencher is nonradiatively scattered in the solution. The temperature dependence of the N N = 0 constant kSV N k Q and, therefore, of the constant N of bimolecular collisions is directly related prima kQ rily with an increase in the diffusion coefficient according to (13), which shows that the diffusion enhances with temperature faster than according to a linear law because of the temperature dependence of the viscosity. Therefore, under the steady state mea surement conditions, an energy excess of the molecu lar motion in the solution (heating or an excitation energy excess) should be rather well sensed by solu tion particles and strongly affect the collision fre quency of luminophore molecules with other solution particles. (To complete the picture, we note that the lifetime of the excited state can also vary with temper ature.) Upon heating from 20 to 80, the Stern Volmer constants calculated directly from the fluores cence intensities of the tautomeric band also increase
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(from 30.9 to 52 M1) and do not differ strongly from these constants for the main UV band of the fluoro phore. We note here that this calculation is only esti mative, whereas, to correctly determine this quantity, the method described in [15] should be used. The next experiment shows the possibility of iden tifying internal proton transfer reaction of the thermo dynamic type. Figure 3 presents the fluorescence spectra of an acetonitrile solution of 4' (diethy lamino) 3 hydroxyflavone. It can be seen from this figure that the irradiation of the solution is accompa nied by the appearance of two fluorescence bands with maxima at 506 (short wavelength band) and 572 nm (long wavelength band). The first band is more intense than the second one and, in accordance with the scheme of Fig. 1, is identified as the fluorescence of the normal form. The second fluorescence band is attributed to the S1P state of the tautomeric form, which is a photoproduct of the normal form, arising upon photoexcitation. The positions of the two bands correspond to those previously observed in the spectra of this compound [19, 20]. The band of the tautomer is shifted with respect to the normal spectrum by 66 nm, which is half that of its structural analog, 3 hydroxyflavone, in the same solvent. The tautomer has the same chemical formula as the initial parent molecule, and, as in the case of 3 hydroxyflavone, the difference is related to the proton transfer from the hydroxyl group of the molecule to the oxygen atom of the carbonyl group. As distinct from 3 hydroxyfla vone, for 4 (diethylamino) 3 hydroxyflavone, the intensity ratio of the two bands IN/I is greater than unity, which can be a result of a high rate k of the reverse proton transfer reaction and, consequently, of the thermodynamic character of this reaction. Using the same solution, we performed experiments on fluo rescence quenching by the TEMPO spin quencher and the results of the measurement of the ratio IN/I as a function of the quencher concentration are shown on the top of Fig. 3. It can be seen that, in this case, the band intensity ratio does not depend on the degree of quenching, which indicates that, for this solution, in the excited state, conditions for the satisfaction of relation (14) between the band intensities (i.e., the independence on the degree of quenching) are imple mented, the condition of which is the thermodynamic character of the reaction of internal proton transfer in the excited state. The data of independent kinetic measurements [16] also indicate that the character of this reaction in these compounds is also thermody namic. Therefore, the results of experiments on fluo rescence quenching in solutions of 3 hydroxyflavone and 4 (diethylamino) 3 hydroxyflavone make it pos sible to conclude that, based on conclusions of model calculations presented in the first part of this work, the character of the internal proton transfer reaction can be determined rather simply by steady state spectros copy methods.

48 Ifl 105, pulses

TOMIN

IN/IP 0.4

O OH

0.3 0.2 0.1

O

4 0

4 Q, mM

C2H5 N C2H5

N 0 400 500

P 600 700 , nm

Fig. 3. Fluorescence spectra of solutions of 4 (diethylamino) 3 hydroxyflavone in acetonitrile (the excitation wavelength is 390 nm). The inset shows the temperature dependence of the intensity ratio IN/I of the normal and tautomeric forms.

Let us show that these conclusions can also be made based on experiments on the temperature fluo rescence quenching in such objects, as was substanti ated in the first part of this work. Figure 4 presents intensity ratios of the fluorescence bands of 3 hydrox yflavone and 4 (diethylamino) 3 hydroxyflavone molecules obtained for different temperatures from temperature experiments.
IN/IP 0.06 0.04 0.02 0 1.6 1.5 1.4 1.3 0 30 50 T, C 70 90 ()

As was previously mentioned, the different probes chosen for investigation were previously well studied by kinetic laser spectroscopy methods and the type of the reaction that proceeds in each of them was deter mined. The experimental investigations performed showed that, for probes with a reaction of the kinetic type, such as 3 hydroxyflavone, the intensity ratio between the fluorescence bands increases according to a law that weakly differs from the linear behavior (Fig. 4a). At room temperature, the band intensity ratio IN/I for 3 hydroxyflavone molecules is substan tially smaller than unity, 0.028, which indicates that the proton transfer reaction rate k+ is high. The data of Fig. 4b show that, in contrast to 3 hydroxyflavone, the band intensity ratio IN/I for 4 (diethylamino) 3 hydroxyflavone molecules is greater than unity, which alone indicates that the rate constant k of the reverse proton transfer reaction is high and, therefore, can be indicative of the thermodynamic character of the proton transfer reaction, which occurs according to the scheme presented in Fig. 1a with a low barrier between the N and states and a high rate k of the reverse reaction. This conclusion is also sup ported by the data of kinetic studies with a high time resolution and fluorescence excitation with ultrashort laser pulses. The fluorescence decay laws of the two forms have identical time components 38 and 310 ps (for ethyl acetate), which is indicative of an intense energy exchange between both forms in the excited state [21, 22]. Upon heating the solution, the mea sured values of the band intensity ratio IN/I dot increase, but, rather, slightly decrease. At the same time, either of these bands, as is always the case with
OPTICS AND SPECTROSCOPY Vol. 113 No. 1 2012

30

50 (b)

70

90

Fig. 4. Temperature dependences of the fluorescence bands intensity ratio IN/IP for solutions of (a) 3 hydroxy flavone and (b) 4 (diethylamino) 3 hydroxyflavone in acetonitrile. For 3 hydroxyflavone, the dependences are given for two excitation wavelengths: (+) 305 and () 340 nm.

FLUORESCENCE QUENCHING AS A METHOD

49

organic molecules, is rather efficiently quenched [23]. A small decrease in the ratio IN/IT for 4 (diethy lamino) 3 hydroxyflavone with increasing tempera ture is most likely related to an increase in the rate of the proton transfer reaction. In accordance with the dependences of Fig. 4, the temperature quenching strongly changes the intensity distribution for the reaction of the kinetic type (3 hydroxyflavone), increasing it, whereas, for reactions of the thermodynamic character (4 (diethylamino) 3 hydroxyflavone), this ratio remains unchanged, which is consistent with the conclusions obtained pre viously. The method can be used for comparatively simple express selection of molecular probes, which are candidates for new applications. The natural fluo rescence quenching process with heating (temperature quenching) proceeds in almost all molecular systems, under this approach, there is no necessity to introduce additional substances into the sample as a quencher. This method is especially valuable for studies of deli cate systems and objects, such as, e.g., living cells, for which the introduction of a quencher is difficult or impossible. In this case, in order to determine the mechanism of the reaction of a probe under examina tion, it suffices to record the spectra of dual fluores cence at several temperatures in a rather narrow and easily available temperature range between room tem perature and 6080. Therefore, the presented results of verifying the method using molecules of the 3 hydroxyflavone familythe parent 3 hydroxyfla vone molecule and 4 (diethylamino) 3 hydroxyfla voneas well as the data from [21] for solutions of 1 methyl 2 (4 methoxy)phenyl 3 hydroxy 4(1H) qui nolone (QMOM), which were studied by other inde pendent methods and the character of the reaction for which is well known, indicate that the obtained tem perature dependences allow one to reliably identify the character of the internal proton transfer reaction in this system. We note that, using temperature dependences sim ilar to those presented in Fig. 4a, one can estimate the probability of temperature quenching for kinetic reac tions, as was done in [21] for the particular case of QMOM molecules. Upon heating this dye from room temperature to 80, the value of k(t0) was determined to be 3 108 s1. This is a rather large quantity, and it is contributed by intramolecular quenching mecha nisms, by solvent quenching, and by molecular oxygen, which is always contained in the solution. The contri bution from the latter factor can also be estimated, since the bimolecular quenching constant for it is known, and, at room temperature, it is 1010 L M1 s1. Under normal conditions, the concentration of oxy gen is about 103 M and, in this case, the product of the concentration into the bimolecular quenching constant will be 107 s1 [2, 3], which is 30 times smaller than the determined value of the probability k(t 0). Therefore, we can conclude that, most likely, an
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Ka, Ifl 1.0 () 0.8 0.6 0.4 0.2 0 300 1.0 0.8 0.6 0.4 0.2 0 400 500 , nm 600 700 400 1 2 3 500 600 (b) 700 1 2 3

Fig. 5. Fluorescence spectra of solutions of: (a) 3 ami nophthalimide (1) in water and (2) the same solution with an addition of a quencher (potassium iodide, C = 2.6 M); (b) 3 aminophthalimide (1) in ethanol upon excitation in the fundamental absorption band and (2) upon irradiation by quenching light from a ruby laser at the fundamental frequency.

increase in the probability of temperature quenching of the tautomer of QMOM and the solution close to it is related to intramolecular channels and energy exchange processes with nearest solvent molecules. Similar conclusions can also be applied to solutions of 3 hydroxyflavone, because these are molecules from the same family and their characteristics and temper ature dependences do not differ greatly. In principle, using different variants of the diffu sion theory of bimolecular collisions, one of which gives relation (12) for the bimolecular quenching con stant, the obtained data make it possible to calculate microlocal values of diffusion coefficients under dif ferent excitation conditions based on data on the quenching of dual fluorescence. We also note that the application of quenchers can yield useful information on the character of processes in excited states in such systems in which a photophys ical reaction or process proceed with no barrier and has an irreversible character. Photophysical reactions of this kind can be exemplified by processes in polar solutions with fluorophores, the electric dipole moment of which changes as a result of an electronic transition [2426]. In these solutions, during the life time of the excited state, a continuous Stokes shift of the fluorescence spectrum is observed. Figure 5 pre sents data on the fluorescence quenching in solutions

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TOMIN

of 3 aminophthalimide. The quenching leads not only to a considerable decrease in the fluorescence yield, but also to a short wavelength shift of the spectrum as a whole. The dipole moment of 3 aminophthalimide molecules experiences a considerable change in the electric dipole moment upon excitation; namely, this moment changes from 2.6 D in the ground state to 4.9 D in the excited state, to which molecules pass prior to the fluorescence event [24, 25]. Changes in the magnitude and orientation of the dipole violate the equilibrium electric configuration, which is formed by fluorophore molecules in the ground state and by dipole moments of solvent molecules in the immediate environment. This violation initiates a molecular rear rangement process or the intermolecular relaxation process in the fluorophore solvate. To a good approxi mation, this process has a barrierless character and, conventionally, can be represented by a transition of the solvate to an equilibrium state with a lower energy in the system of continuous orientational sublevels on the scheme of Fig. 1c. For molecules in a strongly polar solvent, the electric dipole moments of which considerably change as a result of the electronic tran sition, the Stokes shift is especially strongly pro nounced, causing considerable long wavelength shifts of steady state spectra (sometimes, by several thou sands of reciprocal centimeters, i.e., almost the same shift as that observed as a result of the proton transfer reaction in 3 hydroxyflavone) or time long wave length shifts of instantaneous fluorescence spectra that proceed during the intermolecular relaxation times in the excited state. If the lifetime is decreased by, e.g., a quencher or with a laser field, the shift will be decreased. This pattern is shown in Fig. 5. Upon quenching by a diffusion quencher, potassium iodide, at a concentration of 2.6 M of an aqueous solution of 3 aminophthalimide, as can be seen from Fig. 5a, a short wavelength shift of the fluorescence spectrum by almost 8 nm is observed, whereas the shape of the spectrum during the shift changes weakly. This effect can be obtained using a different method of quenching by means of induced transitions in a field of laser radiation. Figure 5b presents the spectra of 3 aminophthalimide in ethanol that were obtained upon excitation by the second harmonic of a ruby laser at a wavelength of 347 nm and upon the simultaneous action of radiation at the fundamental frequency with a wavelength of 694 nm, which falls into the range of the long wavelength wing of the broad fluorescence band. As can be seen from this fig ure, the quenching action of the fundamental fre quency leads to a short wavelength shift by 8.5 nm. Therefore, irrespective of the method of quenching in polar solutions, the degree of the Stokes shift of the fluorescence spectrum can be decreased. Interestingly, changes in the spectral properties of fluorescence also cause corresponding transformations of amplification spectra (and, consequently, the possible frequency range in which lasing conditions are fulfilled) of active

solutions; therefore, in the regime of lasing of, e.g., dye solutions, the shortening of the lifetime of the excited state due to induced transitions in the field of laser radiation also leads to a shift of lasing spectra as a result of amplification of shorter wavelength forms. These phenomena were observed in [27] in glycerol solutions of 3 dimethylamino 6 monomethylamino N methylphthalimide. Using these experiments in polar solutions, one can determine intermolecular relaxation times and, in the cases where they are known (these data are scarce), the probabilities of bimolecular quenching or the probabilities of induced transitions in the light field for particular systems. Finally, let us describe another interesting and practically useful application of the method of fluores cence quenching by impurities for obtaining a more pronounced spectrum of dual fluorescence in the case in which it is overlapped with the fluorescence spec trum of another form. This situation is characteristic of solutions of molecules of the families of 3 hydroxy flavones and 3 hydroxyquinolones, when solutions or interstitial matrices contain sites with proton acceptor properties or solvents are characterized by consider able values of the KamletTaft constant , which is responsible for the proton acceptor properties of sol vent molecules. In this case, as is known, an anionic form of initial molecules is formed, which exhibits intense fluorescence the spectrum of which is located between the two fluorescence bands, which impedes the use of the signal of these probes for the study of some important objects. This situation cannot be obviated in studies of, e.g., various proteins and membranes. Problems caused by the signal from this form manifest themselves espe cially significantly upon application of selective exci tation methods, in studies of edge excitation effects, and in analysis of the fluorescence decay kinetics of the basic probe. We will illustrate one of the possibili ties of suppressing this interfering signal in a solution of 3 hydroxyflavone. Figure 6 shows fluorescence signals from 3 hydroxyflavone in acetonitrile that were obtained at 20, 50, and 80 upon UV irradiation at a wavelength of 340 nm of (a) pure solvent and (b) with an addition of the TEMPO quencher. At room temperature, the fluorescence spectrum consists of a short wavelength blue band (N form) and a green, more intense, band with a maximum near 525 nm, which belongs to the tautomer. An increase in temperature is accompanied by the occurrence of another band, which is located near 450 nm and which is overlapped with the fluores cence of the normal and tautomeric forms. As the temperature is increased, the intensity of this band increases and, at 80C, it already appreciably exceeds the fluorescence intensity of the normal form. This radiation belongs to the anionic form of 3 hydroxyfla vone molecules, which is created in the ground state as a result of detachment of a proton from the hydroxyl
OPTICS AND SPECTROSCOPY Vol. 113 No. 1 2012

FLUORESCENCE QUENCHING AS A METHOD Ifl, rel. units ()

51

8 6

2 4 2 0 350 8 (b) 6 1 2 N* 450 24 3 3 P* 550

22

2 N* 0 350 450 P* 550

, nm

Fig. 6. Fluorescence spectra of solutions of 3 hydroxyflavone in acetonitrile (C = 105 M) recorded at temperatures of (1) 20, (2) 50, and (3) 80C in (a) pure solvent and (b) with an addition of the TEMPO quencher at a concentration of 5 103 M at the same temperatures. The arrows show the behavior of the spectra with increasing temperature.

group and its passage into the solvent. The fluores cence of this form was thoroughly studied; it can be easily distinguished if the excitation was chosen cor rectly; its lifetimes were measured, which, for 3 hydroxyflavone, amount to 24 ns (depending on the type of the solvent) [28]. Introduction of the TEMPO quencher in a rather small concentration of 5 103 M (Fig. 6c) completely suppresses this form and does not appear even at a temperature of 80; now, the signal of the dual fluorescence of 3 hydroxyflavone is observed in the pure form. This result is physically clear, since the lifetime of the normal form is very short and, therefore, the SternVolmer constants for the quenching of the A form will be considerably higher than for the normal form. The lifetime of the tautomer is also shorter, being about 1 ns; in addition, its fluo rescence is much more intense than the remaining forms. Therefore, in solutions with the quencher, it is possible to measure the fluorescence of the N form with a higher accuracy and, consequently, the inten
OPTICS AND SPECTROSCOPY Vol. 113 No. 1 2012

sity ratio IN/I, which is one of the basic characteris tics of probes with dual fluorescence. A detailed description of these experiments was given in our work [29]. ACKNOWLEDGMENTS This study was supported by Pomeranian Univer sity, S l upsk, Poland, grant no. BW 6/20/05. REFERENCES
1. R. P. Haugland, Handbook of Fluorescent Probes and Research Products, 10th ed. (Molecular Probes, Eugene, OR, 2003). 2. J. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, New York, 2006). 3. B. Valeur, Molecular Fluorescence: Principles and Appli cations, 4th ed. (Wiley VCH, Weinheim, 2007).

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TOMIN 17. P. K. Sengupta and M. Kasha, Chem. Phys. Lett. 68, 382 (1979). 18. M. Kasha, J. Chem. Soc., Faraday Trans. 282, 2379 (1986). 19. A. S. Klymchenko, V. G. Pivovarenko, and A. P. Dem chenko, J. Phys. Chem. A 107, 4211 (2003). 20. A. S. Klymchenko and A. P. Demchenko, Phys. Chem. Chem. Phys. 5, 461 (2003). 21. V. I. Tomin, R. Jaworski, and D. A. Yushchenko, Opt. Spectrosc. 109 (3), 336 (2010). 22. D. A. Yushchenko, V. V. Shvadchak, M. D. Bilokin, A. S. Klymchenko, G. Duportail, Y. Mely, and V. G. Pivovarenko, Photochem. Photobiol. 5, 1038 (2006). 23. V. I. Tomin and R. Jaworski, Opt. Spectrosc. 109 (2), 279 (2010). 24. N. G. Bakhshiev, Photophysics of DipoleDipole Inter actions (S. Peterb. Gos. Univ., St. Petersburg, 2005) [in Russian]. 25. N. G. Bakhshiev, Spectroscopy of Intermolecular Inter actions (Nauka, Leningrad, 1970) [in Russian]. 26. N. A. Nemkovich, A. N. Rubinov, and V. I. Tomin, in Topics in Fluorescence Spectroscopy, Vol. 2: Principles, Ed. by J. R. Lakowicz (Plenum, New York, 1991), p. 367428. 27. A. V. Aristov, N. G. Bakhshiev, V. A. Kuzin, and I. V. Piterskaya, Opt. Spektrosk. 30, 143 (1971). 28. V. I. Tomin and R. Jaworski, Opt. Spectrosc. 103 (6), 952 (2007). 29. V. I. Tomin, Opt. Spectrosc. 106 (3), 355 (2009).

4. A. P. Demchenko, Introduction to Fluorescence Sensing (Springer, 2009). 5. V. I. Tomin, in Hydrogen Bonding and Transfer in the Excited State, Ed. by Ke Li Han and Guang Jiu Zhao (Wiley, New York). 6. V. I. Tomin, in Springer Series on Fluorescence, Methods and Applications. V.8. Advanced Fluorescence Reporters in Chemistry and Biology I. Fundamentals and Molecular Design, Ed. by A. Demchenko (Springer, New York, 2010), pp. 189223. 7. A. P. Demchenko, Anal. Biochem. 343, 1 (2005). 8. A. P. Demchenko, A. S. Klymchenko, V. G. Pivo varenko, S. Ercelen, and G. Duportail, J. Fluorescence 13, 291 (2003). 9. E. I. Kapinus, Photonics of Molecular Complexes (Naukova Dumka, Kiev, 1988) [in Russian]. 10. W. Rettig, V. Bonacic Koutecky, F. Heisel, and J. A. Miehe, Adv. Chem. Phys. 68, 1 (1987). 11. Z. R. Grabowsky, K. Rotkiewicz, and W. Rettig, Chem. Rev. 103, 3899 (2003). 12. A. P. Demchenko, in Fundamental Photoprocesses and Inhomogeneous Broadening of Electronic Spectra of Organic Molecules, Ed. by V. I. Tomin (Wyd. PAP, S l upsk, 2006), p. 79. 13. L. G. Arnaut and S. J. Formosinho, Photochem. Pho tobiol. A 75, 1 (1993). 14. L. G. Arnaut and S. J. Formosinho, Photochem. Pho tobiol. A 75, 21 (1993). 15. V. I. Tomin, Opt. Spectrosc. 105 (4), 496 (2008). 16. V. I. Tomin, S. Oncul, G. Smolarczyk, and A. P. Dem chenko, Chem. Phys. 342, 126 (2007).

Translated by V. Rogovoi

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Vol. 113

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2012