ISSN 0030-400X, Optics and Spectroscopy, 2008, Vol. 105, No. 2, pp. 217222. Pleiades Publishing, Ltd., 2008.

. Original Russian Text V.I. Tomin, 2008, published in Optika i Spektroskopiya, 2008, Vol. 105, No. 2, pp. 238244.

CONDENSED-MATTER SPECTROSCOPY

Dynamic Fluorescence Quenching and the Highest Excited States of Molecules

V. I. Tomin
Institute of Physics, Pomeranian Pedagogical University, S l upsk, 76-200 Poland e-mail: tomin@if.pap.edu.pl
Received January 22, 2008

AbstractThe dynamic uorescence quenching in organic molecules, or quenching of the second kind according to Vavilovs classication, is an efcient method of investigating excited states in solutions and is widely used in various elds. The effect of quenching on the intensity of the uorescence from the rst and higher singlet states of organic molecules is studied. The results may serve as a basis for determining the nature of the short-wavelength luminescence and can be used to distinguish the Sn uorescence from the comparably intense luminescence of impurities, which is a very important problem when investigating such emissions. A method for obtaining dynamic quenching by specially chosen quenchers is proposed. The method is based on an experimentally found strong increase in the constants of bimolecular collisions of luminophore and quencher molecules when the luminophore is excited through the highest singlet states. PACS numbers: 33.50.Dq DOI: 10.1134/S0030400X08080092

INTRODUCTION In recent years, various luminescent methods are extensively used not only in physics and chemistry, where they are traditional, but also in biology, medicine, biomedical optics, biophotonics, and diagnostics of processes in complex systems, including active living structures. To appreciate the spectrum of possible applications, it is sufcient to look through, for example, the latest issue of the catalogue of luminescing molecular probes [1]. The absorption and uorescence in complex molecules most frequently occur due to transitions between the ground (S0) and the rst excited (S1) singlet states. The S1 energy state is the nal state in the process of light absorption from the ground state and simultaneously is the initial level of uorescence both in vapors and in the condensed phase. In some cases, the uorescence can occur from the highest excited singlet states, but it is difcult to observe due to a very high probability of competing nonradiative transitions. This fact is directly reected in the empirical Vavilov law [2] and in the Kasha rule [3]. According to Vavilov, the luminescence quantum yield of organic molecules does not depend on the excitation wavelength, while the Kasha rule holds that the light emission in complex molecules always occurs from the lowest excited state of a given multiplicity. Satisfaction of these two laws implies that the rates knR of nonradiative transition from the Sn (n 2) state to the S1 level are more than three orders of magnitude higher than the radiative transition rates kR, which are usually 108 109 s1 [3, 4]. In addition, experimental observation of

such emission is rather difcult due to a low quantum yield and to the presence of some amount of impurities in available commercial materials and reagents that emit in the same short-wavelength region and require laborious and long procedures to get rid of them. The highest singlet states are comparatively poorly studied mainly because such investigations are rather complicated and can be performed only by a limited number of methods. In addition to the direct measurements of the uorescence spectra and quantum yields upon the single-phonon excitation of the highest states and to the measurements of the lifetimes using pico- and femtosecond spectroscopy, there are some original methods of investigation of the Sn uorescence. These methods include the stepwise excitation of Sn states, triplettriplet annihilation, spectroscopy of hole burning in the absorption spectra at low temperatures, measurement of the widths of spectral lines in the excitation spectra in supersonic jets, etc. [4]. As a rule, the Sn states have pico- and femtosecond lifetimes, because of which it is difcult to populate these levels high enough to observe their uorescence or induced absorption. At the same time, there are grounds to think that the knowledge of some of these states can be important from the fundamental viewpoint. In addition, such information can be very useful for certain applications. For example, photoreactions that are initiated from different excited states may proceed differently, because the reactivity and other physicochemical characteristics of excited states with different (bonding and antibonding) types of orbitals can be considerably different.

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TOMIN Sn knR2

S1

S0

Fig. 1. Energy level diagram of a complex molecule, illustrating transitions between vibronic levels.

The yield and lifetimes of the S2 uorescence of aromatic thiones were studied in [5, 6]. It was found that the intramolecular interconversion S2 S1 plays a key role in the decay of the S2 states in inert solvents (peruorohydrocarbons), while the intramolecular photophysical and photochemical processes dominate in more active solvents. These data directly indicate that the highest singlet states (higher then S1) can be strongly reactive. Various photophysical and photochemical processes in organic molecules, such as formation of excimers and exciplexes, electronic density redistribution, transitions to the triplet metastable states, a change in the molecule geometry, phototautomerization, etc., are directly related to the singlet S1 state. As a rule, investigations of these processes do not take into account the highest excited states. At the same time, as was shown in, for example, [5, 6], the role of the high singlet states is not restricted by the processes of intramolecular interconversion. Very frequently, both the initial excited molecules and the products of their photoreactions exhibit pronounced luminescence, and then the luminescent methods are very effective and are widely used in various elds of physics, chemistry, and biology. Recent publications demonstrate rather noticeable photoreactions occurring through the S2 singlet state in 3-hydroxyavone (3HF) [7, 8] and 2-butylamino-6methyl-4-nitropyridine N-oxide [9]. This stimulates interest in the processes related to the S2 states and in experimental methods of their investigation. An important process used to investigate the highest singlet states

is their uorescence, which most frequently has a very low quantum yield and, hence, is difcult to investigate experimentally, especially using standard steady-state devices (spectrouorimeters). In the days when the Kasha rule was being formulated, no uorescence of the Sn states was recorded (except for the uorescence in azulene), these days, owing to the progress in the development of modern methods of luminescence recording and to the new potentials provided by lasers, the luminescence from the S2 and S3 states was observed and studied in various molecular objects [4]. In this study, the effect of dynamic quenching on the intensity of the uorescence of the Sn and S1 excited singlet states is studied in general. The dependences of the uorescence intensity on the quencher concentration are studied, and it is shown that dynamic quenching can be successfully used to determine the nature of uorescence from the highest excited states and to identify the luminescence type, which is a topical problem when investigating such emissions. In addition, we propose a method to obtain quenching of the highest excited states by specially chosen materials. The method is based on the fact, experimentally found when investigating the 3HF uorescence, that the constants of bimolecular collisions of luminophore and quencher molecules strongly increase when the luminophore molecules are excited through the highest singlet states. MODEL Figure 1 shows the energy level diagram describing the uorescence from the rst (S1) and an arbitrary (Sn) singlet states. Let us consider the main transitions of light absorption and excitation energy relaxation in this scheme. After absorption of light with the frequency 1 in the main absorption band, the molecule passes from the ground S0 state to the excited S1 state, which is responsible for the long-wavelength uorescence band (the S1 S0 transition with the probability kR1). Nonradiative transitions with a rate of knR1 also occur from this state. Let the molecule also have a detectable uorescence from the state Sn and the solution have a foreign quenching substance with the concentration Q, which decreases the uorescence yield due to diffusion collisions. In the simplest case of the standard excitation of a uorophore in the rst absorption band by radiation with a density ue1, the detailed equilibrium equation for the S1 level can be written as B a u e 1 N 0 = ( k R 1 + k nR 1 + k Q Q ) N 1 , (1) where Ba is the Einstein absorption coefcient, N0 and N1 are the concentrations of luminophore molecules in the ground and the rst excited states, and kQQ is the diffusion quenching probability. Eq. (1) can be rewritten in the form N 1 = B a u e 1 N 0 / ( k R 1 + k nR 1 + k Q Q ) .
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knR1

kR1

3 kR2

(2)
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DYNAMIC FLUORESCENCE QUENCHING AND THE HIGHEST EXCITED STATES

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As is seen from this equation, the concentration of molecules in the excited singlet state is inversely proportional to the quencher concentration Q and the bimolecular quenching constant kQ. The intensity of the uorescence bands is directly proportional to the population of corresponding energy states and, hence, we can assume that the uorescence yield decreases as soon as the product kQQ becomes comparable with the sum of the radiative and nonradiative deactivation constants of the S1 excited state, kQQ kR1 + knR1. (2b) Formula (2a) reects the essence of the quenching process and allows one to easily obtain the well-known expressions [3] for the relative decrease in the uorescence yield and in the lifetime of the uorescing state (the SternVolmer law): B 0 / B = 1 + k SV Q , (3) where B0 is the uorescence yield in the absence of a quencher and kSV is the SternVolmer constant, which is related to the bimolecular quenching constant and the excited state lifetime 0 by the expression k SV = k Q .
0

cence. In this case, the balance equation will be written as B 2 u e 2 N 0 = ( k R 2 + k nR 2 + k Q 2 Q ) N 2 . (7) From this relation, the number of molecules in the excited state can be expressed as N 2 = B 2 u e 2 N 0 / ( k R 2 + k nR 2 + k Q 2 Q ) . (8) Similar to the previous case for the S1 uorescence, we can use the lifetime of uorescence from the second level S2 = 1/ ( k R 2 + k nR 2 + k Q 2 Q ) and rewrite (8) in the form N 2 = S2 B 2 u e 2 N 0 . (10) This relation is formally analogous to (2a), with the difference that a noticeable quenching of the second singlet level with the concentration N2 can be observed only at very high quencher concentrations Q. Indeed, the effect of the quenching factor kQQ can be detected only in the case of fulllment of the inequality k Q 2 Q k R 2 + k nR 2 . Usually, the lifetime of the uorescing state S2 does exceed several picoseconds and, hence, kR2 + knR2 ~ 1012 s1 [1, 2, 4]. For the diffusion mechanism of quenching, this means that the quenching effect will be noticeable in the case of very high quencher concentrations (Q ~ 102 M), which evidently cannot be reached in practice without changing the solution nature because the bimolecular quenching constant is kQ ~ 1010 M1 s1 [3]. At the same time, as is seen from (2b), the quenching of the S1 uorescence usually occurs at concentrations Q ~ 102 M because kR1 knR1 ~ 109 s1. Thus, it is obvious that, under the standard conditions of single-photon excitation, in the solutions of molecules uorescing from the S1 and Sn singlet levels, the quencher will affect only the long-wavelength uorescence S1 S0. Under the action of a quencher with a concentration Q ~ 102101 M, typical quenching in solutions with the Sn and S1 uorescence will have the following pattern. The quantum yield of the S1 uorescence and its lifetime will decrease with increasing quencher concentration Q, while the same characteristics of the Sn uorescence will remain unchanged. The physical meaning of such a pattern is clear. An extremely short lifetime of the Sn level does not allow the luminophore molecule to experience bimolecular collisions with quencher molecules in a number sufcient for uorescence quenching. When observing and studying the blue uorescence from the Sn state, it is important to distinguish this uorescence from the uorescence of impurities [4]. For this purpose, the method of dynamic quenching can be very helpful because it allows one to separate the uo(9)

(4)

Expressions (3) and (4) are widely and fruitfully used for investigating luminescing solutions. We will also use them in this study. The value in the parentheses in the denominator of expression (2a) can be considered as inverse to the experimentally found luminescence decay constant (lifetime) of the S1 level if it is excited by a pulse, S1 = 1/ ( k R 1 + k nR 1 + k Q Q ) . (5) Expression (5) is convenient because S1 can be determined by means of kinetic uorescent spectroscopy. Taking (5) into account, expression (2a) can be rewritten in the form N 1 = S1 B a u e 1 N 0 , (6) and, hence, the population of the uorescing state is proportional to its uorescent lifetime and to the excitation rate Baue1N0. From this relation, it is possible, in principle, to calculate the excited level population N1 from the known characteristics in the right-hand side (they all are comparatively easily measured or calculated from experimental data). Note that, for efciently luminescing luminophores, we can assume that kR1 ~ knR1 and, under usual experimental conditions, the number of transitions with quenching is comparable to the sum of radiative and nonradiative transitions or higher than it by a factor of several; i.e., condition (2b) is fullled. Let us complicate the problem and excite the uorophore (the probability B2u2) through a higher singlet state Sn (Fig. 1), which exhibits a detectable uoresOPTICS AND SPECTROSCOPY Vol. 105 No. 2 2008

220 ka, cm1 2 2

TOMIN

1
290 305 340

excited molecule with molecules of a foreign substance (quencher) per unit time. During the contact, the luminophore molecule loses the excitation energy and passes to the ground state. The energy transferred to the quencher nonradiatively scatters in the solution. Under the steady-state conditions, the excess excitation energy should be rather well sensed by the solution particles and strongly affect the diffusion coefcient, which is expressed by the formula [3] D = kT /6 ( T ) R . (11)

1 0 220 280 340 400 , nm

Fig. 2. Absorption spectra of the 3HF molecule in (1) pure acetonitrile and (2) acetonitrile with addition of TEMPO with a concentration of 5 103 M. The wavelengths used for the uorescence excitation (290, 305, and 340 nm) are shown by vertical arrows.

rescence of impurities (it will be quenched) and the uorescence of the nth excited singlet using simple experiments. The impurity uorescence quenching simplies recording of the Sn uorescence. Thus, the method of dynamic quenching can be used to investigate the Sn uorescence in solutions of organic molecules. QUENCHING OF THE HIGHEST EXCITED STATES It is desirable to nd a way of quenching of the highest excited states, which could be used as an additional method for investigating these states. As follows from the consideration in the previous section, such quenching can be obtained in the following ways: (i) by working with large concentrations of ordinary quenchers; (ii) by using specially chosen selective quenchers, for example, closely spaced acceptor pairs, which quench uorescence as a result of nonradiative energy transfer; (iii) by nding conditions for obtaining much higher SternVolmer constants. The rst way obviously is not feasible because the nature and physicochemical properties of solutions strongly change in the presence of high concentrations (110 M) of foreign substances. The second way is also not simple and requires laborious preliminary selection of appropriate acceptor pairs, which cannot always be found for a particular material due to some principle reasons, which will not be discussed here. However, we can try to achieve in practice the third way. To do this, let us turn to the physical meaning of the SternVolmer constant and recall how it is dened and to which is related. According to classical concepts, this constant characterizes the number of contacts of the

Here, T is the temperature; R is the collision radius, which is usually taken to be equal to the sum of the radii of the luminophore and quencher molecules; and (T) is the viscosity of the medium, which strongly decreases with increasing temperature for many liquid solvents. Thus, as is seen from (11), the diffusion coefcient should strongly depend on temperature. Upon excitation and relaxation of a molecule, some excess energy is always released in the environmental medium, which causes heating of the solvates. The instantaneous heating strongly depends on the excitation frequency and can be rather signicant (3050), but this thermal energy excess is rapidly scattered for times considerably shorter than the excited state lifetime and, hence, almost does affect the spectra. Therefore, the energy excess and its effect on the environment can be used only in the solvate characteristics that retain a longer response to heating. Additional mobility of molecules in solvates that absorbed a high energy will be retained for a time exceeding the time of heat transfer from the luminophore to the solvate shell (these times are comparable with the period of molecular vibrations). Thus, an increase in the diffusion coefcient in the solvate of a light-absorbing luminophore should be expected in the case of a rather high heat evolution, which increases with the excitation frequency. This effect can be rather simply detected by determining the SternVolmer constants kSV upon excitation through different Sn states. We performed such experiments on the uorescence of 3HF solutions excited into the S2 and S1 absorption bands and quenched by potassium iodide and 2,2,6,6tetramethyl-1-piperidinylooxy (TEMPO) spin quencher. The absorption spectra of the 3HF solution in acetonitrile are shown in Fig. 2 (the excitation wavelengths are shown by vertical arrows). Our experimental results are described in detail in [10, 11]. The TEMPO quencher was found to be the most efcient. Processing of the measured data on the uorescence of 3HF in solutions with addition of this quencher allowed us to determine the SternVolmer constant under different excitation conditions. These constants for different experimental temperatures are listed in Table 1. A very important circumstance is that these constants strongly increase with the excited level energy, i.e., with decreasing wavelength of the excitation light. As is seen from Table 1, the SternVolmer constant for excitation
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Table 1. SternVolmer constants for quenching of the fluorescence of 3HF in acetonitrile by TEMPO at different temperatures and excitation wavelengths Excitation wavelength, nm 290 305 340 SternVolmer constant, M1 t = 20C 430.05 61.55 4.65 t = 30C 355.47 80.59 27.22 t = 40C 447.47 101.93 34.07 t = 50C 351.45 74.83 46.81 t = 60C 756.84 229.77 87.92 t = 70C 526.45 184.57 92.45 t = 80C 595.77 249.24 87.57

Table 2. Calculated quencher concentrations necessary for quenching the highest excited states upon stepwise excitation Excitation band (wavelength, nm) S0 S2 (290) B/B0 1.1 1.05 1.01 1.1 1.05 1.01 1.1 1.05 1.01 kQ 109, l M1 s1 430 Q, M l1 0.1 n 109, s 2.32 103 1.16 103 0.23 103 1.62 102 0.81 102 1.62 103 2.15 1.09 101 2.15 101
0

Q, M l1 1

n 109, s 2.32 104 1.16 104 0.23 104 1.62 103 0.81 103 1.62 104 2.15 102 1.09 103 2.15 103

S0

S2 (305)

61.6

0.1

S0

S1 (340)

4.65

0.1

in the long-wavelength band is 13.3 M1 at room temperature and increases almost sevenfold as temperature increases to 80C. The excitation into the second absorption band (S2) yields 61.5 1 at T = 20, which then continuously increases to 249 1 at 80. Finally, the excitation at the shortest wavelength used in our experiment (290 nm), which corresponds to the edge of the S2 absorption band, yields even higher constants, 430 1 at room temperature and 596 1 at 80. From these data, we can estimate the constant of bimolecular quenching or of bimolecular collisions kQ at different temperatures, knowing its relation to the SternVolmer constant (4). Since the lifetime of the short-wavelength form of 3HF is ~1 ns, the kQ constant at room temperature is approximately 4.65 109 M1 s1. It is natural to assume that the lifetime remains the same upon the excitation through the higher absorption bands. Then, for excitation in the second band, this constant is an order of magnitude higher, kQ = 6.16 1010 M1 s1. At the excitation at the shortest wavelength, this constant will be even higher, increasing from ~4 1011 M1 s1 at room temperature to ~6 1011 M1 s1. Thus, we can see the direct effect of heat evolution in nonradiative transitions on the dynamics of molecules in the nearest environment of the luminophore. Note that the kQ constant is also affected by heating due to absorption in the quencher. As is seen from
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Fig. 2, the excitation wavelengths of 305 and 290 nm lie within the S2 absorption band, the absorption coefcient of 3HF decreases with decreasing wavelength, but the quencher begins efciently absorb in the region of 290 nm. Since the quencher does not uoresce, the total energy absorbed in it transforms into heat and stimulates additional thermal motion of quencher molecules. In principle, using the data obtained and the diffusion theory of bimolecular collisions, it is possible to calculate diffusion coefcients under different excitation conditions. The high constants of bimolecular contacts allow one to hope that the dynamic quenching can be realized using the TEMPO spin quencher or similar substances. Let us evaluate possible effects and see what they may cause. We performed corresponding calculations for the obtained constants kQ at room temperature (see Table 2). Is it seen that, even at room temperature and excitation at a wavelength of 290 nm, it is possible to easily obtain measurable (5% or B0 /B = 1.05) quenching of the uorescence yield at the quencher concentration of 101 M in the systems where the excited level lifetime is of the order of 1 ps. Using precision methods of uorescence recording, which can detect a 1% decrease in the uorescence signal, we can measure quenching of the states with even shorter lifetimes (~0.23 ps). In the cases when it is possible to use higher quencher concentrations (1 M), this effect will be an

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TOMIN

order of magnitude stronger and we can expect quenching of the states with even femtosecond lifetimes. The data given in Table 2 suggest the possibility of estimating the lifetimes of the Sn states from the quenching efciency using the SternVolmer relation (3) and formula (4) in the case of high SternVolmer constants kSV in the solution. This cannot be realized under conditions of ordinary single-photon excitation because the high kSV are related to the heat evolution in the Sn S1 channel, preceding the measurement event. In the case of the direct excitation S0 Sn and following the S1, recording of the uorescence in the channel Sn such heat evolution in uorescing solvates does not occur. However, the idea proposed can be realized using a scheme of combined excitation by two pulses with different frequencies or by stepwise excitation. First, a high Sn singlet is populated by a photon with the frequency 2 (Fig. 1) and then rapidly (as a rule, for a time shorter than 1 ns) relaxes to the S1 states, thus heating the excited solvate with the luminophore molecule in the core. In such a solvate, the diffusion motion of all molecules occurs according to the microcharacteristics of the heated state, which are different from the characteristics of the initial solvate before heating. Hence, it is these solvates that should be used for investigating the uorescence from the Sn state and they can be selected by exciting the Sn state once again, by the second photon with the frequency 3 directly from the S1 state. Then, the excited luminophore molecules in such solvates will interact with the quencher molecules according to the coefcients of bimolecular collisions, characteristic for the heated solvates. Thus, we can increase the rate of diffusion collisions (the constant of bimolecular contacts) of quencher molecules with the luminophore molecules in the excited Sn state, whose luminescence we want to study, and record the quenching of the Sn fluorescence. CONCLUSIONS The effect of quenching on the intensity of the uorescence from the rst and higher singlet states of organic molecules is studied. The relations obtained can be used for determining the nature of the shortwavelength emission and for separating the Sn uorescence from the comparably intense emission of impurities, which is important when investigating such uorescence.

It is experimentally found and substantiated that the SternVolmer constants are considerably higher when the uorescence is excited through the states higher than the rst excited singlet state. In particular, the SternVolmer constant at room temperature is 13.3 M1 in the case of excitation in the long-wavelength band and increases to 430 M1 for excitation through the second absorption band. A method of dynamic quenching of the highest excited states by specially chosen quenchers is proposed. The method is based on the use of the experimentally found strong increase in the constants of bimolecular contacts of the luminophore and quencher molecules, when the luminophore is excited through the highest singlet states. ACKNOWLEDGMENTS I acknowledge nancial support from the Pomeranian Pedagogical University (S l upsk) (project no. BW 6/20/06). REFERENCES
1. R. P. Haugland, Handbook of Fluorescent Probes and Research Products, 10th ed. (Molecular Probes, Eugene, OR, 2003). 2. S. I. Vavilov, Structure of Light in Collected Scientic Papers (Akad. Nauk SSSR, Moscow, 1952), Vol. 2 [in Russian]. 3. J. Lakowicz, Principles of Fluorescent Spectroscopy (Plenum, New York, 1984). 4. V. L. Ermolaev, Usp. Khim. 70, 471 (2001). 5. A. Maciejewski, D. R. Demmer, D. R. James, et al., J. Am. Chem. Soc. 107, 2831 (1985). 6. M. Lorenc, A. Maciejewski, M. Ziolek, et al., Chem. Phys. Lett. 346, 224 (2001). 7. V. I. Tomin and R. Yavorski, Opt. Spektrosk. 103 (5), 769 (2007). 8. V. I. Tomin and R. Jaworski, EPJ Special Topics. May, 123 (2007). 9. J. S. de Klerk, A. Szemik-Hojniak, F. Ariese, and C. Gooijer, J. Phys. Chem. A 111, 5828 (2007). 10. V. I. Tomin, Opt. Spectrosc. 104 (6), 838 (2008). 11. V. I. Tomin and G. Smolarczyk, Opt. Spectrosc. 104 (6), 832 (2008).

Translated by M. Basieva

OPTICS AND SPECTROSCOPY

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