- Email: [email protected]
Features of absorption and fluorescence spectra of prodan
Spectrochimica Acta Part A 68 (2007) 36–42
Features of absorption and fluorescence spectra of prodan Viktor Ya. Artukhov, Oksana M. Zharkova ∗ , Julia P. Morozova Tomsk State University, Russia, Tomsk-50, Lenina 36, Russian Federation Received 30 May 2006; received in revised form 12 October 2006; accepted 31 October 2006
Abstract Some important and essential features of absorption and fluorescence spectra of prodan in homogeneous and binary mixes are studied. According to results obtained from experimental and quantum-chemical researches we show that the absorption spectrum of prodan in nonpolar solvent within 25,000–50,000 cm−1 is formed by eight electronic transitions. Quantum-chemical calculations are performed in the geometry of both the ground and exited states of prodan. The rate constants of photoprocesses and the quantum yield of fluorescence are determined for the prodan and its complexes with water. A dramatic shift of the fluorescence band at changing from nonpolar solvent to isopropyl alcohol and water is explained. The roles of general solvent effects and specific interactions are separated. According to values of molecular electrostatic potential and charges on atoms the centers of possible interaction of prodan with a solvent are obtained. Possible models of prodan complexes in water are offered. The results of quantum-chemical calculation for offered complexes of prodan in water are compared with those for the free prodan molecule. The presence of the second band (about 24,000 cm−1 ) in fluorescence spectra of prodan in isopropyl (ethyl) alcohol–water solvents is explained. © 2006 Elsevier B.V. All rights reserved. Keywords: Prodan; Quantum yield; Fluorescence; Hydrogen bond; Ion hydroxonium
1. Introduction Fluorescence is used for a wide variety of biomedical and biophysical purposes. There are fluorophores which display high sensitivity to the polarity on the local environment. As an example can be selected prodan (6-propionyl-2dimethylaminnaphtalene) (Fig. 1) and its derivatives. Prodan can monitor the properties of the membrane surface. When bound to membranes, prodan and its derivatives display large spectral shifts at the membrane phase-transition temperature [1]. The attractiveness of using prodan in membrane studies resides in the possibility detecting the properties of the bilayer surface, eventually in parallel with the use of laurdan (6-lauroyl2-dimethylaminnaphtalene), that is located more deeply in the bilayer, by steady-state measurements of emission intensity [2]. Prodan may be useful as an uncharged fluorescent probe for the determination of surface hydrophobisity of proteins in the presence or absence of k-carrageenan and as a function of heating at different pH [3].
∗
Corresponding author at: 634028 Tomsk, Karpova st. 18–50, Russian Federation. Tel.: +8 3822 41 89 53. E-mail address: [email protected] (O.M. Zharkova). 1386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2006.10.048
Despite the wide use of prodan as fluorescent probe [1,3,4] there were no any systematic and profound studies to determinate nature of absorption bands, nature of fluorescent state, photophysical processes in a free prodan molecule, possible centers of prodan complexing with a solvent. In reference books only the long-wavel absorption band of prodan from 25,000 to 35,000 cm−1 is usually given [1]. Authors [5] compare experimental absorption spectrum of prodan recorded in the solvent cyclohexane from 25,000 to 45,000 cm−1 with the theoretical data obtained different methods (AM1/CISD, DFT/CISD, ZINDO/S). Location of the maximum of prodan fluorescence strongly depends on its surroundings. According to [6] a band of prodan fluorescence in reverse micelles system formed by sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) in heptane at different water additions has three according maxima at 390, 413, and 503 nm. It is due to the prodan molecules are distributed in three regions of the AOT reverse micelles [6]. The authors of [4] note that the prodan fluorescence maximum depends on a phase state of phospholipins. The double-band fluorescence was observed in small unilamellar vesicules composed of dipalmitoylphosphatidylcholine at 298 K [7]. Along with the prodan, as a fluorescent probe for investigation of a large series of biological objects, the laurdan (6-lauroyl-2-dimethylaminnaphtalene) is widely used [1,8–10].
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 1. Prodan.
The fluorescence spectrum of laurdan in glycerin contains two bands with maxima at 425 and 500 nm [11,12]. The strong dependence on excitation wavelength is observed for a spectrum. At short-wave excitation the blue component of spectrum gives considerable contribution to a spectrum of total fluorescence. The shift of excitation to the red region results in decreasing a role of blue component and more long-wave spectrum [12]. The fluorescence properties of both molecules are explained by existence of two states: locally excited LE and twisted internal charge transfer TICT [13]. According to [14], it is possible to talk of localized LE and TICT states very conditionally. This term is valid for very low temperatures only. At room and higher temperatures strong delocalization and overlap of these states take place. In our paper analysis of spectral-luminescence properties of prodan was realized on the basis of experimental and theoretical investigations. The interpretation of absorption spectrum of prodan in an inert solvent was performed. The rate constants of photoprocesses are determined for the prodan and its complexes with hydroxonium ion. Quantum-chemical calculations are performed in the geometry of both the ground and exited states of prodan. In work [15] is considered only ground state geometry of prodan. At that authors [15] offer different variants of nonplanar ground state geometry of prodan. In our paper the possible centers of interaction of the prodan with a solvent have been detected. The role of hydrogen bonding and nonspecific interactions in formation of fluorescent capacity of prodan have been studied. An estimation of contribution of general solvent effects in shift of absorption and fluorescence bands has been performed.
37
ter and SDL-2 spectrofluorometer respectively. For all emission measurements the excitation wavelength was 360 nm. All spectral measurements were carried out at the ambient temperature of 298 K. The quantum-chemical analysis of prodan and its complexes was performed by INDO method (intermediate neglect of differential overlap) [16,17]. The centers of interaction were selected using MEP method (molecular electrostatic potential) [17,18]. The geometric parameters of the molecule (bond lengths and angles) were taken as averaged over related compounds discussed in [19,20]. The structural parameters of our calculation almost coincide with data from [15]. Considering a significant negative effective charge on the nitrogen atom in the dimethylamino group we suggested that the methyl groups in the dimethylamino group are to deflect from the molecule plane through the angle of 15◦ . In paper [15] this deviation angle equals to 20.6◦ . 3. Results and discussion The interpretation of absorption spectrum was performed comparing results of an experimental research and quantumchemical calculation. Parametrization of the INDO method was made on a basis of experimental data in “inert” hydrocarbon solvents; therefore, results of absorption spectrum calculation should reproduce an experimental spectrum of prodan in isooctane. The Fig. 2 represent the experimental absorption spectrum of prodan in the solvent isooctane. This spectrum will be quite agreed an electronic spectrum from [5]. The long-wave absorption band of prodan in isooctane (Fig. 2) has poorly allowable vibrational structure and is characterized by a half-width (1/2 ) of 5000 cm−1 . The graphic partition of this band gives two bands with maxima at 27,500 and 30,500 cm−1 . The method of 2nd derivative gives three maxima, but one of them concerns to electronic-vibrational transition of S0 → S1 band. The quantumchemical calculation of a spectrum in this region has shown three electronic transitions (Fig. 2). The first state of the above three states is nπ* one (28,920 cm−1 ) while the last two are ππ* states (29,500 and 32,020 cm−1 ). The nπ* state (f (oscilla-
2. Experimental and computational details Prodan was obtained from Fluka. Fluorophore concentrations in the solutions were kept in the range of 2.2 × 10−6 –1.1 × 10−4 M. All using solvents were of spectroscopic grade and were obtained from joint-stock company Ekros. The solvents were used without further purification after confirming the absence of absorption and fluorescent impurities. Binary solutions were prepared using volumetric percent. Absorption and fluorescence emission measurement were carried out with the model SF-26 automatized spectrophotome-
Fig. 2. Absorption spectra of prodan (1.1 × 10−4 M) in isooctane. The digits on the diagram indicate results of quantum-chemical calculation.
38
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 3. The charges on atoms of free prodan molecule at the states S0 and S2 (ππ* ).
tor strength) = 10−6 ) in experimental absorption spectrum is not observed. Within 35,000–49,000 cm−1 spectral region the method of 2nd derivative exhibits five electronic transitions (Fig. 2). As a result of quantum-chemical calculation five electronic transitions are also obtained. The first two states of the above five are πσ * ones while the last three are ππ* states. Comparing results of quantum-chemical calculation and experimental data we have concluded that the spectral region from 35,000 up to 37,500 cm−1 is formed by only πσ * state, the spectral region from 37,500 up to 42,000 cm−1 is formed by one πσ * state and two ππ* states, and the spectral region from 42,000 up to 49,000 cm−1 is formed by one ππ* state. Thus, the absorption spectrum within 25,000–49,000 cm−1 is determined by eight electronic transitions (S0 → Si , i = 1–8). Let us mark that the results of our calculations on a position of energy levels and nature of states, will be quite agreed results of calculation of prodan by a ZINDO/S method [5]. The Fig. 3 represents the charges on atoms of free prodan molecule at the states S0 and S2 (ππ* ). Note, that at ππ* state the considerable charge redistribution from nitrogen atom (−0.097) to oxygen atom (−0.589) and to some atoms of an aromatic ring is observed. Thus, the S0 → S2 transition is determined by intramolecular charge transfer. Considerable decreasing of negative charge on nitrogen atom indicates that at ππ* state the
Fig. 4. Absorption spectra of prodan (1.1 × 10−4 M) in different solvents: isooctane (1), isopropyl alcohol (2), water (3).
interaction via dimethylamino group is less probable than at the ground state. The calculated data of prodan dipole moment at S0 , S1 (nπ* ), S2 (ππ* ) and S3 (ππ* ) states are 18.25 × 10−30 , 9.41 × 10−30 , 27.09 × 10−30 , and 34.03 × 10−30 C × m, respectively. The authors [5] have received a calculated data of prodan dipole moment at S1 (8.34 × 10−30 C × m), S2 (28.38 × 10−30 C × m) and S3 (50.04 × 10−30 C × m) states using ZINDO/S method. Changing from isooctane to isopropyl alcohol the shift to lower frequency region is observed in absorption spectra (Fig. 4). In spectral region from 22,000 to 35,000 cm−1 the shift is more of that in spectral region from 35,000 to 49,000 cm−1 with the half-width of this band being increased. Within 35,000–49,000 cm−1 region the redistribution of intensity is observed. Similar behavior of the shifts takes place in water. However to reveal structural features of this spectrum appeared to be impossible because of weak solubility of prodan in water. Changing from isooctane to isopropyl alcohol (Fig. 5) the shift of fluorescence band to lower frequency region (from 24,840 to 21,050 cm−1 ) and tenfold increase of intensity are observed. In water the shift is more considerable (from 24,840 to 19,320 cm−1 ) with fluorescence capacity being very weak (Fig. 5).
Fig. 5. Fluorescence spectra of prodan (1.1 × 10−5 M) in different solvents. (Inset: fluorescence spectrum of prodan in water.)
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
39
Fig. 7. Shift of band (evaluated from prodan fluorescence spectra for isopropyl alcohol–isooctane and isooctane–isopropyl alcohol mixtures) vs. concentration of solvent (isopropyl alcohol or isooctane).
Fig. 6. The scheme of energy levels and photoprocesses of free prodan with exited state geometry.
In order to explain the high sensitivity of prodan fluorescence to solvent it is necessary to calculate the geometry of molecule at an exited state. The method for definition of an exited state geometry is described in [21]. The greatest changes of electron density occur on the C1 –C2 , C3 –C4 , C5 –C6 , and C7 –C8 bonds (Fig. 1). Taking into account considerable negative charges on C3 (Q = −0.109) and C7 (Q = −0.076) carbon atoms of aromatic ring we further assumed that these two atoms can partially come from the sp2 -hybridization to sp3 one, that is, go outside the plane of aromatic part of the molecule. The declination of the C3 and C7 atoms from the plane is 0.28 and ˚ respectively. According to our suppositions this changed 0.26 A, geometry corresponds to exited state geometry (equilibrium) of prodan. The results of quantum-chemical calculation of prodan with exited state geometry are shown in Fig. 6. At transition to this geometry the significant shift of ππ* states to lower frequency region and small shift of nπ* state to higher frequency region are observed. Thus at this geometry S1 state is ππ* state. The quantum yield of fluorescence from S1 (ππ* ) state is estimated to be of 0.07 [21]. This calculation allowed us to describe an experimental fluorescence spectrum of prodan in isooctane (Fig. 5). The shift in absorption spectra and dramatic shift in fluorescence spectra at changing from nonpolar to polar solvent are not explained only by large difference in the dipole moment of the ground and exited states as demonstrated [1]. In Table 1 the
experimental shifts of absorption and fluorescence bands and the shifts estimated according to general solvent effects [1,22] are shown. Note, that the contribution of general solvent effects to the shift of bands is no more than 30%. Therefore, the remaining shift is caused by specific interactions. An influence of dielectric permittivity () on a band shift is confirmed by the fact of the prodan fluorescence band shift for isopropyl alcohol–isooctane solvents (Fig. 7). On addition of isooctane to the solution of prodan in isopropyl alcohol (less than 30%) the shift of the maximum of fluorescence band is small. With increasing of isooctane concentration up to 90% the shift ups to 26%. Therefore, the contribution of dielectric permittivity to shift of a band is comparable with the data represented in [1] (Table 1). On addition of isopropyl alcohol (5%) to the solution of prodan in isooctane the shift of the band is 40%. The value in this case has changed by 30%, therefore, the shift is caused by not only specific interactions but also by dielectric permittivity of environment. Indicated alcohol concentration corresponds to small value of dielectric permittivity. Note, that at smalls the function f() changes especially dramatically [23]. The contribution of general effects of the solvent at changing to isopropyl alcohol gives the shift of 770 cm−1 (Table 1) that is certainly higher than that obtained even at 5% alcohol concentration. On a basis of the data [1] we have received maxima of the prodan fluorescence band max at various polarity parameters f() (Fig. 8). This dependence appeared to be non-linear. The MEP calculations show that prodan at the ground state has two proton-acceptor centers (Fig. 9a). The strongest one is
Table 1 Shifts of absorption and fluorescence bands of prodan Solvent
abs , experiment (cm−1 )
abs , general solvent effectsa (cm−1 )
fl , experiment (cm−1 )
fl , general solvent effectsa (cm−1 )
Isooctane–isopropyl alcohol Isooctane–water
1300 1000
410 430
3790 5520
770 800
a ␣ (Onsager sphere radius) = 5.6 A. ˚ The dipole moments of prodan in ground state (18.35 × 10−30 C × m) and exited state (34.03 × 10−30 C × m) are taken from quantum-chemical calculation, realized by us.
40
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 8. Maxima of fluorescence bands (νmax ) of prodan in different solvents vs. f(ε).
related to the oxygen atom of the carbonyl group and is localized in the molecule plane. The second one is related to the nitrogen atom of dimethylamino group and is localized lower ˚ At S1 n* state only the minimum the molecule plane by 1.5 A. related to nitrogen atom of dimethylamino group is observed (Fig. 9b). At S2 and S3 ππ* states the MEP minima are observed near oxygen atom of carbonyl group and near 3rd, 19th and 20th carbon atoms of aromatic ring and hydrocarbon chain (Fig. 9c). The MEP results for exited state geometry of prodan (S1 (ππ* )) have also shown the minima relating to the oxygen atom of C=O group and carbon atoms of hydrocarbon chain. Thus the prodan molecule in equilibrium state is possible to interact with a solvent via several centers. The prodan contains hydrophylic and hydrophobic parts. The hydrophylic part of prodan involve two proton-acceptor centers. According to MEP results the interaction with a solvent (alcohol, water) via these centers is possible through hydrogen bonding. The MEP results also show that an interaction via hydrophobic part of prodan (hydrocarbon chain) is possible too. Kessler and Zaycev [24] have shown that between solvent and solute molecules the interaction is possible without charge transfer and formation of a chemical bond that is called the solvophobic or hydrophobic interaction. That is the hydrophobic interaction via hydrocarbon chain of prodan is possible. As it was noted, the MEP minimum is also observed near the 3rd carbon atom of aromatic ring. However, via this center the interactions both hydrogen bonding type and van der Waals type are possible; therefore, it is difficult to relate this center either to hydrophobic or to hydrophylic part of a molecule. Returning to the Fig. 8 we should note that water and alcohols correspond to complexes with a hydrogen bonding. In prodan
Fig. 10. Fluorescence spectra of prodan (1.1 × 10−5 M) in isooctane–isopropyl alcohol mixture.
with acetone or acetonitrile the hydrogen bonding is possible to be formed but considerably weaker than with water or alcohol. There are different solute-environment space configurations which contribute to a band broadening. The authors [25] call this phenomenon as inhomogeneous broadening. Probably such configurations are complexes formed both via carbonyl group and via carbon atoms. We have estimated the value of inhomogeneous broadening by formula from [25]. The values obtained for prodan in isooctane, isopropyl alcohol, and water are 310, 465, and 495 cm−1 , respectively. In binary solutions of isopropyl alcohol in isooctane (Fig. 10) at isopropyl alcohol concentration from 5 to 90% the fluorescence increases from 2 to 7 times in intensity. On addition of isopropyl alcohol (5–90%) to solution of prodan in isooctane the curves intersect at 22,900 cm−1 . The intersection point shows that the fluorophore is in various environment. According to MEP results the interaction with alcohol at ππ* exited state both via carbonyl group and via carbon atoms of prodan is possible. The most deep minimum is observed near the carbonyl group that means this interaction to be determinant. The prodan complex via other centers is likely to give a weaker fluorescence. Therefore, complex of prodan via carbonyl group gives a modest contribution in fluorescence increasing. Let us consider the prodan in the solvents: isopropyl alcohol–water and ethyl alcohol–water. On addition to the prodan solutions in alcohol less than 90% of water (the water is more strong donor of protons) small decreasing of optical density (about 10%) in the first absorption band was observed. At 90% water concentration in the solution the value of optical density decreased by 4 times and the maxi-
˚ (b) S1 (np* ), z = −1,5 A; ˚ (c) S1 (pp* ) (exited state geometry), z = −1,5 A. ˚ (z) distance Fig. 9. Minima obtained by a MEP method for a prodan: (a) S0 , z = −1.5 A; from plane of prodan.
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 11. Fluorescence spectra of prodan (1.1 × 10−5 M) in isopropyl alcohol– water mixture.
mum of absorption band shifted to a higher frequency region by 500 cm−1 . The analysis of fluorescence spectra obtained on addition of different water concentrations to prodan solution in isopropyl (ethyl) alcohol has shown that the shift of the band (about 2000 cm−1 ) to higher frequency region and fluorescence quenching (at 90% of water) by 8 times are observed (Fig. 11). Besides, at 90% water concentration the second band is observed near 24,000 cm−1 (Fig. 11). It occurs in a case of both isopropyl alcohol and ethyl alcohol. To explain the results obtained we consider MEP data. In the fluorescent state the alcohol molecule interacts through all possible centers detected by MEP method. Being added to solution water substitutes alcohol through all centers. We refute the supposition that the shift of a band to lower frequency region is caused by the effect of dielectric permittivity only. It is the case because the contribution of general solvent effects to the band shift, according to [1], at changing from isopropyl alcohol to water is 50 cm−1 . Shift of the fluorescence band to lower frequency region and appearance of the second band at 24,000 cm−1 we have assigned to the interaction both via the oxygen of carbonyl group and via the carbon atom of aromatic ring of prodan. As the consideration of specific interactions with water molecules via selected centers has not allowed us to reproduce our experimental data. Therefore, the necessity of new model of a complex is appeared. According to [26] there are ionic forms H3 O+ , H5 O2 + , and H9 O4 + in water, the most stable of them being H5 O2 + . In the case of solution of the organic proton-acceptor molecule (B) in water there occurs a substitution reaction: the water molecule in H5 O2 + is submitted by the molecule B, that is, B forms a complex with H3 O+ : (H2 O· · ·H· · ·OH2 )+ + B ↔ (H2 O· · ·H· · ·B)+ + H2 O.
41
Fig. 12. The scheme of energy levels and photoprocesses of prodan–H3 O+ complex with exited state geometry.
level to higher frequency region up to 29,800 cm−1 is observed. The hydrogen bonding with an H3 O+ ion shifts the ππ* -levels to lower frequency region both in absorption (by 1000 cm−1 ) and in fluorescence (by 5000 cm−1 ) that quite corresponds to the experimental data. The calculated quantum yield of fluorescence for this complex is about 0.013. At interaction with hydroxonium ion via 3rd carbon atom of the aromatic ring of prodan the shift to higher frequency region up to 25,000 cm−1 is observed. This fact makes it possible to conclude that the second fluorescence band corresponds to this complex. An analysis of a set of spectra for high prodan concentrations (2.2 × 10−4 M) in ethyl alcohol–water solvents has shown presence of the second band already at 70% water concentration (Fig. 13). Thus, according to our opinion the second band exists even at smaller water concentrations but it cannot be visible because of high fluorescence intensity of the first band. Intensity decreasing of the first band results in the second band to show itself. The curves 4–7 (Fig. 13) have an intersection point that allows us to consider that these bands belong to two
(1)
Therefore, we decided to examine the prodan-H3 O+ complex. To do so the exited state geometry of prodan was used. The interaction with hydroxonium ion via oxygen atom of carbonyl group of prodan shifts the maximum of a band to lower frequency region (Fig. 12). For this complex a considerable shift of the nπ* -
Fig. 13. Fluorescence spectra of prodan (2.2 × 10−4 M) in ethanol–water mixture.
42
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
types of prodan complexes. It confirms our supposition about interaction with water via various centers of prodan.
This investigation was carried out with financial support from the Russian Foundation for Basic Research (project 0403-81017).
4. Conclusion References In this work a series experimental and theoretical investigations of a prodan molecule are realized. Quantum-chemical calculations are performed in the geometry of both the ground and exited states of prodan. Possible models of prodan complexes in water are offered. The rate constants of radiative and nonradiative processes are determined for the free prodan and its complexes with water. A dramatic shift of the fluorescence band at changing from nonpolar solvent to isopropyl alcohol and water is explained. The estimation of general solvent effects was given. Our studies on the features of absorption and fluorescence spectra of prodan by experimental and theoretical methods suggest the following conclusions: 1. The non-linear dependence of solvatofluorochromic shift on the component concentration in isooctane–isopropyl alcohol and isopropyl alcohol–isooctane solvents is determined by nonspecific (dielectric permittivity) and specific (hydrogen bonding) solvation. 2. At the fluorescent state the centers of specific solvation (hydrogen bonding) are the carbonyl group and carbon atoms of aromatic ring and the hydrocarbon chains. The MEP value near the oxygen atom of the carbonyl group at the ground state is considerably more than at ππ* exited state. 3. At the ground state the centers of specific solvation are dimethylamino- and carbonyl groups. 4. The prodan fluorescence intensity increasing in isooctane– isopropyl alcohol is caused by hydrogen bonding between the oxygen atom of carbonyl group of prodan and alcohol. 5. The prodan fluorescence quenching in isopropyl alcohol– water solvents is conditioned by presence of the hydrogen bonding complex with hydroxonium ion via carbonyl group of prodan. 6. The second fluorescence band of prodan (24,000 cm−1 ) in isopropyl alcohol–water solvents occurs due to interaction of a molecule with hydroxonium ion via the 3rd carbon atom of aromatic ring.
[1] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic Plenum Publishers, New York, 1999. [2] E.K. Krasnowska, L.A. Bagatolli, E. Gratton, T. Parasasii, Biochem. Biophys. Acta 1511 (2001) 330. [3] N. Alizadeh-Pasdar, E.C.Y. Li-Chan, Food Hydrocoll. 15 (2001) 285. [4] M. Kusube, H. Matsuki, S. Kaneshina, Coll. Surf. 42 (2005) 79. [5] A.B.J. Parusel, W. Nowak, S. Grimme, G. K¨ohler, J. Phys. Chem. A 102 (1998) 7149. [6] B. Sengupta, J. Guharay, P.K. Sengupta, Spectrochim. Acta 56 (2000) 1433. [7] K.A. Kozyra, J.R. Heldt, M. Engelke, H.A. Diehl, Spectrochim. Acta 61 (2005) 1153. [8] G. Webber, F.J. Farris, Biochemistry 18 (1979) 3073. [9] M. Viard, J. Gallay, M. Vinsent, M. Paternostre, Biophys. J. 80 (2001) 347. [10] T. Parassi, E.K. Krasnowska, L. Bagatolli, E. Gratton, J. Fluor. 8 (1998) 365. [11] V.I. Tomin, M. Brozis, J. Heldt, Z. Naturforsch. 58a (2003) 109. [12] M. Brozis, V.I. Tomin, J. Appl. Spectrosc. 70 (2003) 465. [13] M. Brozis, K.A. Kozyra, V.I. Tomin, J. Heldt, J. Appl. Spectrosc. 69 (2002) 412. [14] M. Brozis, V.I. Tomin, J. Heldt, J. Appl. Spectrosc. 69 (2002) 589. [15] A.B.J. Parusel, R. Schamschule, G. K¨ohler, J. Mol. Struct. (Theochem.) 544 (2001) 253. [16] V.Ya. Artukhov, A.I. Galeeva, Russ. Phys. J. 29 (1986) 949. [17] G.V. Maier, V.Ya. Artukhov, O.K. Bazyl, Excited Electronic States and Photochemistry of Organic Compounds, Nauka, Novosibirsk, 1997. [18] V.Ya. Artukhov, J. Struct. Chem. 19 (1978) 418. [19] A.J. Gordon, R.A. Ford, The Chemist’s Companion, Mir, Moscow, 1976. [20] A. Domenicano, I. Hargittai, Accurate Molecular Structures. Their Determination and Importance, Oxford University Press, Oxford, 1992. [21] V.Ya. Artukhov, O.M. Zharkova, Yu.P. Morozova, J. Appl. Spectrosc. 72 (2005) 326. [22] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Mir, Moscow, 1986. [23] Yu.P. Morozova, O.N. Chaykovskaya, N.Yu. Vasil’eva, Russ. J. Phys. Chem. 72 (1998) 272. [24] Yu.M. Kessler, A.L. Zaycev, Solvophobic Effects. Theory, Experiment, Practice, Himiya, Leningrad, 1989. [25] M. Jozefowicz, K.A. Kozyra, J.R. Heldt, J. Heldt, Chem. Phys. 320 (2005) 45. [26] G.V. Yukhnevich, E.G. Tarakanova, V.D. Mayorov, N.B. Librovich, Russ. Chem. Rev. 64 (1995) 963.
Features of absorption and fluorescence spectra of prodan Viktor Ya. Artukhov, Oksana M. Zharkova ∗ , Julia P. Morozova Tomsk State University, Russia, Tomsk-50, Lenina 36, Russian Federation Received 30 May 2006; received in revised form 12 October 2006; accepted 31 October 2006
Abstract Some important and essential features of absorption and fluorescence spectra of prodan in homogeneous and binary mixes are studied. According to results obtained from experimental and quantum-chemical researches we show that the absorption spectrum of prodan in nonpolar solvent within 25,000–50,000 cm−1 is formed by eight electronic transitions. Quantum-chemical calculations are performed in the geometry of both the ground and exited states of prodan. The rate constants of photoprocesses and the quantum yield of fluorescence are determined for the prodan and its complexes with water. A dramatic shift of the fluorescence band at changing from nonpolar solvent to isopropyl alcohol and water is explained. The roles of general solvent effects and specific interactions are separated. According to values of molecular electrostatic potential and charges on atoms the centers of possible interaction of prodan with a solvent are obtained. Possible models of prodan complexes in water are offered. The results of quantum-chemical calculation for offered complexes of prodan in water are compared with those for the free prodan molecule. The presence of the second band (about 24,000 cm−1 ) in fluorescence spectra of prodan in isopropyl (ethyl) alcohol–water solvents is explained. © 2006 Elsevier B.V. All rights reserved. Keywords: Prodan; Quantum yield; Fluorescence; Hydrogen bond; Ion hydroxonium
1. Introduction Fluorescence is used for a wide variety of biomedical and biophysical purposes. There are fluorophores which display high sensitivity to the polarity on the local environment. As an example can be selected prodan (6-propionyl-2dimethylaminnaphtalene) (Fig. 1) and its derivatives. Prodan can monitor the properties of the membrane surface. When bound to membranes, prodan and its derivatives display large spectral shifts at the membrane phase-transition temperature [1]. The attractiveness of using prodan in membrane studies resides in the possibility detecting the properties of the bilayer surface, eventually in parallel with the use of laurdan (6-lauroyl2-dimethylaminnaphtalene), that is located more deeply in the bilayer, by steady-state measurements of emission intensity [2]. Prodan may be useful as an uncharged fluorescent probe for the determination of surface hydrophobisity of proteins in the presence or absence of k-carrageenan and as a function of heating at different pH [3].
∗
Corresponding author at: 634028 Tomsk, Karpova st. 18–50, Russian Federation. Tel.: +8 3822 41 89 53. E-mail address: [email protected] (O.M. Zharkova). 1386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2006.10.048
Despite the wide use of prodan as fluorescent probe [1,3,4] there were no any systematic and profound studies to determinate nature of absorption bands, nature of fluorescent state, photophysical processes in a free prodan molecule, possible centers of prodan complexing with a solvent. In reference books only the long-wavel absorption band of prodan from 25,000 to 35,000 cm−1 is usually given [1]. Authors [5] compare experimental absorption spectrum of prodan recorded in the solvent cyclohexane from 25,000 to 45,000 cm−1 with the theoretical data obtained different methods (AM1/CISD, DFT/CISD, ZINDO/S). Location of the maximum of prodan fluorescence strongly depends on its surroundings. According to [6] a band of prodan fluorescence in reverse micelles system formed by sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) in heptane at different water additions has three according maxima at 390, 413, and 503 nm. It is due to the prodan molecules are distributed in three regions of the AOT reverse micelles [6]. The authors of [4] note that the prodan fluorescence maximum depends on a phase state of phospholipins. The double-band fluorescence was observed in small unilamellar vesicules composed of dipalmitoylphosphatidylcholine at 298 K [7]. Along with the prodan, as a fluorescent probe for investigation of a large series of biological objects, the laurdan (6-lauroyl-2-dimethylaminnaphtalene) is widely used [1,8–10].
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 1. Prodan.
The fluorescence spectrum of laurdan in glycerin contains two bands with maxima at 425 and 500 nm [11,12]. The strong dependence on excitation wavelength is observed for a spectrum. At short-wave excitation the blue component of spectrum gives considerable contribution to a spectrum of total fluorescence. The shift of excitation to the red region results in decreasing a role of blue component and more long-wave spectrum [12]. The fluorescence properties of both molecules are explained by existence of two states: locally excited LE and twisted internal charge transfer TICT [13]. According to [14], it is possible to talk of localized LE and TICT states very conditionally. This term is valid for very low temperatures only. At room and higher temperatures strong delocalization and overlap of these states take place. In our paper analysis of spectral-luminescence properties of prodan was realized on the basis of experimental and theoretical investigations. The interpretation of absorption spectrum of prodan in an inert solvent was performed. The rate constants of photoprocesses are determined for the prodan and its complexes with hydroxonium ion. Quantum-chemical calculations are performed in the geometry of both the ground and exited states of prodan. In work [15] is considered only ground state geometry of prodan. At that authors [15] offer different variants of nonplanar ground state geometry of prodan. In our paper the possible centers of interaction of the prodan with a solvent have been detected. The role of hydrogen bonding and nonspecific interactions in formation of fluorescent capacity of prodan have been studied. An estimation of contribution of general solvent effects in shift of absorption and fluorescence bands has been performed.
37
ter and SDL-2 spectrofluorometer respectively. For all emission measurements the excitation wavelength was 360 nm. All spectral measurements were carried out at the ambient temperature of 298 K. The quantum-chemical analysis of prodan and its complexes was performed by INDO method (intermediate neglect of differential overlap) [16,17]. The centers of interaction were selected using MEP method (molecular electrostatic potential) [17,18]. The geometric parameters of the molecule (bond lengths and angles) were taken as averaged over related compounds discussed in [19,20]. The structural parameters of our calculation almost coincide with data from [15]. Considering a significant negative effective charge on the nitrogen atom in the dimethylamino group we suggested that the methyl groups in the dimethylamino group are to deflect from the molecule plane through the angle of 15◦ . In paper [15] this deviation angle equals to 20.6◦ . 3. Results and discussion The interpretation of absorption spectrum was performed comparing results of an experimental research and quantumchemical calculation. Parametrization of the INDO method was made on a basis of experimental data in “inert” hydrocarbon solvents; therefore, results of absorption spectrum calculation should reproduce an experimental spectrum of prodan in isooctane. The Fig. 2 represent the experimental absorption spectrum of prodan in the solvent isooctane. This spectrum will be quite agreed an electronic spectrum from [5]. The long-wave absorption band of prodan in isooctane (Fig. 2) has poorly allowable vibrational structure and is characterized by a half-width (1/2 ) of 5000 cm−1 . The graphic partition of this band gives two bands with maxima at 27,500 and 30,500 cm−1 . The method of 2nd derivative gives three maxima, but one of them concerns to electronic-vibrational transition of S0 → S1 band. The quantumchemical calculation of a spectrum in this region has shown three electronic transitions (Fig. 2). The first state of the above three states is nπ* one (28,920 cm−1 ) while the last two are ππ* states (29,500 and 32,020 cm−1 ). The nπ* state (f (oscilla-
2. Experimental and computational details Prodan was obtained from Fluka. Fluorophore concentrations in the solutions were kept in the range of 2.2 × 10−6 –1.1 × 10−4 M. All using solvents were of spectroscopic grade and were obtained from joint-stock company Ekros. The solvents were used without further purification after confirming the absence of absorption and fluorescent impurities. Binary solutions were prepared using volumetric percent. Absorption and fluorescence emission measurement were carried out with the model SF-26 automatized spectrophotome-
Fig. 2. Absorption spectra of prodan (1.1 × 10−4 M) in isooctane. The digits on the diagram indicate results of quantum-chemical calculation.
38
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 3. The charges on atoms of free prodan molecule at the states S0 and S2 (ππ* ).
tor strength) = 10−6 ) in experimental absorption spectrum is not observed. Within 35,000–49,000 cm−1 spectral region the method of 2nd derivative exhibits five electronic transitions (Fig. 2). As a result of quantum-chemical calculation five electronic transitions are also obtained. The first two states of the above five are πσ * ones while the last three are ππ* states. Comparing results of quantum-chemical calculation and experimental data we have concluded that the spectral region from 35,000 up to 37,500 cm−1 is formed by only πσ * state, the spectral region from 37,500 up to 42,000 cm−1 is formed by one πσ * state and two ππ* states, and the spectral region from 42,000 up to 49,000 cm−1 is formed by one ππ* state. Thus, the absorption spectrum within 25,000–49,000 cm−1 is determined by eight electronic transitions (S0 → Si , i = 1–8). Let us mark that the results of our calculations on a position of energy levels and nature of states, will be quite agreed results of calculation of prodan by a ZINDO/S method [5]. The Fig. 3 represents the charges on atoms of free prodan molecule at the states S0 and S2 (ππ* ). Note, that at ππ* state the considerable charge redistribution from nitrogen atom (−0.097) to oxygen atom (−0.589) and to some atoms of an aromatic ring is observed. Thus, the S0 → S2 transition is determined by intramolecular charge transfer. Considerable decreasing of negative charge on nitrogen atom indicates that at ππ* state the
Fig. 4. Absorption spectra of prodan (1.1 × 10−4 M) in different solvents: isooctane (1), isopropyl alcohol (2), water (3).
interaction via dimethylamino group is less probable than at the ground state. The calculated data of prodan dipole moment at S0 , S1 (nπ* ), S2 (ππ* ) and S3 (ππ* ) states are 18.25 × 10−30 , 9.41 × 10−30 , 27.09 × 10−30 , and 34.03 × 10−30 C × m, respectively. The authors [5] have received a calculated data of prodan dipole moment at S1 (8.34 × 10−30 C × m), S2 (28.38 × 10−30 C × m) and S3 (50.04 × 10−30 C × m) states using ZINDO/S method. Changing from isooctane to isopropyl alcohol the shift to lower frequency region is observed in absorption spectra (Fig. 4). In spectral region from 22,000 to 35,000 cm−1 the shift is more of that in spectral region from 35,000 to 49,000 cm−1 with the half-width of this band being increased. Within 35,000–49,000 cm−1 region the redistribution of intensity is observed. Similar behavior of the shifts takes place in water. However to reveal structural features of this spectrum appeared to be impossible because of weak solubility of prodan in water. Changing from isooctane to isopropyl alcohol (Fig. 5) the shift of fluorescence band to lower frequency region (from 24,840 to 21,050 cm−1 ) and tenfold increase of intensity are observed. In water the shift is more considerable (from 24,840 to 19,320 cm−1 ) with fluorescence capacity being very weak (Fig. 5).
Fig. 5. Fluorescence spectra of prodan (1.1 × 10−5 M) in different solvents. (Inset: fluorescence spectrum of prodan in water.)
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
39
Fig. 7. Shift of band (evaluated from prodan fluorescence spectra for isopropyl alcohol–isooctane and isooctane–isopropyl alcohol mixtures) vs. concentration of solvent (isopropyl alcohol or isooctane).
Fig. 6. The scheme of energy levels and photoprocesses of free prodan with exited state geometry.
In order to explain the high sensitivity of prodan fluorescence to solvent it is necessary to calculate the geometry of molecule at an exited state. The method for definition of an exited state geometry is described in [21]. The greatest changes of electron density occur on the C1 –C2 , C3 –C4 , C5 –C6 , and C7 –C8 bonds (Fig. 1). Taking into account considerable negative charges on C3 (Q = −0.109) and C7 (Q = −0.076) carbon atoms of aromatic ring we further assumed that these two atoms can partially come from the sp2 -hybridization to sp3 one, that is, go outside the plane of aromatic part of the molecule. The declination of the C3 and C7 atoms from the plane is 0.28 and ˚ respectively. According to our suppositions this changed 0.26 A, geometry corresponds to exited state geometry (equilibrium) of prodan. The results of quantum-chemical calculation of prodan with exited state geometry are shown in Fig. 6. At transition to this geometry the significant shift of ππ* states to lower frequency region and small shift of nπ* state to higher frequency region are observed. Thus at this geometry S1 state is ππ* state. The quantum yield of fluorescence from S1 (ππ* ) state is estimated to be of 0.07 [21]. This calculation allowed us to describe an experimental fluorescence spectrum of prodan in isooctane (Fig. 5). The shift in absorption spectra and dramatic shift in fluorescence spectra at changing from nonpolar to polar solvent are not explained only by large difference in the dipole moment of the ground and exited states as demonstrated [1]. In Table 1 the
experimental shifts of absorption and fluorescence bands and the shifts estimated according to general solvent effects [1,22] are shown. Note, that the contribution of general solvent effects to the shift of bands is no more than 30%. Therefore, the remaining shift is caused by specific interactions. An influence of dielectric permittivity () on a band shift is confirmed by the fact of the prodan fluorescence band shift for isopropyl alcohol–isooctane solvents (Fig. 7). On addition of isooctane to the solution of prodan in isopropyl alcohol (less than 30%) the shift of the maximum of fluorescence band is small. With increasing of isooctane concentration up to 90% the shift ups to 26%. Therefore, the contribution of dielectric permittivity to shift of a band is comparable with the data represented in [1] (Table 1). On addition of isopropyl alcohol (5%) to the solution of prodan in isooctane the shift of the band is 40%. The value in this case has changed by 30%, therefore, the shift is caused by not only specific interactions but also by dielectric permittivity of environment. Indicated alcohol concentration corresponds to small value of dielectric permittivity. Note, that at smalls the function f() changes especially dramatically [23]. The contribution of general effects of the solvent at changing to isopropyl alcohol gives the shift of 770 cm−1 (Table 1) that is certainly higher than that obtained even at 5% alcohol concentration. On a basis of the data [1] we have received maxima of the prodan fluorescence band max at various polarity parameters f() (Fig. 8). This dependence appeared to be non-linear. The MEP calculations show that prodan at the ground state has two proton-acceptor centers (Fig. 9a). The strongest one is
Table 1 Shifts of absorption and fluorescence bands of prodan Solvent
abs , experiment (cm−1 )
abs , general solvent effectsa (cm−1 )
fl , experiment (cm−1 )
fl , general solvent effectsa (cm−1 )
Isooctane–isopropyl alcohol Isooctane–water
1300 1000
410 430
3790 5520
770 800
a ␣ (Onsager sphere radius) = 5.6 A. ˚ The dipole moments of prodan in ground state (18.35 × 10−30 C × m) and exited state (34.03 × 10−30 C × m) are taken from quantum-chemical calculation, realized by us.
40
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 8. Maxima of fluorescence bands (νmax ) of prodan in different solvents vs. f(ε).
related to the oxygen atom of the carbonyl group and is localized in the molecule plane. The second one is related to the nitrogen atom of dimethylamino group and is localized lower ˚ At S1 n* state only the minimum the molecule plane by 1.5 A. related to nitrogen atom of dimethylamino group is observed (Fig. 9b). At S2 and S3 ππ* states the MEP minima are observed near oxygen atom of carbonyl group and near 3rd, 19th and 20th carbon atoms of aromatic ring and hydrocarbon chain (Fig. 9c). The MEP results for exited state geometry of prodan (S1 (ππ* )) have also shown the minima relating to the oxygen atom of C=O group and carbon atoms of hydrocarbon chain. Thus the prodan molecule in equilibrium state is possible to interact with a solvent via several centers. The prodan contains hydrophylic and hydrophobic parts. The hydrophylic part of prodan involve two proton-acceptor centers. According to MEP results the interaction with a solvent (alcohol, water) via these centers is possible through hydrogen bonding. The MEP results also show that an interaction via hydrophobic part of prodan (hydrocarbon chain) is possible too. Kessler and Zaycev [24] have shown that between solvent and solute molecules the interaction is possible without charge transfer and formation of a chemical bond that is called the solvophobic or hydrophobic interaction. That is the hydrophobic interaction via hydrocarbon chain of prodan is possible. As it was noted, the MEP minimum is also observed near the 3rd carbon atom of aromatic ring. However, via this center the interactions both hydrogen bonding type and van der Waals type are possible; therefore, it is difficult to relate this center either to hydrophobic or to hydrophylic part of a molecule. Returning to the Fig. 8 we should note that water and alcohols correspond to complexes with a hydrogen bonding. In prodan
Fig. 10. Fluorescence spectra of prodan (1.1 × 10−5 M) in isooctane–isopropyl alcohol mixture.
with acetone or acetonitrile the hydrogen bonding is possible to be formed but considerably weaker than with water or alcohol. There are different solute-environment space configurations which contribute to a band broadening. The authors [25] call this phenomenon as inhomogeneous broadening. Probably such configurations are complexes formed both via carbonyl group and via carbon atoms. We have estimated the value of inhomogeneous broadening by formula from [25]. The values obtained for prodan in isooctane, isopropyl alcohol, and water are 310, 465, and 495 cm−1 , respectively. In binary solutions of isopropyl alcohol in isooctane (Fig. 10) at isopropyl alcohol concentration from 5 to 90% the fluorescence increases from 2 to 7 times in intensity. On addition of isopropyl alcohol (5–90%) to solution of prodan in isooctane the curves intersect at 22,900 cm−1 . The intersection point shows that the fluorophore is in various environment. According to MEP results the interaction with alcohol at ππ* exited state both via carbonyl group and via carbon atoms of prodan is possible. The most deep minimum is observed near the carbonyl group that means this interaction to be determinant. The prodan complex via other centers is likely to give a weaker fluorescence. Therefore, complex of prodan via carbonyl group gives a modest contribution in fluorescence increasing. Let us consider the prodan in the solvents: isopropyl alcohol–water and ethyl alcohol–water. On addition to the prodan solutions in alcohol less than 90% of water (the water is more strong donor of protons) small decreasing of optical density (about 10%) in the first absorption band was observed. At 90% water concentration in the solution the value of optical density decreased by 4 times and the maxi-
˚ (b) S1 (np* ), z = −1,5 A; ˚ (c) S1 (pp* ) (exited state geometry), z = −1,5 A. ˚ (z) distance Fig. 9. Minima obtained by a MEP method for a prodan: (a) S0 , z = −1.5 A; from plane of prodan.
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
Fig. 11. Fluorescence spectra of prodan (1.1 × 10−5 M) in isopropyl alcohol– water mixture.
mum of absorption band shifted to a higher frequency region by 500 cm−1 . The analysis of fluorescence spectra obtained on addition of different water concentrations to prodan solution in isopropyl (ethyl) alcohol has shown that the shift of the band (about 2000 cm−1 ) to higher frequency region and fluorescence quenching (at 90% of water) by 8 times are observed (Fig. 11). Besides, at 90% water concentration the second band is observed near 24,000 cm−1 (Fig. 11). It occurs in a case of both isopropyl alcohol and ethyl alcohol. To explain the results obtained we consider MEP data. In the fluorescent state the alcohol molecule interacts through all possible centers detected by MEP method. Being added to solution water substitutes alcohol through all centers. We refute the supposition that the shift of a band to lower frequency region is caused by the effect of dielectric permittivity only. It is the case because the contribution of general solvent effects to the band shift, according to [1], at changing from isopropyl alcohol to water is 50 cm−1 . Shift of the fluorescence band to lower frequency region and appearance of the second band at 24,000 cm−1 we have assigned to the interaction both via the oxygen of carbonyl group and via the carbon atom of aromatic ring of prodan. As the consideration of specific interactions with water molecules via selected centers has not allowed us to reproduce our experimental data. Therefore, the necessity of new model of a complex is appeared. According to [26] there are ionic forms H3 O+ , H5 O2 + , and H9 O4 + in water, the most stable of them being H5 O2 + . In the case of solution of the organic proton-acceptor molecule (B) in water there occurs a substitution reaction: the water molecule in H5 O2 + is submitted by the molecule B, that is, B forms a complex with H3 O+ : (H2 O· · ·H· · ·OH2 )+ + B ↔ (H2 O· · ·H· · ·B)+ + H2 O.
41
Fig. 12. The scheme of energy levels and photoprocesses of prodan–H3 O+ complex with exited state geometry.
level to higher frequency region up to 29,800 cm−1 is observed. The hydrogen bonding with an H3 O+ ion shifts the ππ* -levels to lower frequency region both in absorption (by 1000 cm−1 ) and in fluorescence (by 5000 cm−1 ) that quite corresponds to the experimental data. The calculated quantum yield of fluorescence for this complex is about 0.013. At interaction with hydroxonium ion via 3rd carbon atom of the aromatic ring of prodan the shift to higher frequency region up to 25,000 cm−1 is observed. This fact makes it possible to conclude that the second fluorescence band corresponds to this complex. An analysis of a set of spectra for high prodan concentrations (2.2 × 10−4 M) in ethyl alcohol–water solvents has shown presence of the second band already at 70% water concentration (Fig. 13). Thus, according to our opinion the second band exists even at smaller water concentrations but it cannot be visible because of high fluorescence intensity of the first band. Intensity decreasing of the first band results in the second band to show itself. The curves 4–7 (Fig. 13) have an intersection point that allows us to consider that these bands belong to two
(1)
Therefore, we decided to examine the prodan-H3 O+ complex. To do so the exited state geometry of prodan was used. The interaction with hydroxonium ion via oxygen atom of carbonyl group of prodan shifts the maximum of a band to lower frequency region (Fig. 12). For this complex a considerable shift of the nπ* -
Fig. 13. Fluorescence spectra of prodan (2.2 × 10−4 M) in ethanol–water mixture.
42
V.Ya. Artukhov et al. / Spectrochimica Acta Part A 68 (2007) 36–42
types of prodan complexes. It confirms our supposition about interaction with water via various centers of prodan.
This investigation was carried out with financial support from the Russian Foundation for Basic Research (project 0403-81017).
4. Conclusion References In this work a series experimental and theoretical investigations of a prodan molecule are realized. Quantum-chemical calculations are performed in the geometry of both the ground and exited states of prodan. Possible models of prodan complexes in water are offered. The rate constants of radiative and nonradiative processes are determined for the free prodan and its complexes with water. A dramatic shift of the fluorescence band at changing from nonpolar solvent to isopropyl alcohol and water is explained. The estimation of general solvent effects was given. Our studies on the features of absorption and fluorescence spectra of prodan by experimental and theoretical methods suggest the following conclusions: 1. The non-linear dependence of solvatofluorochromic shift on the component concentration in isooctane–isopropyl alcohol and isopropyl alcohol–isooctane solvents is determined by nonspecific (dielectric permittivity) and specific (hydrogen bonding) solvation. 2. At the fluorescent state the centers of specific solvation (hydrogen bonding) are the carbonyl group and carbon atoms of aromatic ring and the hydrocarbon chains. The MEP value near the oxygen atom of the carbonyl group at the ground state is considerably more than at ππ* exited state. 3. At the ground state the centers of specific solvation are dimethylamino- and carbonyl groups. 4. The prodan fluorescence intensity increasing in isooctane– isopropyl alcohol is caused by hydrogen bonding between the oxygen atom of carbonyl group of prodan and alcohol. 5. The prodan fluorescence quenching in isopropyl alcohol– water solvents is conditioned by presence of the hydrogen bonding complex with hydroxonium ion via carbonyl group of prodan. 6. The second fluorescence band of prodan (24,000 cm−1 ) in isopropyl alcohol–water solvents occurs due to interaction of a molecule with hydroxonium ion via the 3rd carbon atom of aromatic ring.
[1] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic Plenum Publishers, New York, 1999. [2] E.K. Krasnowska, L.A. Bagatolli, E. Gratton, T. Parasasii, Biochem. Biophys. Acta 1511 (2001) 330. [3] N. Alizadeh-Pasdar, E.C.Y. Li-Chan, Food Hydrocoll. 15 (2001) 285. [4] M. Kusube, H. Matsuki, S. Kaneshina, Coll. Surf. 42 (2005) 79. [5] A.B.J. Parusel, W. Nowak, S. Grimme, G. K¨ohler, J. Phys. Chem. A 102 (1998) 7149. [6] B. Sengupta, J. Guharay, P.K. Sengupta, Spectrochim. Acta 56 (2000) 1433. [7] K.A. Kozyra, J.R. Heldt, M. Engelke, H.A. Diehl, Spectrochim. Acta 61 (2005) 1153. [8] G. Webber, F.J. Farris, Biochemistry 18 (1979) 3073. [9] M. Viard, J. Gallay, M. Vinsent, M. Paternostre, Biophys. J. 80 (2001) 347. [10] T. Parassi, E.K. Krasnowska, L. Bagatolli, E. Gratton, J. Fluor. 8 (1998) 365. [11] V.I. Tomin, M. Brozis, J. Heldt, Z. Naturforsch. 58a (2003) 109. [12] M. Brozis, V.I. Tomin, J. Appl. Spectrosc. 70 (2003) 465. [13] M. Brozis, K.A. Kozyra, V.I. Tomin, J. Heldt, J. Appl. Spectrosc. 69 (2002) 412. [14] M. Brozis, V.I. Tomin, J. Heldt, J. Appl. Spectrosc. 69 (2002) 589. [15] A.B.J. Parusel, R. Schamschule, G. K¨ohler, J. Mol. Struct. (Theochem.) 544 (2001) 253. [16] V.Ya. Artukhov, A.I. Galeeva, Russ. Phys. J. 29 (1986) 949. [17] G.V. Maier, V.Ya. Artukhov, O.K. Bazyl, Excited Electronic States and Photochemistry of Organic Compounds, Nauka, Novosibirsk, 1997. [18] V.Ya. Artukhov, J. Struct. Chem. 19 (1978) 418. [19] A.J. Gordon, R.A. Ford, The Chemist’s Companion, Mir, Moscow, 1976. [20] A. Domenicano, I. Hargittai, Accurate Molecular Structures. Their Determination and Importance, Oxford University Press, Oxford, 1992. [21] V.Ya. Artukhov, O.M. Zharkova, Yu.P. Morozova, J. Appl. Spectrosc. 72 (2005) 326. [22] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Mir, Moscow, 1986. [23] Yu.P. Morozova, O.N. Chaykovskaya, N.Yu. Vasil’eva, Russ. J. Phys. Chem. 72 (1998) 272. [24] Yu.M. Kessler, A.L. Zaycev, Solvophobic Effects. Theory, Experiment, Practice, Himiya, Leningrad, 1989. [25] M. Jozefowicz, K.A. Kozyra, J.R. Heldt, J. Heldt, Chem. Phys. 320 (2005) 45. [26] G.V. Yukhnevich, E.G. Tarakanova, V.D. Mayorov, N.B. Librovich, Russ. Chem. Rev. 64 (1995) 963.