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Solvent effect on the ground and excited state dipole moments of fluorescein
Journal of Molecular Structure (Theochem) 548 (2001) 165±171
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Solvent effect on the ground and excited state dipole moments of ¯uorescein Bilal AcemiogÆlu a,1, Mustafa Arõk a, Hasan EfeogÆlu b, Yavuz Onganer a,* a
Department of Chemistry, Faculty of Arts and Sciences, AtatuÈrk University, 25240 Erzurum, Turkey b Department of Physics, Faculty of Arts and Science, AtatuÈrk University, 25240 Erzurum, Turkey Received 22 February 2001; revised 18 April 2001; accepted 18 April 2001
Abstract The ground-state and excited-state dipole moments of ¯uorescein were studied at room temperature in n-alcohols (methanol± n-hexanol) and acetonitrile and acetonitrile±benzene solvent mixtures. The excited-state dipole moments were estimated from Lippert's, Bakhshiev's and Chamma±Viallet's equations by using the variation of the Stokes' shift with the solvent dielectric constant and refractive index. Experimental ground-state dipole moments for ¯uorescein in n-alcohols, acetonitrile and acetonitrile±benzene solvent mixtures were estimated by the Guggenheim±Smith method (GSM). It was determined that dipole moments of the excited-state were higher than those of the ground-state in n-alcohols while they were lower than those of the ground-state in acetonitrile and acetonitrile±benzene solvent mixtures. The solute±solvent interactions on the ground-state and the excited-state dipole moments of ¯uorescein are discussed. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Fluorescein; Stokes' shift; Dipole moment; Solute±solvent interactions
1. Introduction Determination of the ground-state and excited-state dipole moments of dye molecules is important, because the values of dipole moments provide information about the change in electronic distrubition upon excitation. Fluorescein, a dye molecule, is a highly ¯uorescent probe (Fig. 1), and it has been ®rst synthesized by Von Bayer in 1871 [1]. The dye is commonly used in laser industry [2±4] as well as used for ¯uorescein angiography in medical treatment * Corresponding author. Tel.: 190-442-233-1970; fax: 190-442233-1062. E-mail address: [email protected] (Y. Onganer). 1 Present address: Department of Chemistry, Faculty of Arts and Sciences, KahramanmarasË SuÈtcËuÈimam University, 46100 K.MarasË, Turkey.
[5]. It has been increasingly used in energy transfer processes, nowadays [6,7]. Moreover, it is widely used to label proteins in biochemistry [8] and to understand ¯uorescence-quenching processes as well [9±11]. A large number of usage of ¯uorescein makes it popular, therefore, it has been subject to most of the scienti®c investigations. Zanker and Peter ®rst showed that ¯uorescein exhibited several prototropic form in the groundstate as a function of pH [12] Then, the ®rst spectroscopic behavior in the excited-state was reported by measuring its ¯uorescence spectra in water as a function of pH by Rozwadowski [13] and Leonhardt et al. [14]. They showed that ¯uorescein had four prototropic forms (neutral, cation, monoanion and dianion). Yguerabide et al. have also studied excited state proton reactions of ¯uorescein in phosphate buffers of varying concentrations [15]. In another work,
0166-1280/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0166-128 0(01)00513-9
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
166
and the excited-state dipole moments of ¯uorescein with the steady-state ¯uorescence spectroscopy technique and Guggenheim±Smith method (GSM) in nalcohols which are polar protic solvents and in acetonitrile (AN) and acetonitrile±benzene (AN±BN) mixtures which are polar aprotic solvents. The molecular inretactions between dye and n-alcohol solvents, AN, and AN±BN solvent mixtures were interpreted in terms of previously proposed equations [18±20]. Fig. 1. Structure of ¯uorescein.
Rohatgi and Singhal found out that ¯uorescein have a dimeric form as a function of concentration in alkaline aqueous solution at pH 12 [16]. On the other hand, the absorption and emission spectroscopic properties of ¯uorescein in n-alcohols, micellar and microemulsion media have been studied by BiswasË et al. [17]. But, there has been no work done with regard to the determination of the ground-state and excited-state dipole moments of ¯uorescein in n-alcohols and acetonitrile±benzene solvent systems. The goal of this paper is to determine and compare the ground-state
2. Experimental Fluorescein was purchased from Sigma and showed a single spot on a TLC plate. Therefore, it was directly used without further puri®cation. Solvents were spectroscopic grade of Merck and used after distilling over CaH2. Fluorescein was stored in the dark as a concentrated stock solution of 5 mM in methanol. Samples were prepared by evaporating 20 and 100 ml of stock solution for n-alcohols, AN and AN±BN solvent mixtures, respectively, and then redissolving with 5 ml of solvent. For the measurements, the ®nal
Table 1 Some physical constants of solvents and spectral data of ¯uorescein in solvents Solvent
l a a (nm)
l f b (nm)
nÅ a c (cm 21)
nÅ f d (cm 21)
nD25e
D 25f
ET(30) g
Methanol Ethanol n-propanol n-butanol n-pentanol n-hexanol Acetonitrile 90%AN±10%BN h 80%AN±20%BN 70%AN±30%BN 60%AN±40%BN 50%AN±50%BN 40%AN±60%BN 30%AN±70%BN 20%AN±80%BN
456 454 453 452 460 452 279 279 279 280 280 281 281 282 282
515 508 507 506 508 504 307 307 306 307 307 308 307 307 308
21930 22026 22075 22123 21739 22124 35842 35842 35842 35714 35714 35587 35587 35461 35336
19417 19685 19724 19763 19685 19841 32573 32573 32680 32573 32573 32468 32573 32573 32468
1.3280 1.3602 1.3833 1.3974 1.4064 1.4165 1.3420 1.3521 1.3642 1.3722 1.3903 1.4044 1.4245 1.4427 1.4616
32.58 24.64 20.15 17.44 14.00 13.30 37.12 34.41 30.46 26.06 24.30 22.17 15.18 12.36 10.67
55.13 52.00 50.35 49.53 49.12 48.60 45.41 44.31 44.00 43.60 43.00 42.40 42.00 40.80 39.96
a b c d e f g h
Wavelength of absorption maxima (uncertainty is ^1 nm). Wavelength of ¯uoresence emission maxima (uncertainty is ^1 nm). Wavenumber of absorption maxima. Wavenumber of ¯uorescence emission maxima. Refractive index. Dielectric constant. Solvent polarity in the unit of kcal/mol. Volume percentages of AN: acetonitrile and BN: benzene.
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171 Table 2 Dipole moments of ¯uorescein in the ground and excited-states for the solvents Solvent
m g a (D)
m e b (D)
m e c (D)
m e d (D)
Methanol Ethanol n-propanol n-butanol n-pentanol n-hexanol Acetonitrile 90%AN±10%BN 80%AN±20%BN 70%AN±30%BN 60%AN±40%BN 50%AN±50%BN 40%AN±60%BN 30%AN±70%BN 20%AN±80%BN
3.97 7.40 10.18 12.15 16.04 18.16 5.25 6.85 7.37 8.07 8.37 9.42 9.86 10.23 10.77
8.82 12.15 15.03 17.00 20.89 23.01 0.48 2.08 2.60 3.30 3.60 4.65 5.09 5.46 6.00
5.00 8.00 10.63 12.53 16.33 18.41 4.41 6.23 6.79 7.54 7.87 8.98 9.44 9.82 10.38
4.56 7.73 10.42 12.35 16.19 18.30 ± ± ± ± ± ± ± ± ±
a The experimental ground-state dipol moments calculated the GSM. b The excited-state dipole moments calculated with Dm Lippert's equation. c The excited-state dipole moments calculated with Dm Bakhshiev's equation. d The excited-state dipole moments calculated with Dm Chamma±Viallet's equation.
from from from from
concentration was 2 £ 10 25 and 1 £ 10 24 M in n-alcohols and in AN and AN±BN solvent mixtures, respectively. Absorption spectra were recorded on a Shimadzu UV±Vis 160A spectrophotometer. Fluorescence spectra were taken by using a Shimadzu RF 5301PC Spectro¯uorophotometer. The capacitance values (the values of C and C0) for the dielectric constants (D) were measured with a capacitory by means of Hewlett Packard 4284 A 20 Hz±1 MHz Precision LCR meter. The refraction indices (n) were determined by using a jena refractometer. All measurements were recorded at room temperature. Some spectral data and solvent related physical parameters are given in Table 1.
3. Results and discussion 3.1. Experimental and theoretical ground-state dipole moments We found that the theoretical dipole moment of
167
¯uorescein was mg 8:83 D by using SCF MO LCAO method in MNDO approximation [21]. This kind of calculation assumes that molecules are involved in the gas phase and does not include solvent interactions. To obtain much more appropriate values of dipole moments for the ground and excited state, spectroscopic techniques, which re¯ect the molecule's electronic state and interactions with the solvent molecules, were employed. The ground-state dipole moments (m g) of ¯uorescein in n-alcohols, AN and AN±BN solvent mixtures with different polarity were estimated according to the GSM [22] that is expressed as 27kT 1 mg 1 AD 2 An M 4pN d D 1 22 where k is the Boltzman constant, T the absolute temperature, N Avagadro's number, d and D are the density and dielectric constant of the solvent, respectively. AD and An are the numerical values obtained from the solution dielectric constant and refractive index measurements, respectively, and M is molecular weight of the solute. The dielectric constants of the solvents and solutions were calculated from the ratio of C/C0. C and C0 are capacitances which are measured when capacitor was full with solvent or solution and empty, respectively. For n-alcohols and acetonitrile, these results are in good agreement with those reported in the literature [23]. The experimental ground-state dipole moment of ¯uorescein according to the GSM has been estimated for each solvent system. The excited-state dipole moments (m e) have been calculated by using the values of m g from the GSM and the values of Dm from Lippert's, Bakhshiev's and Chamma±Viallet's equations. Their values are presented in Table 2. 3.2. Excited-state dipole moments Three formulae were used for the treatment of solvent spectral shift to determine the excited-state dipole moments of ¯uorescein. Lippert's equation [18] is given as " # 2Dm2 D 2 1 n2 2 1 Dn na 2 nf 3 2 2 2 2n 1 1 a0 hc 2D 1 1 where na and nf are the absorption and emission wavenumbers in cm 21 at maxima, respectively. Dm2 is
168
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
Table 3 Solvent polarity functions (for de®nitions, see text. Eqs. (6)±(8)) Solvent
F1
F2
F3
Methanol Ethanol n-Propanol n-Butanol n-Pentanol n-Hexanol Acetonitrile 90%AN±10%BN 80%AN±20%BN 70%AN±30%BN 60%AN±40%BN 50%AN±50%BN 40%AN±60%BN 30%AN±70%BN 20%AN±80%BN
0.309 0.290 0.275 0.264 0.250 0.245 0.307 0.301 0.294 0.287 0.279 0.270 0.249 0.233 0.218
0.854 0.813 0.779 0.751 0.703 0.692 0.861 0.852 0.836 0.817 0.802 0.785 0.715 0.655 0.652
0.651 0.649 0.650 0.645 0.627 0.629 0.663 0.665 0.666 0.662 0.666 0.667 0.645 0.628 0.626
equal to me 2 mg 2 and m e and m g are the excitedstate and ground-state dipole moments, respectively. h is the Planck constant and c is the speed of light. a0 is the solute cavity radius. D and n are the solvent dielectric constant and refractive index, respectively. Bakhshiev's equation [19] is given as " # 2Dm2 D 2 1 n2 2 1 2n2 1 1 Dn na 2 nf 3 2 2 n 1 2 n2 1 2 a0 hc D 1 2 3
and Chamma±Viallet's equation [20] is Dn n 1 nf a 2 2 2Dm2 2 3 a0 hc 1
2n2 1 1 2 n2 1 2 #
D21 n2 2 1 2 2 D12 n 12
! 4
3 n4 2 1 2 n2 1 22
The symbols in Eqs. (3) and (4) are the same as in Eq. (2). The value of the solute cavity radius (a0 ) was calculated from the molecular volume of ¯uorescein according to the following equation [24] a0 3M=4pdN1=3
5
where d is the density of solute molecule, M the molecular weight of the solute and N is the Avagadro's Ê. number. For ¯uorescein, a0 was found to be 5.88 A For Eqs. (2)±(4), the solvent polarity functions or reaction ®eld factors [25,26], F1, F2 and F3, are de®ned as follows: " # D21 n2 2 1 2 2 F1 6 2D 1 1 2n 1 1 2n2 1 1 F2 n2 1 2 F3
Fig. 2. The variation of Stokes' shift with F1 by using Lippert's equation. (X) in AN and AN±BN solvent mixtures; (W) in n-alcohols.
"
"
D21 n2 2 1 2 2 D12 n 12
1 3 n4 2 1 F2 1 2 2 n2 1 22
# 7
8
F1 is used for the Lippert's equation, F2 is used for the Bakhshiev's equation, and F3 is used for the Chamma±Viallet's equation. Their values are listed in Table 3 for the solvent systems employed. When the spectral data of ¯uorescein in Table 1 were analyzed it was found that absorption and ¯uorescence emission maxima are red-shifted in n-alcohols while absorption maxima are blue-shifted in AN and AN±BN solvent mixtures as solvent polarity is increased. But there is no important change of ¯uorescence emission maximum in AN and AN±BN solvent mixtures with increasing solvent polarity. To determine Stokes' shifts in n-alcohols, AN and AN± BN solvent mixtures, we have used the wavenumbers of absorption and ¯uorescence emission at maxima given in Table 1. In order to obtain excited-state
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
Fig. 3. The variation of Stokes' shift with F2 by using Bakhshiev's equation. (X) in AN and AN±BN solvent mixtures; (W) in n-alcohols.
dipole moment values, Stokes' shift (Dn) and the arithmetic mean of (Dn=2) values have been plotted against the solvent polarity functions that are F1, F2, and F3. By taking into account Eqs. (2)±(4); (Dn) vs F1, (Dn) vs F2, and (Dn=2) vs F3 plots were prepared for ¯uorescein in n-alcohols and they were given in Fig. 2 by using Eq. (2), in Fig. 3 by using Eq. (3), and in Fig. 4 by using Eq. (4). The same procedure was applied for the data obtained by using AN and AN±
Fig. 4. The variation of arithmetic mean of Stokes' shift with F3 in n-alcohols by using Chamma±Viallet's equation.
169
BN solvent systems and their plots were given in Figs. 2, 3 and 5. From the slopes of these plots, Dm values were calculated. Since Dm is (m e 2 m g) [27], one can obtain m e. We found that the excited-state dipole moment values were higher than those of groundstate (m e . m g) in n-alcohols according to all the three equations while the excited-state dipole moments were lower than those of ground-state (m e , m g) in AN and AN±BN solvent mixtures, except for Chamma±Viallet's equation. These results suggest more stable excited singlet-states relative to the ground-state in n-alcohols [28]. In AN and AN± BN solvent mixtures this situation is opposite to those in n-alcohols. According to the three equations, the Stokes' shifts versus solvent polarity functions should be linear in the presence of general solvent effects as a function of the dielectric constant and the refractive index. The deviations from the linearity imply the speci®c solute±solvent interactions [29]. These deviations are related to the extent of the interactions between solute and solvent molecules and these interactions lead to the energy difference changes between the ground and the excited-state. Another approach is to estimate excited-state dipole moment values by using the values of Dm from the Lippert's, Bakshiev's and Chamma±Viallet's equations with the value of theoretical dipole moment, mg 8:83 D; for n-alcohols, AN and AN±BN solvent mixtures. In n-alcohols, the excited-state dipole moment values from the Lippert's, Bakshiev's and Chamma±Viallet's equations are 13.68, 9.34, 9.11 D, respectively. The correlation coef®cents are 0.77 and 0.79 (Figs. 2 and 3, respectively). The poor correlation between the Stokes' shifts and solvent polarity functions is attributed to the contribution of hydrogen bonding in n-alcohols. This situation has also been demonstrated in Ref. [30]. Chamma±Viallet's equation has shown a very poor correlation, even no correlation, and the correlation coef®cient is equal to the value of 0.08 (Fig. 4). This scattering suggests speci®c interactions between solute and solvent molecules (in general, H-bonding). Moreover, the excitedstate dipole moment values compared to the groundstate values are higher according to Eqs. (2)±(4), implying more polar excited-state structure than that of ground-state structure. This H-bonding results from the interactions between n-alcohol molecules and C± OH and CyO groups of ¯uorescein molecule. It has
170
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
Fig. 5. The variation of arithmetic mean of Stokes' shift with F3 in AN and AN±BN solvent mixtures by using Chamma±Viallet's equation.
been demonstrated that such interactions between methanol and ¯uorescein molecules were present by X-ray crystallography analysis elsewhere [31]. In acetonitrile and acetonitrile±benzene solvent mixtures, the excited-state dipole moment values from Lippert's and Bakhshiev's equations were found to be 4.06 and 8.35 D, respectively. Stokes' shift plots vs solvent polarity functions are linear with correlation values of 0.93 (Fig. 2) and 0.91 (Fig. 3) according to Lippert's and Bakhshiev's
Fig. 6. The variation of Dn with ET(30). (X) in AN and AN±BN solvent mixtures; (W) in n-alcohols.
equations, respectively. This shows the presence of general solvent effects as a function of dielectric constant and refractive index. Based on Eq. (4), Dn=2 vs F3 plot does not obey such a relationship (Fig. 5) for AN and AN±BN solvent mixtures. Moreover, the excited-state dipole moment values in AN and AN±BN solvent mixtures compared to the ground-state values are lower than those of groundstate dipole moment, indicating more polar groundstate structure than the excited-state structure. If one considers the excited-state dipole moment values from ¯uorescence spectroscopy technique and ground-state dipole moments from the GSM as shown in Table 2, it can be seen that Lippert's equation is more sensitive to the speci®c solute±solvent interactions while Bakhshiev equation is more sensitive to the general solvent effects [29]. Somehow, for both solvent systems, Chamma±Viallet's equation does not indicate any trend regarding interactions. If the dipole moment changes of ¯uorescein were dependent only on solvent polarity, the plot of Dn vs. ET(30) should have indicated a linear relationship for all solvents employed [32,33]. But this has not been observed for our system, as shown in Fig. 6, indicating speci®c solute±solvent interactions. Therefore, the protic solvents (n-alcohols) have a different slope than those for aprotic solvents (AN and AN±BN solvent mixtures). As a result, the dipole moment changes of ¯uorescein result from both solvent
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
polarity effect and H-bonding effect as a function of solvent used. 4. Conclusions We have studied dipole moment variations of ¯uorescein in the ground and excited-states as a function of the solute±solvent interactions by using the GSM and the solvatochromism method. We found that ¯uorescein posseses higher dipole moments in the excitedstate than in the ground-state in n-alcohols while it posseses lower dipole moment values in the excitedstate than in the ground-state in AN and AN±BN solvent mixtures. This demonstrates that the excitedstate of ¯uorescein is more polar than the ground-state in n-alcohols while it shows opposite to this situation in AN and AN±BN solvent mixtures. Moreover, one can say that relative to the ground-state the excitedstate in n-alcohols is more stable with solvent effects while the ground-state relative to the excited-state is more stable with solvent effects in the AN and AN± BN solvent mixtures. The speci®c interactions due to H-bonding between ¯uorescein and n-alcohol molecules and the presence of general solute±solvent interactions in AN and AN±BN solvent mixtures were con®rmed by means of Lippert's and Bakhshiev's equations. Acknowledgements We are grateful to the Department of Chemistry and Research Fund of AtatuÈrk University for technical device and support (Project Number: 1998/44). References [1] V. Sun, K.R. Gee, D.H. Klaubert, R.P. Haugland, J. Org. Chem. 62 (1997) 6469.
[2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]
[32] [33]
171
F.P. Schafer (Ed.), Dye Lasers Springer, Berlin, 1973. L.I. Grossweiner, Radiat. Res. Rew. 2 (1970) 345. J.M. Demas, G.A. Grossby, J. Phys. Chem. 75 (1971) 911. U.M.D. Martin, Optahalmic Surgery Lasers: Clin. Sci. 27 (1996) 3. H. Xuebau, Sci. Chin. 50 (1992) 7. Y. Xiaobin, M. Weng, Z. Manhua, T. Shen, Dyes Pigm. 35 (1997) 87. F.F. Chen, Arch. Biochem. Biophys. 133 (1969) 265±276. I. Lopez Arbeloa, J. Photochem. 18 (1982) 161±168. I. Lopez Arbeloa, J. Photochem. 14 (1980) 97. J. Guilford, Q. Xiaohua, J. Photochem. Photobiol., A: Chem. 113 (1998) 125. V. Zanker, W. Peter, Chem. Ber. 91 (1958) 572±580. M. Rozwadowski, Acta Phys. Pol. 20 (1961) 1005±1017. H. Leonhardt, L. Gordon, R. Livingston, J. Phys. Chem. 75 (1971) 245±249. J. Yguerabide, E. Talavera, J.M. Alvarez, B. Quintero, Photochem. Photobiol. 60 (1994) 435±441. K.K. Rohatgi, G.S. Singhal, J. Phys. Chem. 70 (1966) 1695. S. Biswas, S.Ch. Bhattacharya, P.K. Sen, S.P. Moulik, J. Photochem. Photobiol. A: Chem. 123 (1999) 121±128. E. Lippert, Z. Elektrochem. 61 (1957) 962. N.G. Bakhshiev, Opt. Spektrosk. 16 (1964) 821. A. Chamma, P. Viallet, C. R. Acad. Sci. Paris, Ser. C 270 (1970) 1901. J.A. Pople, G.A. Segal, J. Chem. Phys. 44 (1966) 3289. F. Moll, E. Lippert, Z. Elektrochem. 58 (1954) 853. D.R. Besirre, E.L. Quitevis, J. Phys. Chem. 98 (1998) 13088. P. Suppan, Chem. Phys. Lett. 3 (1983) 272. S.M. Khopde, K.I. Priyadarsini, D.K. Palit, T. Mukherjee, Photochem. Photobiol. 72 (2000) 625. S. Kothainayaki, M. Swaminathan, J. Photochem. Photobiol., A: Chem. 84 (1994) 84 . N.G. Bakhshiev, Opt. Spectrosc. 13 (1962) 24. C. Reichardt, Solvents and Solvent Effects in Organic Chemistry, VCH, Weinhwim, 1988. J.R. Lakowicz, Principle of Fluorescence Spectroscopy, Plenum Press, New York, 1983. E.V. Donckt, Bull. Soc. Chim. Belg. 78 (1969) 69. I.N. Polyakova, Z.A. Starikova, B.V. Parusnikov, I.A. Krasavin, G.M. Dobryakova, B.V. Zhadanov, J. Struct. Chem. 25 (1984) 98 (Engl. Transl.). T.C. Werner, R.M. Hoffman, J. Phys. Chem. 77 (1973) 1611. H.M. Rosenberg, E. Eimutus, Spectrochim. Acta 22 (1966) 1751.
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Solvent effect on the ground and excited state dipole moments of ¯uorescein Bilal AcemiogÆlu a,1, Mustafa Arõk a, Hasan EfeogÆlu b, Yavuz Onganer a,* a
Department of Chemistry, Faculty of Arts and Sciences, AtatuÈrk University, 25240 Erzurum, Turkey b Department of Physics, Faculty of Arts and Science, AtatuÈrk University, 25240 Erzurum, Turkey Received 22 February 2001; revised 18 April 2001; accepted 18 April 2001
Abstract The ground-state and excited-state dipole moments of ¯uorescein were studied at room temperature in n-alcohols (methanol± n-hexanol) and acetonitrile and acetonitrile±benzene solvent mixtures. The excited-state dipole moments were estimated from Lippert's, Bakhshiev's and Chamma±Viallet's equations by using the variation of the Stokes' shift with the solvent dielectric constant and refractive index. Experimental ground-state dipole moments for ¯uorescein in n-alcohols, acetonitrile and acetonitrile±benzene solvent mixtures were estimated by the Guggenheim±Smith method (GSM). It was determined that dipole moments of the excited-state were higher than those of the ground-state in n-alcohols while they were lower than those of the ground-state in acetonitrile and acetonitrile±benzene solvent mixtures. The solute±solvent interactions on the ground-state and the excited-state dipole moments of ¯uorescein are discussed. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Fluorescein; Stokes' shift; Dipole moment; Solute±solvent interactions
1. Introduction Determination of the ground-state and excited-state dipole moments of dye molecules is important, because the values of dipole moments provide information about the change in electronic distrubition upon excitation. Fluorescein, a dye molecule, is a highly ¯uorescent probe (Fig. 1), and it has been ®rst synthesized by Von Bayer in 1871 [1]. The dye is commonly used in laser industry [2±4] as well as used for ¯uorescein angiography in medical treatment * Corresponding author. Tel.: 190-442-233-1970; fax: 190-442233-1062. E-mail address: [email protected] (Y. Onganer). 1 Present address: Department of Chemistry, Faculty of Arts and Sciences, KahramanmarasË SuÈtcËuÈimam University, 46100 K.MarasË, Turkey.
[5]. It has been increasingly used in energy transfer processes, nowadays [6,7]. Moreover, it is widely used to label proteins in biochemistry [8] and to understand ¯uorescence-quenching processes as well [9±11]. A large number of usage of ¯uorescein makes it popular, therefore, it has been subject to most of the scienti®c investigations. Zanker and Peter ®rst showed that ¯uorescein exhibited several prototropic form in the groundstate as a function of pH [12] Then, the ®rst spectroscopic behavior in the excited-state was reported by measuring its ¯uorescence spectra in water as a function of pH by Rozwadowski [13] and Leonhardt et al. [14]. They showed that ¯uorescein had four prototropic forms (neutral, cation, monoanion and dianion). Yguerabide et al. have also studied excited state proton reactions of ¯uorescein in phosphate buffers of varying concentrations [15]. In another work,
0166-1280/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0166-128 0(01)00513-9
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
166
and the excited-state dipole moments of ¯uorescein with the steady-state ¯uorescence spectroscopy technique and Guggenheim±Smith method (GSM) in nalcohols which are polar protic solvents and in acetonitrile (AN) and acetonitrile±benzene (AN±BN) mixtures which are polar aprotic solvents. The molecular inretactions between dye and n-alcohol solvents, AN, and AN±BN solvent mixtures were interpreted in terms of previously proposed equations [18±20]. Fig. 1. Structure of ¯uorescein.
Rohatgi and Singhal found out that ¯uorescein have a dimeric form as a function of concentration in alkaline aqueous solution at pH 12 [16]. On the other hand, the absorption and emission spectroscopic properties of ¯uorescein in n-alcohols, micellar and microemulsion media have been studied by BiswasË et al. [17]. But, there has been no work done with regard to the determination of the ground-state and excited-state dipole moments of ¯uorescein in n-alcohols and acetonitrile±benzene solvent systems. The goal of this paper is to determine and compare the ground-state
2. Experimental Fluorescein was purchased from Sigma and showed a single spot on a TLC plate. Therefore, it was directly used without further puri®cation. Solvents were spectroscopic grade of Merck and used after distilling over CaH2. Fluorescein was stored in the dark as a concentrated stock solution of 5 mM in methanol. Samples were prepared by evaporating 20 and 100 ml of stock solution for n-alcohols, AN and AN±BN solvent mixtures, respectively, and then redissolving with 5 ml of solvent. For the measurements, the ®nal
Table 1 Some physical constants of solvents and spectral data of ¯uorescein in solvents Solvent
l a a (nm)
l f b (nm)
nÅ a c (cm 21)
nÅ f d (cm 21)
nD25e
D 25f
ET(30) g
Methanol Ethanol n-propanol n-butanol n-pentanol n-hexanol Acetonitrile 90%AN±10%BN h 80%AN±20%BN 70%AN±30%BN 60%AN±40%BN 50%AN±50%BN 40%AN±60%BN 30%AN±70%BN 20%AN±80%BN
456 454 453 452 460 452 279 279 279 280 280 281 281 282 282
515 508 507 506 508 504 307 307 306 307 307 308 307 307 308
21930 22026 22075 22123 21739 22124 35842 35842 35842 35714 35714 35587 35587 35461 35336
19417 19685 19724 19763 19685 19841 32573 32573 32680 32573 32573 32468 32573 32573 32468
1.3280 1.3602 1.3833 1.3974 1.4064 1.4165 1.3420 1.3521 1.3642 1.3722 1.3903 1.4044 1.4245 1.4427 1.4616
32.58 24.64 20.15 17.44 14.00 13.30 37.12 34.41 30.46 26.06 24.30 22.17 15.18 12.36 10.67
55.13 52.00 50.35 49.53 49.12 48.60 45.41 44.31 44.00 43.60 43.00 42.40 42.00 40.80 39.96
a b c d e f g h
Wavelength of absorption maxima (uncertainty is ^1 nm). Wavelength of ¯uoresence emission maxima (uncertainty is ^1 nm). Wavenumber of absorption maxima. Wavenumber of ¯uorescence emission maxima. Refractive index. Dielectric constant. Solvent polarity in the unit of kcal/mol. Volume percentages of AN: acetonitrile and BN: benzene.
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171 Table 2 Dipole moments of ¯uorescein in the ground and excited-states for the solvents Solvent
m g a (D)
m e b (D)
m e c (D)
m e d (D)
Methanol Ethanol n-propanol n-butanol n-pentanol n-hexanol Acetonitrile 90%AN±10%BN 80%AN±20%BN 70%AN±30%BN 60%AN±40%BN 50%AN±50%BN 40%AN±60%BN 30%AN±70%BN 20%AN±80%BN
3.97 7.40 10.18 12.15 16.04 18.16 5.25 6.85 7.37 8.07 8.37 9.42 9.86 10.23 10.77
8.82 12.15 15.03 17.00 20.89 23.01 0.48 2.08 2.60 3.30 3.60 4.65 5.09 5.46 6.00
5.00 8.00 10.63 12.53 16.33 18.41 4.41 6.23 6.79 7.54 7.87 8.98 9.44 9.82 10.38
4.56 7.73 10.42 12.35 16.19 18.30 ± ± ± ± ± ± ± ± ±
a The experimental ground-state dipol moments calculated the GSM. b The excited-state dipole moments calculated with Dm Lippert's equation. c The excited-state dipole moments calculated with Dm Bakhshiev's equation. d The excited-state dipole moments calculated with Dm Chamma±Viallet's equation.
from from from from
concentration was 2 £ 10 25 and 1 £ 10 24 M in n-alcohols and in AN and AN±BN solvent mixtures, respectively. Absorption spectra were recorded on a Shimadzu UV±Vis 160A spectrophotometer. Fluorescence spectra were taken by using a Shimadzu RF 5301PC Spectro¯uorophotometer. The capacitance values (the values of C and C0) for the dielectric constants (D) were measured with a capacitory by means of Hewlett Packard 4284 A 20 Hz±1 MHz Precision LCR meter. The refraction indices (n) were determined by using a jena refractometer. All measurements were recorded at room temperature. Some spectral data and solvent related physical parameters are given in Table 1.
3. Results and discussion 3.1. Experimental and theoretical ground-state dipole moments We found that the theoretical dipole moment of
167
¯uorescein was mg 8:83 D by using SCF MO LCAO method in MNDO approximation [21]. This kind of calculation assumes that molecules are involved in the gas phase and does not include solvent interactions. To obtain much more appropriate values of dipole moments for the ground and excited state, spectroscopic techniques, which re¯ect the molecule's electronic state and interactions with the solvent molecules, were employed. The ground-state dipole moments (m g) of ¯uorescein in n-alcohols, AN and AN±BN solvent mixtures with different polarity were estimated according to the GSM [22] that is expressed as 27kT 1 mg 1 AD 2 An M 4pN d D 1 22 where k is the Boltzman constant, T the absolute temperature, N Avagadro's number, d and D are the density and dielectric constant of the solvent, respectively. AD and An are the numerical values obtained from the solution dielectric constant and refractive index measurements, respectively, and M is molecular weight of the solute. The dielectric constants of the solvents and solutions were calculated from the ratio of C/C0. C and C0 are capacitances which are measured when capacitor was full with solvent or solution and empty, respectively. For n-alcohols and acetonitrile, these results are in good agreement with those reported in the literature [23]. The experimental ground-state dipole moment of ¯uorescein according to the GSM has been estimated for each solvent system. The excited-state dipole moments (m e) have been calculated by using the values of m g from the GSM and the values of Dm from Lippert's, Bakhshiev's and Chamma±Viallet's equations. Their values are presented in Table 2. 3.2. Excited-state dipole moments Three formulae were used for the treatment of solvent spectral shift to determine the excited-state dipole moments of ¯uorescein. Lippert's equation [18] is given as " # 2Dm2 D 2 1 n2 2 1 Dn na 2 nf 3 2 2 2 2n 1 1 a0 hc 2D 1 1 where na and nf are the absorption and emission wavenumbers in cm 21 at maxima, respectively. Dm2 is
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Table 3 Solvent polarity functions (for de®nitions, see text. Eqs. (6)±(8)) Solvent
F1
F2
F3
Methanol Ethanol n-Propanol n-Butanol n-Pentanol n-Hexanol Acetonitrile 90%AN±10%BN 80%AN±20%BN 70%AN±30%BN 60%AN±40%BN 50%AN±50%BN 40%AN±60%BN 30%AN±70%BN 20%AN±80%BN
0.309 0.290 0.275 0.264 0.250 0.245 0.307 0.301 0.294 0.287 0.279 0.270 0.249 0.233 0.218
0.854 0.813 0.779 0.751 0.703 0.692 0.861 0.852 0.836 0.817 0.802 0.785 0.715 0.655 0.652
0.651 0.649 0.650 0.645 0.627 0.629 0.663 0.665 0.666 0.662 0.666 0.667 0.645 0.628 0.626
equal to me 2 mg 2 and m e and m g are the excitedstate and ground-state dipole moments, respectively. h is the Planck constant and c is the speed of light. a0 is the solute cavity radius. D and n are the solvent dielectric constant and refractive index, respectively. Bakhshiev's equation [19] is given as " # 2Dm2 D 2 1 n2 2 1 2n2 1 1 Dn na 2 nf 3 2 2 n 1 2 n2 1 2 a0 hc D 1 2 3
and Chamma±Viallet's equation [20] is Dn n 1 nf a 2 2 2Dm2 2 3 a0 hc 1
2n2 1 1 2 n2 1 2 #
D21 n2 2 1 2 2 D12 n 12
! 4
3 n4 2 1 2 n2 1 22
The symbols in Eqs. (3) and (4) are the same as in Eq. (2). The value of the solute cavity radius (a0 ) was calculated from the molecular volume of ¯uorescein according to the following equation [24] a0 3M=4pdN1=3
5
where d is the density of solute molecule, M the molecular weight of the solute and N is the Avagadro's Ê. number. For ¯uorescein, a0 was found to be 5.88 A For Eqs. (2)±(4), the solvent polarity functions or reaction ®eld factors [25,26], F1, F2 and F3, are de®ned as follows: " # D21 n2 2 1 2 2 F1 6 2D 1 1 2n 1 1 2n2 1 1 F2 n2 1 2 F3
Fig. 2. The variation of Stokes' shift with F1 by using Lippert's equation. (X) in AN and AN±BN solvent mixtures; (W) in n-alcohols.
"
"
D21 n2 2 1 2 2 D12 n 12
1 3 n4 2 1 F2 1 2 2 n2 1 22
# 7
8
F1 is used for the Lippert's equation, F2 is used for the Bakhshiev's equation, and F3 is used for the Chamma±Viallet's equation. Their values are listed in Table 3 for the solvent systems employed. When the spectral data of ¯uorescein in Table 1 were analyzed it was found that absorption and ¯uorescence emission maxima are red-shifted in n-alcohols while absorption maxima are blue-shifted in AN and AN±BN solvent mixtures as solvent polarity is increased. But there is no important change of ¯uorescence emission maximum in AN and AN±BN solvent mixtures with increasing solvent polarity. To determine Stokes' shifts in n-alcohols, AN and AN± BN solvent mixtures, we have used the wavenumbers of absorption and ¯uorescence emission at maxima given in Table 1. In order to obtain excited-state
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
Fig. 3. The variation of Stokes' shift with F2 by using Bakhshiev's equation. (X) in AN and AN±BN solvent mixtures; (W) in n-alcohols.
dipole moment values, Stokes' shift (Dn) and the arithmetic mean of (Dn=2) values have been plotted against the solvent polarity functions that are F1, F2, and F3. By taking into account Eqs. (2)±(4); (Dn) vs F1, (Dn) vs F2, and (Dn=2) vs F3 plots were prepared for ¯uorescein in n-alcohols and they were given in Fig. 2 by using Eq. (2), in Fig. 3 by using Eq. (3), and in Fig. 4 by using Eq. (4). The same procedure was applied for the data obtained by using AN and AN±
Fig. 4. The variation of arithmetic mean of Stokes' shift with F3 in n-alcohols by using Chamma±Viallet's equation.
169
BN solvent systems and their plots were given in Figs. 2, 3 and 5. From the slopes of these plots, Dm values were calculated. Since Dm is (m e 2 m g) [27], one can obtain m e. We found that the excited-state dipole moment values were higher than those of groundstate (m e . m g) in n-alcohols according to all the three equations while the excited-state dipole moments were lower than those of ground-state (m e , m g) in AN and AN±BN solvent mixtures, except for Chamma±Viallet's equation. These results suggest more stable excited singlet-states relative to the ground-state in n-alcohols [28]. In AN and AN± BN solvent mixtures this situation is opposite to those in n-alcohols. According to the three equations, the Stokes' shifts versus solvent polarity functions should be linear in the presence of general solvent effects as a function of the dielectric constant and the refractive index. The deviations from the linearity imply the speci®c solute±solvent interactions [29]. These deviations are related to the extent of the interactions between solute and solvent molecules and these interactions lead to the energy difference changes between the ground and the excited-state. Another approach is to estimate excited-state dipole moment values by using the values of Dm from the Lippert's, Bakshiev's and Chamma±Viallet's equations with the value of theoretical dipole moment, mg 8:83 D; for n-alcohols, AN and AN±BN solvent mixtures. In n-alcohols, the excited-state dipole moment values from the Lippert's, Bakshiev's and Chamma±Viallet's equations are 13.68, 9.34, 9.11 D, respectively. The correlation coef®cents are 0.77 and 0.79 (Figs. 2 and 3, respectively). The poor correlation between the Stokes' shifts and solvent polarity functions is attributed to the contribution of hydrogen bonding in n-alcohols. This situation has also been demonstrated in Ref. [30]. Chamma±Viallet's equation has shown a very poor correlation, even no correlation, and the correlation coef®cient is equal to the value of 0.08 (Fig. 4). This scattering suggests speci®c interactions between solute and solvent molecules (in general, H-bonding). Moreover, the excitedstate dipole moment values compared to the groundstate values are higher according to Eqs. (2)±(4), implying more polar excited-state structure than that of ground-state structure. This H-bonding results from the interactions between n-alcohol molecules and C± OH and CyO groups of ¯uorescein molecule. It has
170
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Fig. 5. The variation of arithmetic mean of Stokes' shift with F3 in AN and AN±BN solvent mixtures by using Chamma±Viallet's equation.
been demonstrated that such interactions between methanol and ¯uorescein molecules were present by X-ray crystallography analysis elsewhere [31]. In acetonitrile and acetonitrile±benzene solvent mixtures, the excited-state dipole moment values from Lippert's and Bakhshiev's equations were found to be 4.06 and 8.35 D, respectively. Stokes' shift plots vs solvent polarity functions are linear with correlation values of 0.93 (Fig. 2) and 0.91 (Fig. 3) according to Lippert's and Bakhshiev's
Fig. 6. The variation of Dn with ET(30). (X) in AN and AN±BN solvent mixtures; (W) in n-alcohols.
equations, respectively. This shows the presence of general solvent effects as a function of dielectric constant and refractive index. Based on Eq. (4), Dn=2 vs F3 plot does not obey such a relationship (Fig. 5) for AN and AN±BN solvent mixtures. Moreover, the excited-state dipole moment values in AN and AN±BN solvent mixtures compared to the ground-state values are lower than those of groundstate dipole moment, indicating more polar groundstate structure than the excited-state structure. If one considers the excited-state dipole moment values from ¯uorescence spectroscopy technique and ground-state dipole moments from the GSM as shown in Table 2, it can be seen that Lippert's equation is more sensitive to the speci®c solute±solvent interactions while Bakhshiev equation is more sensitive to the general solvent effects [29]. Somehow, for both solvent systems, Chamma±Viallet's equation does not indicate any trend regarding interactions. If the dipole moment changes of ¯uorescein were dependent only on solvent polarity, the plot of Dn vs. ET(30) should have indicated a linear relationship for all solvents employed [32,33]. But this has not been observed for our system, as shown in Fig. 6, indicating speci®c solute±solvent interactions. Therefore, the protic solvents (n-alcohols) have a different slope than those for aprotic solvents (AN and AN±BN solvent mixtures). As a result, the dipole moment changes of ¯uorescein result from both solvent
B. AcemiogÆlu et al. / Journal of Molecular Structure (Theochem) 548 (2001) 165±171
polarity effect and H-bonding effect as a function of solvent used. 4. Conclusions We have studied dipole moment variations of ¯uorescein in the ground and excited-states as a function of the solute±solvent interactions by using the GSM and the solvatochromism method. We found that ¯uorescein posseses higher dipole moments in the excitedstate than in the ground-state in n-alcohols while it posseses lower dipole moment values in the excitedstate than in the ground-state in AN and AN±BN solvent mixtures. This demonstrates that the excitedstate of ¯uorescein is more polar than the ground-state in n-alcohols while it shows opposite to this situation in AN and AN±BN solvent mixtures. Moreover, one can say that relative to the ground-state the excitedstate in n-alcohols is more stable with solvent effects while the ground-state relative to the excited-state is more stable with solvent effects in the AN and AN± BN solvent mixtures. The speci®c interactions due to H-bonding between ¯uorescein and n-alcohol molecules and the presence of general solute±solvent interactions in AN and AN±BN solvent mixtures were con®rmed by means of Lippert's and Bakhshiev's equations. Acknowledgements We are grateful to the Department of Chemistry and Research Fund of AtatuÈrk University for technical device and support (Project Number: 1998/44). References [1] V. Sun, K.R. Gee, D.H. Klaubert, R.P. Haugland, J. Org. Chem. 62 (1997) 6469.
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