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J. Chem. SOC.,Furuduy Trans. 2, 1989,85( 12), 1935-1943
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A Spectroscopic Investigation of the Temperature and Solvent Sensitivities of Resorufin Lucia Flamigni," Elisabetta Venuti, Nadia Camaioni, and Francesco Barigelletti" Istituto FRAE-CNR, v. de' Castagnoli 1, 40126 Bologna, Italy The electronic absorption spectra, fluorescence spectra and fluorescence lifetimes of resorufin anion in protic (ethanol-methanol 4 : 1 v/v) and aprotic (propiononitrile-butyronitrile 4 :5 v/v) solvents in the temperature range 87-293 K are reported. Interaction of the resorufin probe with protic solvent results in the appearance of a spectroscopically identified species, not detected at room temperature. The resemblance of such a species to the neutral form of resorufin, as detected in water at pH < 5 , indicates the involvement of a strong hydrogen bond between the solvent and the alkoxide ion of resorufin. Formation of such a complex is consistent with 'super-stick' behaviour of resorufin in alcohols. At temperatures <150 K, where both protic and aprotic solvents are rigid, quenching of the emitting state of the resorufin anion is seen. Analysis of the lifetime behaviour us. temperature provides the activation energy, A E,, for the solvent rearrangement affecting the emission in the 110-160 K temperature interval ( A & = 1980 and 3600 cm-' for alcohol and nitrile solvents, respectively). These results are discussed in terms of ion-solvent interaction and of shielding of the anionic charge by primary solvation in alcohols.
The interest for studies investigating solute-solvent interactions derives from the possibility of obtaining insights into the liquid molecular dynamics as well as into the very nature of the interactions. Studies on the rotational dynamics'72of dye m01ecules~-~ like resorufin, thionine and cresyl violet have provided information on the effects of (i) molecular shape of the probe, (ii) charge distribution in ground and excited states of the probe and (iii) site-specific and bulk solvent properties. Models for rotational rearrangements which take into account solute-solvent dielectric friction or stick and slip boundary conditions have been developed.* In general, the solute-solvent interactions involving uncharged or positively charged molecular probes conform to slip boundary conditions while anions like resorufin rotate even slower than predicted by stick limits in hydrogen-bonding s o l v e n t ~ . ~In, ~resorufin ,~
the two end oxygen atoms bear partial negative charges, rendering this molecule a rather sensitive probe to either site-specific (hydrogen-bonding) or bulk dipolar solvent properties. In order to provide new insights into the nature of the interactions between resorufin and solvent environment, we present some spectroscopic data obtained in aprotic polar propiononitrile-butyronitrile (4: 5 v/v equimolar mixture) and protic polar ethanolmethanol (4:l v/v) solvents. The temperature was varied from 293 to 87 K. Although 1935
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Temperature and Solvent Sensitivities of Resorufin
the room-temperature polar properties of the employed solvents are similar (the static dielectric constant, E = 27.7, 20.3, 24.3, and 32.6 for propiononitrile, butyronitrile, ethanol, and methanol, respectively') the alcohols exhibit a well defined protic behaviour.' For T < 150 K, the employed solvents gave transparent glasses, l o so allowing spectral investigations to be extended below that temperature. In alcoholic solvents, substantial changes occur for 150 < T / K < 250 in the absorption and emission spectra of resorufin, consistent with the formation of a species not detected at room temperature (see below). For T < 150 K, the increased rigidity of the medium results in the quenching of the emission intensity and lifetime, both in nitrile and alcohol solvents.
Experimental Resorufin was obtained as Na salt by Aldrich and was used as supplied. Thin layer chromatography of concentrated samples did not show traces of impurity. The employed solvents were of the best grade commercially available. Normex buffers (Carlo Erba) were used to adjust the pH in measuring absorption and emission spectra us. pH. Dilute solutions ( 10-5-10-6 mol dmP3) were sealed under vacuum in a 1 cm quartz cell after repeated freeze-pump-thaw cycles. The results obtained were independent of the concentration of the samples. Absorption spectra were recorded on a PerkinElmer LAMBDA 5 u.v.-visible spectrophotometer and uncorrected emission spectra on a Perkin-Elmer MPF44 B spectrofluorimeter. Absorption and emission spectra at various p H were obtained from air-equilibrated samples. Temperature control (293-87 K, uncertainties *2 K) was achieved with a modified C600 Thor cryostat equipped with a 3050 Thor temperature controller. Emission lifetimes were measured by a modified Applied Photophysics time-correlated single-photon counting apparatus. All lifetime measurements were performed using 540 nm excitation, obtained by a monochromated deuterium lamp (full width at half maximum = 2.5 ns) and selecting the emission using an interference filter with transmission maximum at 590nm. The curves were analysed according to a single exponential decay with non-linear iterative programs. '' The quality of the fit was assessed by the xz value close to unity and a regular distribution of the residuals along the time axis. Standard iterative non-linear programs were also employed to extract pertinent parameters from the temperature dependence of the lifetime. l a
Resu1ts Absorption and fluorescence3-' spectra of resorufin dissolved in the polar nitrile solution for the 293-160 K temperature interval are reported in fig. 1. On cooling, the apparent molar absorption coefficient of resorufin increases, the emission intensity showing a parallel trend. The shape and energy position of the spectra (maxima at 588 and 596 nm for absorption and emission, respectively) are unaffected in the quoted temperature interval. Further cooling below 160 K results in slight hypsochromic shifts of the spectral energies until at 87 K the absorption maximum is found at 576 nm and the emission maximum at 593nm. The spectral shifts are accompanied by a reduced absorbance, and taking into account a 20% solvent contraction it is estimated that the absorbance on the maximum reduces by ca. 1/3 at 87 K with respect to that at 160 K. The emission intensity shows a parallel trend, reducing to ca. 1/2 by passing from 160 to 87 K. The absorption and emission spectra of resorufin dissolved in the polar, protic alcohol solution for the 293-150 K interval are reported in fig. 2 and 3, respectively. On cooling, the intensity of the 571 nm absorption band gradually reduces, while a new broad band appears, peaking at 480 nm. In the investigated spectral range, the two bands show an isosbestic point at 508 nm. At 110 K only a small portion of the absorption peaking at 571 nm contributes to the overall spectrum. The reported behaviour indicates that two
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0.50
e,
5
g
0.25,
4
500
550
600
650
hlnm
Fig. 1. Effect of temperature on the absorption and emission spectra of resorufin in nitrile solvent. ( a ) 293 K; ( b ) 240 K; ( c ) 160 K.
equilibrated species are responsible for the spectral changes along with temperature, the species associated with the lower wavelength portion of the spectrum being predominant at low temperature. Fig. 3 shows the temperature dependence of the emission spectrum, as obtained by light excitation of the alcohol solution at 480 nm. On cooling, the intensity of the emission peaking at 593 nm gradually reduces, while a new broad emission (maximum at 556 nm) gains intensity. At 87 K the two emission bands undergo a slight hypsochromic shift with respect to 160 K. The reported sensitivity of resorufin to the environment was further probed by investigating the temperature dependence of the emission lifetime, T f . In fig. 4 and 5, the obtained l / ~ ,data are plotted against 1 / T for the nitrile and alcohol solvents, respectively. At 293 K, Tf = 5.5 and 4.9 ns, while for T < 160 K, rf reduces to 4.1 and 2.9 ns in nitrile and alcohol solvents, respectively. Table 1 collects some of the obtained spectroscopic and photophysical parameters. In order to analyse the temperature dependence of Tf, we found it convenient to fit eqn ( 1 ) to the experimental points of fig. 4 and 5,
where ko is a temperature-independent term and ki( T ) are temperature-dependent rates. Two functional forms were adopted for ki ( T ) , namely an Arrhenius-type expression, I4 where A and AE represent the frequency factor and activation energy parameters, respectively, and an empirical expression. l 5 The. latter takes into account the stepwise behaviour of the 1/ Tf us. 1/ T experimental points around the glass-to-fluid transition
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Temperature and Solvent Sensitivities of Resorujin
1938
Oh
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OD.
Q)
r
fl2 3
0.2
0.0
400
500
600
h/nm
Fig. 2. Effect of temperature on the absorption spectrum of resorufin in alcohol solvent. Full curves: ( a ) 298-265 K; ( b ) 235 K; (c) 220 K; ( d ) 205 K; ( e ) 190 K; (f)175 K; ( g ) 160 K; ( h ) 150 K. Dashed curve, 110 K, not corrected for solvent contraction.
region of the solvents (approximately 110-160 K for the two ~ o l v e n t s ' ~ ) .
ki( T ) = A exp ( - A E I R T )
(2)
ki( T ) = B / { 1 + exp [ C (1/ TB- 1/ T ) ] }
(3) In eqn (3), B is a deactivation rate constant responsible for the shortening of rf for T < T B ,TBis the temperature around which the step in fig. 4 and 5 is centred, and C is a parameter connected with the activation energy for solvent rearrangements affecting the emission behaviour (see below). While the rate constant in eqn (2) is usually related to non-radiative processes,14 the rate constants, k and B, appearing in eqn (1) and (3) might include radiative and non-radiative contributions. lSc The results of the fitting analysis are listed in table 2. While improvement of the fits, as shown in fig. 4 and 5 , required the use of both eqn (2) and (3), only some parameters appearing in eqn (3) will be used in the following discussion.
Discussion 293-160 K Temperature Interval Comparison of the spectral changes as obtained in nitrileI6 and alcohol solvents for the temperature interval 293-160 K (fig. 1-3) suggests that the species developing by cooling the alcohol solvent is connected to the hydrogen-bonding ability of the employed alcohols. This conclusion is consistent with the solvent dependence of the absorption spectrum of resorufin in a series of alcohols, as recently reported by Blanchard and Cihal.' According to the spectral results of fig. 2 and 3, resorufin anion, Res-, and the species responsible for the higher energy spectra, Res-hy, are thermally equilibrated Res-
Res-hy.
(4)
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L. Flamigni et al.
0.0 500
550
1939
600
650
A/nm
Fig. 3. Effect of temperature on the emission spectrum (not corrected for absorbance changes) of resorufin in alcohol solvent. A,,, = 480 nm. Full curves: ( a ) 298-265 K; ( b ) 215 K; (c) 190 K; ( d ) 175 K; ( e ) 160 K. Dashed curve (not corrected for solvent contraction), 87 K.
Table 1. Spectroscopic and photophysical parameters for resorufin
nitrile alcohol'
588 57 1
595 593
5.5 4.gd
576 480', 571'
593 556', 593'
4.1 2.9
'
Unless otherwise stated. Propiononitrile-butyronitrile(4:5 v/v). Ethanol-methanol (4:1 v/v). The emission quantum yield, & = 0.63, and the radiative lifetime, T: = 7.8 ns ( bf= Tf/7:). 7: was calculated according to l / ~ y= 2.88 x n 2 V ~ , x ~ , , , V 1 / 2 , 1 2 * 1 3 where n is the averaged refracis the frequency maximum of the absorption spectrum, tive index of the solvent mixture, V, E,,, = 45 000 dm3 mol-' cm-I is the extinction coefficient of resorufin at 571 nm and VljZ is the absorption bandwidth at half maximum. At 160 K. a
In order to discuss the nature of Res-hy, spectral measurements were performed on resorufin dissolved in water solutions at different pH values. Fig. 6 shows the observed absorption spectral changes. As one can see, the absorption spectrum of the protonated species, ResH, as obtained for pH < 5 , resembles that of Res-hy, the absorption spectrum of ResH showing an additional shoulder at 400 nm, not present in the spectrum of Res-hy. A possible explanation for the reported behaviour in acidic solution relies on aggregate formation." While charged Res- species are unlikely to aggregate because of unfavourable electrostatic interactions, protonation results in formation of neutral ResH species which could conceivably undergo aggregation processes. The spectra in
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Temperature and Solvent Sensitivities of ResoruJin
1940
2.80 2.58
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2.37 2.15 1.93
1.72
2.00
3.25
4.50
5.75
7.00
8.25
9.50
10.75 12.00
10' K/ T
Fig. 4. Temperature dependence of Tf for resorufin in nitrile solvent, A,,, = 540 nm, A,, = 590 nm. The full line results from numerical fitting according to eqn (1)-(3) of the text.
3.47 3.13 X
I
2 x
2.80
I-
12.47
2.13 1.801 2.00
I
I
I
I
I
I
3.25
4.50
5.75
7.00
8.25
9.50
I
10.75 12.00
lo3 K I T
Fig. 5. Temperature dependence of Tf for resorufin in alcohol solvent. A,,, = 540 nm, A,, = 590 nm. The full line results from numerical fitting according to eqn (1)-(3) of the text.
fig. 6 for pH < 5 could result from the overlap of spectra corresponding to ResH and to aggregates? responsible for the absorption shoulder at 400'nm. In the light of the above arguments and according to previous suggestions, Res-hy can possibl be described as an adduct involving Res- and tightly held alcohol molecules. 6 1 1 8 ?At pH = 3-4, the 400 nm absorption shoulder maximizes and excitation performed at that wavelength does not result in any emission. This suggests that aggregates are not emissive.
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1941
Table 2. Kinetic parameters for the deactivation of the luminescence of resorufin"
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nitrile' alcohold
A/s-'
A E /cm-'
B/s-'
AE,h/cm-'
TB/K
1.9 x lo8 4.9 x 10'
17 -11
9.7 x 10' 1.1 x lo8
3600 1980
162 160
" Analysis performed according to eqn (1)-(3) of the text. Estimated uncertainties are 20% on energies, 10% on In(rates) and 2 K on temperatures. AE,= C / R , where C is the parameter appearing in eqn (3) of the text and R is the gas constant (see text for further details). ' Propiononitrile-butyronitrile (4:5 v/v). Ethanol-methanol (4: 1 v/v).
500
400
000
A/nm
Fig. 6. Absorption spectrum of lop5mol dm-3 resorufin in water. ( a ) pH 12; (6) pH 8; (c) pH 7; (4 pH 6; ( 4 pH 5; (f)PH 3; (g) PH 1.
In previous studies, the solvent sensitivity of Res- was discussed in terms of sitespecific and universal interactions with the ~ o l v e n t . ~Rotational -~ reorientation times, Trot, of resorufin at room temperature were about 100,180,350 and 460ps in methanol, ethanol, n-propanol and n-butanol, respectively,? while in various aprotic solvents of comparable viscosity, T,,~< 100 Rotational reorientation times are correlated to hydrodynamic volumes, V,,, , according t02*4*6 Trot
= ( V h y d r / ~)/ ( k , TI
?For the listed alcohols, at room temperature pK, = 18-22.19
(5)
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Temperature and Solvent Sensitivities of Resorujin
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where r ) is the viscosity, K is a dimensionless parameter taking into account stick ( K = 1) or slip ( K < 1) model boundary conditions and kBis the Boltzman constant. For resorufin in alcohol solvents a ‘superstick’ behaviour was noticed: as vh,d = 690 A3 ( K = l),against a calculated van der Waals volume, v,d, = 150-200 A’. The results here reported strongly support the view of Res-hy in alcohol solvents as a chemically distinguishable species, necessarily larger than Res-, and seem in good accord with first solvation sphere (stick) models. 2-6 160-90 K Temperature Interval From previous studies it is known that on cooling, fluctuation of the solvent molecules in an ethanol-methanol (1:4 v/v) mixture becomes a slower and slower process until for T < 150 K, the solvent rearrangement time, r, > 10 ns.20 Measurements performed in our laboratories indicated that for T < 150 K, r,> 10 ns also for the propionitrilebutyronitrile (4:5 v/v) and ethanol-methanol (4: 1 v/v) solvents employed in the present study, so that one concludes that both solvents become rigid on the fluorescence time-scale under observation. As discussed above, for T < 160 K the following changes occur (i) hypsochromic shift of the absorption and emission energy peaks, (ii) broadening of absorption and emission spectral shape, (iii) weakening of the emission intensity. These results indicate that resorufin is rather sensitive to the increased rigidity of the employed solvents at low temperature. The weakening of the emission intensity (not reported here on a quantitative basis) and the sharp shortening of rffor T < 150 K (fig. 4 and 5) point to a quenching mechanism of the emission, possibly involving solute-solvent interactions between the negative partial charges localized on the two end oxygen atoms and the solvent. According to the above discussion, adduct formation in alcohols results in a shielding of the oxygen negative charges leading to a smooth interaction between the probe and the bulk solvent. On the contrary, no adduct formation in nitrile is obtained and ion-dipole interactions between unshielded charges of the solute and solvent are expected to take place at shorter distances. As described in previous work,15b7c the changes of rf of a suitable molecular probe can be related to the changes of T, taking place in the glass-to-fluid transition region of the solvent. An activated form may be assumed for rS2’ r, = T: exp ( A E J R T ) where T: is a high temperature limiting time. On this basis, in the empirical eqn (3), C = AE,/R.”“ Numerical treatment of the experimental l/rf vs. 1/ T data (fig. 4 and 5) gave AE, = 1980 and 3600 cm-’ (table 2) for the alcohol and nitrile solvents, respectively. These findings indicate that the ion-dipole interactions between the anion and the bulk solvent are stronger in nitrile than in alcohols, consistent with a shielding effect on the solute negative charge by the first solvation sphere of the alcohol solvent.
Conclusion Resorufin is highly sensitive to the solvation properties of the probed solvents. In alcoholic solvent, the temperature dependence of the spectral data gives evidence for formation of an adduct, Res-Hy, which behaves like a chemical species (first solvation sphere effect). In addition, in both alcohol and nitrile solvents, slowing down of the solvent molecular motions at low temperature ( T < 150 K) results in marked changes of absorption and emission properties. The latter effects apparently arise from interactions involving the negative charges on the end oxygen atoms and the bulk solvent. The work was supported by ‘Progetto Finalizzato Chimica Fine e Secondaria’ of CNR, Italy. Technical assistance by L. Minghetti is acknowledged.
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1943
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12 13 14 15
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P. Madden and D. Kivelson, Annu. Rev. Phys. Chem., 1980, 31, 523. J. L. Dote, D. Kivelson and R. N. Schwartz, J. Phys. Chem., 1981, 85, 2169. D. Kivelson and K. G. Spears, J. Phys. Chem., 1985, 89, 1999. E. F. Gudgin Templeton and G. A. Kenney-Wallace, J. Phys. Chem., 1986, 90, 5441. E. F. Gudgin Templeton, E. L. Quitevis and G. A. Kenney-Wallace, J. Phys. Chem., 1985, 89, 3238. K. G . Spears and K. M. Steinmetz, J. Phys. Chem., 1985, 89, 3623. G. J. Blanchard and C. A. Cihal, J. Phys. Chem., 1988, 92, 5954. (a) S. L. Murov, Handbook of Photochemistry (Marcel Dekker, New York, 1973); (b) Handbook of Chemistry and Physics (CRC Press, Boca Raton, Fla, 64th edn, 1983-1984). M. J. Kamlet, C. Dickinson and R. W. Taft, Chem. Phys. Lett., 1981, 77, 69. (a) W. G. Horkstroeter, in Creation and Detection of the Excited State, ed. A. A. Lamola (Marcel Dekker, New York, 1971), vol. IA, p. 1; (b) S. D. Doneghan and M. F. Fox, EPA Newsletter, June 1982, 46. (a) P. R. Bevington, Data Reduction and Error Analysisfor Physical Sciences (McGraw-Hill, New York, 1969); (b) F. Barigelletti, S. Dellonte and L. Flamigni, Gazz. Chim. Ital. 1982, 112, 543. N. J. Turro, Modern Molecular Photochemistry, (Benjamin Publishing Co., Menlo Park, California, 1975), chap. 5. J. B. Birks, Photophysics of Aromatic Molecules (Wiley-Interscience, London, 1970), chap. 4. ref. (13), chap. 5. (a) F. Barigelletti, A. Juris, V. Balzani, P. Belser and A. von Zelewsky, fnorg. Chem. 1983, 22, 3335; ( b ) F. Barigelletti, A. Juris, V. Balzani, P. Belser and A. von Zelewsky, J. Phys. Chem. 1987, 91, 1095; (c) F. Barigelletti, J. Chem. Soc., Faraday Trans. 2, 1987, 83, 1567. Possible reasons for the marked changes of absorbance in fig. 1 will be examined elsewhere. Similar behaviour has been reported recently for rhodamine 101, see F. Barigelletti, Chem. Phys. Lett., 1987, 140, 603. F. P. Schaefer, in Dye Lasers, ed. F. P. Schaefer (Springer-Verlag, Berlin-Heidelberg-New York, 1973), p. 1 . F. Kokai, H . Tanaka, J. B. Brauman and M. D. Fayer, Chem. Phys. Lett., 1988, 143, 1. I . Lopez Arbeloa and K. K. Rohatgi-Mukherjee, Chem. Phys. Lett., 1986, 128, 474. F. Barigelletti, J. Phys. Chem., 1988, 92, 3679. M. Davis, in Dielectric Properties and Molecular Behaviour, ed. T. M. Sugden (Van Nostrand Reinhold, London, 1969), p. 280.
Paper 9/01404G; Received 5th April, 1989
