Langmuir 1994,10, 326-329
326
Twisted Charge Transfer Process of Nile Red in Homogeneous Solution and in Faujasite Zeolite Nilmoni Sarkar, Kaustuv Das, Deb Narayan Nath, and Kankan Bhattacharyya* Physical Chemistry Department, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700-032, India Received April 26, 1993. I n Final Form: August 5 , 1 9 9 P Steady-state and time-resolved emission studies on Nile Red, a biological fluorescent probe, in homogeneous solutions and in zeolite 13X and LZY are reported. It ia shown that at moderately high polarity the main nonradiative pathway of Nile Red is twisted intramolecular charge transfer (TICT) process, the rate of which increases exponentially with increase in polarity (E~(30)) of the medium. Due to this polarity-dependentTICT process Nile Red is a sensitiveindicator of the micropolarity of biological environments. From the comparison of the emission properties of Nile Red in zeolite X and Y with those ) of zeolite 13X and LZY are estimated to be 55.5 f 1. This in homogeneoua solutions the E ~ ( 3 0values indicates the polarity of the zeolites is similar to that of an aqueous methanol solution. 1. Introduction
The dynamics and the course of many chemical and biological processes are influenced markedly by the micropolarity of the organized assemblies, a few nanometers in 5i~e.l-l~Hence, for several decades there has been a very active interest in determining the micropolarity of pr0teins,1J-~l5c y c l o d e ~ t r i n smicelles,lOJ1 ,~~ reversed micelles,12J3 and more recently zeolites.3Jk17 For this purpose a large number of fluorescent probes have been used quite extensively because of their remarkable sent ~ * ~has been sitivity to the polarity of the m e d i ~ m . l * ~There a considerable debate regarding the cause of this sensitivity. Cowleyls first noticed the structural similarity of these probes with the molecules exhibiting twisted intramolecular charge transfer (TICT) The 0 Abstract published in Advance ACS Abstracts, November 15, 1993. (1) Weber, G.; Farrie, F. J. Biochemistry 1979, 18, 3075. (2) hkowicz, J. R. InPrinciples ofFluorescence Spectroscopy;Plenum Press: New York, 1983; Chapter VII. (3) Turro, N. J. In Molecular Dynamics in Restricted Geometries; Klafter, J., Drake, J. M., Eds.;John Wiley: New York, 1989; pp 387-404; Pure Appl. Chem. 1986,58, 1219. (4) Sacket, D. L.; Knutaon, J. R.; Wolff, J. J. Biol. Chem. 1990,265, 14899. (5) Sacket, D. L.; Wolff, J. A d . Biochem. 1987,167, 228. (6) Ramamurthy, V.; Eaton, D. F. Acc. Chem. Res. 1988,21,300. (7) Cox, G. S.; Hauptman, P. J.; Turro, N. J. Photochem. Photobiol. 1984, 39, 597. (8) Nag, A.; Chakrabarty,T.; Bhattacharyya, K. J. Phys. Chem. 1990, 94,4203.
(9) Sarkar,N.; Das, K.; Nath, D. N.; Bhattacharyya, K. Chem. Phys. Lett. 1992, 196, 491. (10) Chiang, H. C.; Lukton, A. J. Phys. Chem. 1976, 79,1935. (11) Sarkar,N.; Bhattacharyya, K. Chem. Phys. Lett. 1991,180,283. (12) Menger, F.M.;Donohue, J. A.; Williams, R. F.J. Am. Chem. SOC. 1978,95, 286. (13) Yazdi, P.; McFann, G. J.; Fox, M. A.; Johnston, K. P. J. Phys. Chem. 1990,!44,7225. (14) Liu, X.; Iu, K. K.; Thomas, J. K. J.Phys. Chem. 1989,93,4120. (15) (a) Ramamurthy, V.; Eaton, D. F.;Caspar, J. V. Acc. Chem. Res. 1992,25,299. (b) Ramamurthy,V. In Photochemistry in Organized and Conetrained media; Ramamurthy, V., Ed.; VCH New York, 1991. (16) Ramamurthv, V.: Sanderson, D. R.: Eaton, D. F. Photochem. Photobiol. 1992,56,- 297.. (17) Dutta, P. K.; Tubeville, W. J. Phys. Chem. 1991,95, 4087. (18) Cowley, D. J. Nature 1986, 319, 14. (19) Hicks, J.M.;Vandersall, M.; Babarogic, Z.; Eisenthal,K. B. Chem. Phys. Lett. 1985, 116, 18. (20) Rotkiewin, K.; Grellman, K. H.; Grabowaki, Z. R. Chem. Phys. Lett. 1973,19, 315. (21) Rettig, W. Angew. Chem., Znt. Ed. Engl. 1986,25,971. (22) Reichardt, C. Chem. SOC.Rev. 1992,147. (23) Simon, J. D.; Su, S. G. J. Phys. Chem. 1990,94, 3656. (24) Casey, K. G.; Quitevis, E. L. J.Phys. Chem. 1988,92, 6590.
0743-7463/94/2410-0326$04.50/0
TICT molecules on electronic excitation initially form a moderately nonpolar state with geometry similar to that in the ground state. Subsequently in the “nonpolar” excited state the molecules undergo an intramolecular transfer of an electron from a donor (usuallya dialkylamino group) to an acceptor and this is accompanied by a twist about the bond joining the donor and the acceptor. Thus in the course of the TICT process the molecule goes from a nearly planar “nonpolar” state to a highly polar TICT state in which the donor and the acceptor moiety are mutually p e r p e n d i ~ u l a r . ~Hicks ~ ~ ~et * ~al.19 ~ first demonstrated that the activation barrier for the TICT process decreases linearly with increase in polarity parameter ET(30) of the medium so that the rate of TICT increases . ~ ~ recently the quite drammatically with E ~ ( 3 0 ) More concept of a polarity-dependent barrier for TICT is extended to many other molecules, e.g., (dialkylamin0)phenyl~ulfones,2~ rhodamine laser anilinonaphthalenesulfonates,2B*m etc. The strong polarity dependence of the TICT process is proposed to be responsible for the sensitivity of fluorophores to the polarity of the biological environments.26 Later it is demonstrated quantitatively that the popular biological probes, anilinonaphthalenesulfonates, obey the concept of a polarity-dependentTICT rate.26~27 Recently Nile Red, a laser dye, has been shown to be a highly sensitivefluorescent probe for a number of biological problem^.^^^ The structure of Nile Red (I) consists of an ( H s C J 2 N u ~ ~ \
I
electron donor (diethylamino group) and an electronwithdrawing aromatic system, connected by a flexible ~
~~
~
(25) Nag, A.; Bhattacharyya, K. Chem. Phys. Lett. 1990,169,12. (26) Chang,T. L.; Cheung, H. C. Chem. Phys. Lett. 1990,173, 343. (27) Das, K.; Sarkar,N.;Nath, D. N.; Bhattacharyya,K. Spectrochim. Acta 1992, &A, 1701. (28) Basu, S. In Advances in Qunntum Chemietry;Lowdin, P. O., Ed.; Academic Press: San Diego, CA, 1964; Vol. 1, p 145. (29) Majumdar, D.; Sen, R.; Bhattacharyya, K.; Bhattacharyya, S. P. J. Phys. Chem. 1991,95,4324. (30) Bhattacharyya, K.; Chowdhury, M. Chem. Rev. 1993,93,507. (31) Kosower, E. M. Acc. Chem. Res. 1982, 16, 259. (32) Avouris, P.; Gelbert, W. M.;El-Sayed,M. A. Chem. Rev. 1977,77, 597.
0 1994 American Chemical Society
Langmuir, Vol. 10,No. 1, 1994 327
Twisted Charge Transfer Process of Nile Red
single bond about which rotation is free. It is thus quite reasonable to propose that TICT is the main nonradiative process in the excited electronic state of Nile Red and is the cause of the fluorescence sensitivity of Nile Red. In the present work we wish to establish first that the polaritydependent TICT is, indeed, the most important nonradiative process for Nile Red and then the emission properties of Nile Red will be utilized to estimate the polarity of the faujasite supercages of zeolite X and Y. Of late, there has been much interest on the study of solidstate photochemistry and photophysics of small organic molecules encaged in the pores or the cages of ~eolites.~J’~~ Though a majority of the studies16are focused on how the finite size of the zeolite cavity affects different photoprocesses,the “solventlike”properties of zeolites have also attracted the attention of a number of recent ~orkers.1~J6J7 Dutta and Tubeville” used absorption and Raman spectra of a solvatochromic probe, salicylideneaniline, while Ramamurthy et al.I5J6 used the steady-state emission properties of the well-known TICT molecule (dimethylamin0)benzonitrile (DMABN) to ascertain the micropolarity of the zeolite environment. In the present work we will use both steady-state and time-resolved emission spectroscopy to infer the micropolarity of zeolite 13X and LZY using Nile Red as a probe. 2. Experimental Section Nile Red (Eastman Kodak), zeolite 13X (UOP, Si/A1 = 1.2), and LZY 82 (UOP, Si/Al = 2.5) were used as received. Zeolite (400 mg) is calcined a t 600 OC for 6-7 h, cooled, and then stirred with a 10-mL hexane solution containing 0.20 mg of Nile Red for 10-12 h under nitrogen. The solid was filtered. To remove any Nile Red adsorbed at the surface the solid was washed several times with hexane until the washed hexane showed no trace of absorption due to Nile Red between 450 and 550 nm. The solid was then dried under vacuum for 6-7 h. The dried solid is transferred to emission cells under nitrogen and all measurements were done under nitrogen. The emission spectra were recorded in a Perkin-Elmer MPF 44B fluorometer exciting the sample at 500 nm. To eliminate any scattered light a sharp cut red filter (Corning2-62) was used which cuts off all light below 590nm and has a flat transmission (=90% ) above 630 nm. The fluorescence decays were recorded using a single photon counting setup. The excitationwas done at 550-580 nm using a synchronouslypumped cavity dumped Rhodamine 6G dye laser (Coherent 702) pumped mode locked Antares Nd:YAG laser. by a continuous wave (CW) The detector is a Hamamatsu 2809U microchannel plate PM tube. The response time of the setup is about 100ps. For lifetime measurement, the same filter (2-62) also was used. The deconvolution was done usinggloballifetimeanalysissoftware” (Photon Technology International). The E ~ ( 3 0is) measured using the betaine dye supplied by Professor ReichardtJ2 The quantum yields were determined with respect to that of rhodamine 6G in ethanol as 0.95.%
M ‘0
3.00
1( CI
‘5
2.50
.-u a
a 2.00
150
1 ,, , , , , , , ,, , , , , , , , , , , , , , , , , , , , , 0.10
0.00
0.20
0.30
AI
Figure 1. Lippert plot for Nile Red; 1,n-hexane; 2, dioxane; 3, benzonitrile;4, N,N-dimethylformamide;5, acetone. Correlation coefficient = 0.99. Table I. Emission Properties of Nile Red in Different Solvents. solvent acetonitrile (ACN) 1-butanol ethanol 75% aq ACN methanol 1:l aq alcohol 1:l aq methanol
E ~ ( 3 0 ) 4f 45.7 0.78 49.6 0.34 51.5 0.33 54.7 0.18 55.2 0.22 55.3 0.08 56.1 0.06
h,em Tf kr X (ns) (nm) 10-7 (5-1)
knr X
lo-’ (5-1)
4.8 4.2 3.7 2.6 2.8
16.2 615 4.8 624 15.6 8.2 628 18.3 9.0 31.6 6.9 639 635 27.8 7.9 4.2 1.8 647 51.3 1.6 650 58.9 3.6 hx= 500 nm. Concentration of Nile Red is 1.5 X 1od M.
A v = v m m abs -,,v
em
= (2/cha3)Af(p* - pI2 + constant (1)
where Af =---€ - 1 n 2 - 1 26 + 1 2n2 1
+
t and n respectively denote dielectric constant and refractive index of the medium and a is the radius of the cavity around the Nile Red molecule. p* and p are respectively the dipole moments in the excited- and in the ground-state, respectively. Figure 1 shows a plot of Av against Af for Nile Red in a number of solvents. From structural parameters the radius (a) of the Nile Red molecule is estimated to be 5 A. Using this, we estimate that the dipole moment of Nile Red in the excited state ( p * ) is greater than that ( p ) in the ground state by 7.4 D. This indicates the “nonpolar” excited state of Nile Red from which its emission originates is more polar than the ground state. It may be recalled in the case of DMABN, the classic TICT molecule the dipole moment in the ground state is 5.2 D, while those in the “nonpolar” excited state 3. Results and Discussion and in the TICT state are 6 and 16 D, respectively.% 3.1. Steady-State Emission Spectra. Solvent EfAlong with the red shift the emission quantum yield fects. The emission spectrum of Nile Red exhibits an and lifetime of Nile Red decrease drastically with the appreciable red shift with increase in solvent p ~ l a r i t y . ~ increase in polarity of the medium (Tables I and 11).The The emission maximum shifts from 580 nm in dioxane to emission yield (&) and lifetime (rf)of Nile Red in a series 685 nm in water. This large shift indicates that Nile Red of solvents are given in Tables I and 11. From the values is more polar in the excited state than in the ground state. of experimentally determined (Jf and Tf the radiative (k,) The change in dipole moment on excitation may be and the nonradiative rates (k,) are obtained by using the obtained using the Ooshika-Mataga-Lippert equation2728 relations according to which the difference in absorption energy (vm,aba in cm-1) and emission energy (vmmem) is given by2 r& = krTf and k, + k,, = T ; ~ (2) The emission yield of Nile Red decreases from 0.68 in (33) Knutson, J. R.; Beechem, J. M.; Brand, L. Chem. Phys. Lett. dioxane to 0.04 in 40% aqueous dioxane (Table 11),i.e. by 1983,102, 501. 17 times. Similar large changes of &with polarity are also (34)Kubin, R. F.; Fletcher, A. N. J . Lumin. 1982,27, 455.
328 Langmuir, Vol. 10,No. 1, 1994
Sarkar et al.
Table 11. Emission Properties of Nile Red in Dioxane-Water Misturea
~~
100 95
90 80 70 60 50 40 a
36.6 43.8 46.3 49.2 50.8 52.1 53.4 55.8
068 0.50 0.41 0.28 0.20 0.12 0.07 0.04
4.5 4.4 4.3 4.1 3.7 2.8 2.0 1.3
~
580 614 618 630 637 642 646 650
15 11.3 9.5 6.8 5.4 4.4 3.8 3.4
7.2 11.2 13.4 17.5 21.6 31.0 46.7 74.7
bX= 500 nm. Concentration of Nile Red is 1.5 X 10” M.
Scheme I. Photophysical Processes in Nile Red, Where S l n p and Sitiat Denote Respectively “Nonpolar” and “TICT”Excited States
2000
-
1900
-
1800
-
1700
-
L
-
CI
3
1600
~ , , , , , , , , , , , l , , , , I i, , , , , , , , , i , i , , , , , ,
43
observed in other solvents (Table I). The large change in c#y corresponds to an appreciable increase in the nonradiative rates (knr), e.g. from 7.2 X lo7 s-1 in pure dioxane to 74.7 X lo7s-1 in 40% aqueous dioxane. Obviously with increase in polarity nonradiative rates of Nile Red increase quite sharply and this results in the marked decrease in the quantum yield and lifetime of Nile Red with rise in polarity. Such a strong polarity dependence of nonradiative processes cannot be due to internal conversion (IC) or intersystem crossing (ISC) which depend on the energy gap between the excited state and lower triplets/ground ~tates,~2 and those rates under these conditions can change a t most by a few percent. Specific interactionlreaction between excited Nile Red and solvents is ruled out as the polarity dependence is observed in a wide variety of solvents (Tables I and 11). The only process known in the literature which exhibits such a tremendous polarity dependence is the TICT process.20 Since the structure of Nile Red satisfies all the criterion of the TICT process, namely, an electron donor (diethylamino)and an aromatic acceptor moiety joined by a flexible bond, we propose that polarity dependent TICT is the main nonradiative process in the excited “nonpolar” state of Nile Red. To substantiate this we will show that the polarity dependence of the TICT rates for Nile Red is similar to those 0bserved~9~23-2~ for other TICT probes. 3.2. Effect of Polarity on Nonradiative Rates of Nile Red. Polarity-Dependent TICT Model. To explain the polarity dependence of k,, of Nile Red, we use the two-state model (Scheme I) already used in the case of a number of TICT m o l e ~ u l e s According . ~ ~ ~ ~ to this model the photophysical processes of Nile Red are controlled by two excited states. The molecule is initially excited to the “nonpolar” excited state from which the emission of Nile Red originates. The TICT state is nonemissive, presumably because of rapid nonradiative transfer to triplets.32 During the TICT process the molecule makes a transition from the “nonpolar” excited state (reactant) to the highly polar TICT excited state (product). Evidently the transition state leading to the TICT state is more polar than the reactant (i.e. the “nonpolar”excited state). Thus the activation barrier for the TICT process decreases with increase in polarity. Since the polarity
46
48
50 El(30)
5:
55
50
/
ET(30)
Figure 2. (a, top) Semilog plot of k, of Nile Red versus E ~ ( 3 0 ) of the medium: 1-7 are acetonitrile (ACN), 1-butanol,ethanol, 75% (v/v) aqueous ACN, methanol, 1:l (v/v) aqueous ethanol, and 1:l (v/v)aqueous methanol, respectively. (b,bottom)Semilog plot of k, of Nile Red versus E ~ ( 3 0in) water-dioxane medium. parameter E ~ ( 3 0is) related to solvationenergy,the barrier, EB, is suggested to depend linearly on E ~ ( 3 0as19 )
E, = E’, - A[ET(30)- 301 (3) Thus in a simple Arrhenius model, rate of TICT, k ~ is, given by
k, = kTo exp(-E$RT) (4) In the “nonpolar” excited state of Nile Red the nonradiative processes are TICT, internal conversion (IC), and intersystem crossing (ISC). At high polarity because of the low barrier, rate of TICT is much higher than other nonradiative processes so that one can write k, = kT. Under this condition ln(k,) = ln(k,) = constant + A[ET(30)- 301 (5) Thus at high polarity when TICT is the main nonradiative pathway in the excited state, the plot of ln(k,) versus E ~ ( 3 0should ) be a straight line. In the case of Nile Red, it is readily seen from Figure 2 that such a straight line is obtained for E ~ ( 3 0>) 46 for a number of solvents of different types (Figure 2a). Similar results are also obtained for a series of water-dioxane mixtures (Figure
Twisted Charge Transfer Process of Nile Red
Langmuir, Vol. 10, No. 1, 1994 329
L
Figure 3. Emission spectrum of Nile Red in (a) Zeolite 13X and
(b) Zeolite LZY.
2b). It is evident at lower polarity (E~(30) < 46) the TICT rate is low so that the approximation k,, = k~ is not valid. As a result at lower polarity the curve (Figure 2b) deviates from linearity. Such deviations at low polarities are also observed for anilinonaphthalenesulf~lfonates.~~~~~ The linearity of the plots in Figure 2 at E ~ ( 3 0greater ) than 46 lends strong support to our contention that polaritydependent TICT is the main nonradiative pathway in the “nonpolar” excited state of Nile Red. Thus the dramatic decrease of emission yield and lifetime of Nile Red with increase in polarity may be attributed to the increase in TICT rate. The mechanism of action of Nile Red as a biological fluorescent probe can be understood in the same way. When Nile Red is transferred from highly polar aqueous phase to nonpolar interiors of proteins, the TICT process is inhibited due to reduction in polarity. The suppression of the main nonradiativepathway in the excited “nonpolar” state results in considerable increase in lifetime and fluorescence quantum yield. Thus Nile Red exhibits marked increase in emission quantum yield and lifetime on binding to protein. 3.3. Emission Properties of Nile Red in Faujasite Zeolites. The marked polarity dependence of Nile Red may be utilized to probe the polarity of zeolites. The channel type zeolites (ZSM) are too small to encapsulate the Nile Red molecule and hence could not be studied in this method. However, the faujasite supercages of zeolite 13X and LZY are big enough to host Nile Red. The structure of zeolite 13X consists of spherical supercages of diameter about 13 A which are connected to smaller supercagesthrough windows or pores of diameter 8A.3J4J5 The Nile Red molecule is about 10 A long and 7 A wide and hence can pass easily through the pores into the supercage. Since as discussed in section 3.1 emission energy of Nile Red decreases with increase in solvent polarity the emission maxima of Nile Red is a good indicator of the polarity of the medium. The emission maxima of Nile Red in zeolite 13X (Si/Al = 1.2) and LZY @/A1 = 2.5) are respectively 655 and 640 nm (Figure 3). This indicates LZY is less polar than 13X, which is consistent with previous studies.14 It is evident that the emission energy of Nile Red in zeolite 13X and LZY are between those in methanol and 1:l aqueous methanol (Tables I and 11). Thus it is immediately apparent that the E ~ ( 3 0of ) the zeolite environment is about 55.5 f 1.0.
;
;
;
;
x , 1 0 0 ” ~
’
; ; ;J hboKO~b500 1
I
CHANNEL NUMBER
Figure 4. Fluorescence decay of Nile Red in Zeolite 13X (80
ps/channel).
Table 111. Emission Properties of Nile Red in Zeolite zeolite 13X
loading levela 0.021 0.014
x2
LU-
(nm) A1 T I (ns) A2 TZ (ne) 1.042 655 0.22 0.6 0.78 2.3 LZY 0.995 640 0.75 0.7 0.25 2.5 Number of Nile Red molecules per unit cell of the zeolite. The lifetimes remain unchanged at 3 times the loading level quoted in this table.
It may be mentioned that earlier workers16J6suggested that the polarity of the zeolite environment is between those of methanol (ET= 55.2) and water (ET= 63.0). Thus while the present work is consistent with earlier works, the polarity of zeolite estimated in the present work spans a narrower range. The time-resolved studies indicate that the decay of the fluorescence of Nile Red is biexponential with a short (71 = 0.6 ns) and along component (72 = 2.3-2.5 ns) (Figure 4 and Table 111). The relative contribution of the the short-lived emission to the total emission is A ~ T ~ / ( A I T ~ A2721 and that of the long-lived component is AzTd(Al/71 + A~Tz).It is evident from the value of Ai’s and 7s: in Table I11 that the long-lived component contributes predominantly to the total emission (93% for X and 55% for Y type zeolites). If the lifetimes of the major longlived component are compared with the lifetime of Nile Red in homogeneous solution, we once again conclude the E ~ ( 3 0values ) of the zeolites are 55.5 f 1and that zeolite Y is slightly less polar than zeolite X. Thus both steadystate and time-resolved study indicates that polaritywise, zeolites X and Y are similar to aqueous methanol solution. 4. Conclusions This work indicates that the rather strong polarity dependence of the emission properties of Nile Red arisesfrom the polarity-dependentTICT process. The emission maxima and lifetime of Nile Red in zeolite 13X and LZY-82 indicate that the micropolarity of the zeolite environment is close to that of an 1:l (v/v) aqueous solution of methanol.
+
Acknowledgment. Thanks are due to Professor C. Reichardt for the kind gift of the betaine dye, to Dr. V. Ramamurthy and Dr. A. Biswas for useful discussions, and to the Council of Scientific Research for a grant (I(1154)-EMR/II/90) and DST for a grant (SP/S2/L-30/ 89).
