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Energy transfer between fluorescent dyes in alcohols and in nafion membranes
Journal of Luminescence North-Holland
191
46 (1990) 191-199
ENERGY TRANSFER BETWEEN AND IN NAFION MEMBRANES
FLUORESCENT
DYES IN ALCOHOLS
E.-P. NIU a, K.P. GHIGGINO a,1, T.A. SMITH a and A.W.-H. MAU b a Department of Physical Chemistry, University of Melbourne, Parkville, Victoria, 3052, Australia b CSIRO
Division of Chemicals and Polymers, Private Bag IO, Clayton,
Victoria, 3168, Australia
Received 10 July 1989 Accepted 1 November 1989
Singlet state energy transfer between proflavine (donor) and cresyl violet (acceptor) and between acridine orange (donor) and rhodamine B (acceptor) in alcohols and in Nafion (a perfluorosulfonated polymer membrane) has been investigated by steady-state and time-resolved fluorescence spectroscopy. In solution, the fluorescence decay profiles of the donor in the presence of acceptors are non-exponential but follow kinetics predicted from Wrster’s dipole-dipole model for energy transfer. The critical transfer distances obtained from the time-resolved experiments are in good agreement with the values calculated from spectral overlap data in solution. Higher energy transfer efficiencies than expected are observed for the dyes in Nafion membrane. ‘Ihe results suggest that dye molecules are heterogeneously distributed in the micelle-like polymer.
1. Introduction
Singlet state energy transfer in the condensed phase has been a subject of interest in various chemical, physical and biological subdisciplines. Non-radiative transfer of the excited state energy from a donor (D) to an acceptor (A) is primarily a result of dipole-dipole interaction. According to Forster’s model [l], the rate of energy transfer depends on a number of factors including: (i) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, (ii) the relative orientation of the donor and acceptor transition dipoles, and (iii) the distance between these molecules. Non-radiative energy transfer provides a wealth of information concerning the properties and location of a donoracceptor pair, resulting in the widespread application of such measurements. In systems where the distance between donor and acceptor molecules does not change during the lifetime of the donor, the transfer of energy can be described by Fiirster kinetics [l], and
’ Author for correspondence.
Forster’s model has been extended in order to describe the energy transfer that takes place in solution where the distance between the donor and acceptor varies as a result of Brownian motion during the lifetime of the excited donor [2,3]. Time-resolved fluorescence spectroscopy (TRFS) studies are particularly useful for studying such processes, since the fluorescence decay of the donor may be analyzed to determine the energy transfer parameters directly. Nanosecond and picosecond TRFS studies of singlet energy transfer have found that the Forster model is valid over a wide time scale and acceptor concentration range [41. Excitation energy transfer in dye mixtures has been utilized to achieve better dye-laser performance at the desired wavelength. In this paper we report experimental work on several fluorescent dyes in solution and in Nafion, a perfluorosulfonated polymer membrane which is believed to exhibit an inverted micelle-like structure containing both hydrophobic and aqueous cluster regions (fig. 1) [5]. Non-polar aromatic molecules and dyes can be taken up from solutions into Nafion, where they exhibit novel photochemical and nhatonhvcin~l
192
E.-P. Niu et al. / Energy transfer between fluorescent &es
quantum yield of donor emission in the absence of quenching, n is the refractive index of the medium, N is Avogadro’s number, rd is the excited donor lifetime in the absence of acceptor, and J is the overlap integral which expresses the degree of spectral overlap between the donor emission and the acceptor absorption, i.e. J=
=%(J+a(V)
J0
dv
v4
.
~-40.4
d-1OA Fig. 1. Proposed channel and aqueous cluster networks for water-swollen Nafion. 8: SO;. D = 40 A; d = 10 A.
behaviour [6,7]. The purpose of this paper is to report the results of experimental investigations of the mechanism of energy transfer between fluorescent dyes in solution and in Nafion. In addition, the polymer was investigated as a potential useful medium for promoting energy transfer processes. The donor: acceptor systems chosen for study were proflavine (PR): cresyl violet (CV), and acridine orange (AO): rhodamine B (RB) in both alcohol and Nafion membrane. The data show that the FSrster model for energy transfer is valid in these systems. The energy transfer efficiencies are higher in Nafion than in alcohol solutions, which can be attributed to the spectral properties of the dye molecules in the different media and their heterogeneous distributions in the polymer membrane.
In eq. 2, &(Y) is the donor fluorescence intensity at v (normalized to unit area on a wavenumber scale) and Ed is the extinction coefficient of the acceptor at v. R, is the Fijrster “critical transfer distance” at which the transfer rate is equal to the decay rate of the donor in the absence of the acceptor (kT = T;~) and can be derived from eq. (1) (3) R, can thus be calculated from the known spectral properties of D and A. Using this definition of R, and eq. (l), the rate of energy transfer can be simply given by
(4) The energy transfer efficiency, E, is the fraction of photons absorbed by the donor, which are transferred to the acceptor, and is given by
2. Theoretical background By considering the weak coupling of the electronic and vibronic states of donor and acceptor by a dipole-dipole interaction, Forster [l] showed that the rate of energy transfer from a specific donor to a specific acceptor (kr), separated by a fixed distance (r), may be described by eq. (1)
The transfer efficiency is frequently calculated from the relative fluorescence yield in the presence (@da,) and absence of acceptor (@da>,or from the lifetimes under these respective conditions ( rda and
7d)
E=l-(Gdahd’d),
E=l-
(7d,/Td>a
(6)
Substituting eq. (4) into eq. (5) yields the transfer efficiency as a function of distance where ~~ is a factor describing the relative orientation in space of the transition dipoles of D and A (K’ = f assuming a random orientation), +d is the
ES_
R6, R6,+r6'
E.-P. Niu et al. / Energy transfer between jluorescent &es
Measurement of the rate of energy transfer permits the distance between the donor and acceptor to be calculated. These expressions [eq. (1) and eqs. (3-7)] are only applicable to D-A pairs which are separated by a fixed distance but this condition is generally not found for a mixture of D and A in solution. In the latter case more complex expressions, which are normally derived by averaging the transfer rate over the assumed spatial distribution of D-A pairs, are required. The energy transfer rate will decrease with increasing time after excitation, and the donor fluorescence decays non-exponentially according to eq. (8) [4] -k = p0 exp[ -at
-
g:a312n,Ri
- W2],
(8)
where p (t ) is the ensemble averaged donor decay probability, n, is the acceptor number density (molecules cme3) and g is a numerical factor given by g = ( ;(K2))1’2.
(9)
The average orientation factor (tc’) depends on how the orientations are averaged. In this work, we assume g = 1 for all cases [4]. The energy transfer efficiency for the solution system is
EcnV2(g)
exp(+2[I-erf(E)]V
(IO)
where C is the molar concentration of the acceptor, and C-, is the “critical” molar concentration of the acceptor, given by 01)
Co = (2nzR;) and erf(m) = 2/\/;;/r exp(-x2)
dx.
3. Experimental section 3.1. Materials Cresyl violet and rhodamine B were laser grade dyes (Exciton Chemical, Ohio, USA). The purity of proflavine and acridine orange was ascertained
193
by their absorption and fluorescence spectra [8,9]. Concentrations of dye solutions were determined spectrophotometrically. Spectroscopic grade ethanol, methanol and triply distilled water were used throughout these experiments. Nafion (117) membrane (with thickness of 0.018 cm) was kindly donated by the Polymer Division of DuPont, Delaware, USA. 3.2. Procedure for preparation samples
of Nafon
membrane
Prior to use, the Nafion membranes were cleaned by immersion in boiling concentrated nitric acid and in boiling water (2 x 30 minutes each) [lo]. Sodium-form membranes of Nafion were prepared by stirring the pretreated Nafion in concentrated NaOH solution for at least 1 day. Excess base was then removed by stirring the membrane in several portions of pure water. All membrane samples were equilibrated with the desired solution overnight. The amounts of dye taken up by the membranes were determined by spectrophotometric analysis of the dye remaining in solution. Concentrations of the dyes in Nafion were calculated based on the total membrane volume (- 0.054 cm3 for each sample). 3.3. Instrumentation Absorption spectra relative to a solvent reference at room temperature were obtained using a Hitachi, 150-20 spectrophotometer. The molar extinction coefficient (c) of the acceptors (BB and CV) in membranes were obtained by measuring the optical density of those samples which contained known concentrations of the dyes. Fluorescence spectra and corrected fluorescence quantum yields were obtained at room temperature with a Perkin-Elmer MPF-44A spectrofluorometer by using a resolution of 3 run. Fluorescence decay profiles were recorded by the time-correlated single-photon counting technique by using as an excitation source a synchronously mode-locked and cavity-dumped dye laser (Spectra Physics 171 argon ion laser and Spectra Physics 375 dye laser using Bhodamine 6G). Further details of the instrumentation can be found elsewhere [ll]. Fluo-
194
E.-P. Niu et al. / Energy transfer between fluorescent &es
rescence decay data were analyzed on a VAX 11/780 (Digital) computer by using non-linear least-squares iterative reconvolution programs which were developed in our laboratory. Goodness-of-fit to the fluorescence decay data can be judged by inspection of the weighted residuals, autocorrelation function of the weighted residuals, reduced &i--squared (xf) value and the DurbinWatson parameter (DW) [12].
4. Results and discussion 4. I.
Fluorescence spectral properties of ees
Many studies have shown the ability of Nafion to concentrate polycyclic aromatic hydrocarbons when swollen by water or alcohol [13,14]. The solubility of such molecules in Nafion is surprisingly high compared with an aqueous solution. In this work, four laser-dye molecules - AO, PR, CV and RB - have been successfully incorporated into Nafion membranes by the method described above (see section 3.2). Concentrations of 2 X lop3 M can be readily obtained. Table 1 lists the absorption and fluorescence spectral properties of dyes in water-swollen Nafion (H,O-Nafion) membrane together with those in solution for comparison.
The spectral properties of PR, A0 and RB were found to be strongly pH dependent. We have found that the absorption and emission spectra of CV are also concentration dependent. This may reflect the concentration dependence of the acidbase equilibrium of CV in solution as noted for other dyes [8]. In table 1, the steady-state spectral data of the base form for AO, PR and RB in alcohol solutions are presented. The concentrations of AO, PR, RB and CV were approximately lop5 M in aqueous solutions and 10e4 M in Nafion membranes. The spectral properties of these four dyes in water-swollen Nafion are similar to those in aqueous solutions, although they all show some slight spectral shift. We have also found that their spectra were changed by changing the swelling agent from water to alcohols suggesting that the most probable location for these positively charged chromophores is in the solventaccessible cluster region. Our results suggest that, in H,O-Nafion, dye molecules are most likely in their acid form. The corrected fluorescence quantum yields of the dyes in solution and in Nafion membrane were obtained relative to RB and A0 in ethanol (base form, @aa = 0.71, +Ao = 0.17) [8]. The fluorescence quantum yields are generally higher in H,O-Nafion than in water. It has been reported by Szentirmay et al. [14] that when cationic luminescent probes are taken up from aque-
Table 1 Spectral properties of dyes in different solvents Dye
Solvent
Aabs (rw
Proflavine
MeOH
Cresyl violet
Hz0 Nafron MeOH
A&dine orange
Hz0 Nafion EtOH
Rhodamine B
Hz0 Nafion EtOH H,O
a) Average decay lifetime. b, Ref. (151. ‘) Ref. 181.
405 445 448 593 584 582 430 492 493 542 553 553
(X104) (cm-’ M-l)
c
5.0
6.8 1.2 3.4
11 9.8
x flu
h”
7fI” (ns)
0.19 0.27 0.28 0.54 b, 0.18 0.56 0.17 =) 0.11 0.41 0.71=) 0.34 0.96
4.62
(nm) 500 515 499 623 621 626 525 528 515 570 582 514
4.21 ‘) 3.08 3.79 a) 3.62 3.53 a) 3.20 4.31 a)
E.-P. Niu et al. / Energy transfer between fluorescent dyes
ous solution into Nafion membrane, binding of the dye ions to the anionic sites (within the water cluster phase) on polymer chains occurs. These authors found that in some cases . electrostatic th emission and binding results in a blue shift Qf-.?T an increase in emission quantum yield of these incorporated molecules which they attributed to the interactions of the bound molecules with the polymer chains. The same spectral changes were observed in our experiments. These spectral observations also suggest that the dye molecules are primarily bound to the sulfonated head groups in membrane clusters, as mentioned above. The fluorescence decay profiles of the dyes in Nafion were all non-single exponential but could be adequately fitted by a two-exponential function. It is likely that the dye molecules are in a range of environments resulting in the heterogeneous decay behavior. The average lifetimes, as defined in ref. [16], of the dyes in Nafion are listed in table 1. It is interesting to note that the quantum yield of RB, a widely used laser dye, is 2.8 times higher in Nafion than in H,O and 1.4 times higher than in ethanol. Previous studies [17] have indicated that microviscosity and polarity are important in determining the fluorescence quantum yield from RB. Further spectroscopic studies, particularly of the acid-base spectral properties of these dye molecules in Nafion membrane, will be presented elsewhere.
480
520
560
195
The considerable overlap between the donor fluorescence and acceptor absorption spectrum is illustrated in fig. 2. The spectral overlap integral, J [eq. (2)], was evaluated by numerical integration of the donor fluorescence and acceptor absorption spectra shown in fig. 2. The values of R, calculated from J and other known spectral parameters [eq. (3)] are listed in table 3 (see below). It was assumed that K* in eq. (3) has a value of : corresponding to a random orientation in a homogeneous solvent, although in the heterogeneous environment of the membrane, deviations from this value may apply. Because of the non-single exponential decay of the donors in Nafion membrane, it is difficult to determine R, from the decay data accurately. In this case, only the steady-state spectral data were used to determine the energy transfer parameters. 4.2. Energy transfer processes in alcohol systems The fluorescence decays of donors in the presence of the acceptor at different concentrations were measured for both PR: CV and AORB in alcohol. Donor decays in the absence of an acceptor were exponential with lifetimes of 4.62 ns (PR in methanol) and 3.62 ns (A0 in ethanol). The xf values of the single-exponential fits were about 1.0-1.2, and the Durbin-Watson parameters were larger than 1.8 which indicates an acceptable fit
480
520
560
Fig. 2. The donor fluorescence and acceptor absorption spectra in the spectral overlap region for: left, proflavine: cresyl violet in Nafion, and, right, acridine orange: rhodamine B in Nafion.
E.-P. Niu et al. / Energy transfer between fluorescent dyes
196
represents the one-component
P(Q’Po
Time
(ns)
Fig. 3. Fluorescence decay curves of acridine orange (1 X 10m5 M) in ethanol containing: (a) 0, (b) 1.3X10m3 M and (c) 2.4 x lo- 3 M of rhodamine B.
[12]. In the presence of acceptors the donor fluorescence decay deviated from mono-exponential behaviour. The fluorescence decay curves of the excited donors are shown as a function of acceptor concentrations (fig. 3), displaying the qualitative features expected for dipole-dipole energy transfer. Figure 4 shows the fluorescence decay of A0 in the presence of RB. In fig. 4(a) the solid line
exP
fit using eq. (13)
(2 1*
03)
From the very high values of xf (xf = 2.1), the non-random residuals and low Durbin-Watson parameter (DW = 0.94), it is clear that a single exponential function is not suitable. The curves decay faster and become more non-exponential as the acceptor concentration increases. Equation (8) was used to fit all the fluorescence decay curves of the donors in the presence of acceptors in both solution systems. The quality of the fits were excellent over the entire range of acceptor concentration in each system, as indicated by the improved values of the reduced &i-squared and Durbin-Watson parameters (x: = 0.945, DW = 2.13). As an example, fig. 4(b) shows the fit of the decay curve of A0 in the presence of 1.3 x 10e3 M RB. It can be seen that there is very good agreement between the Fiirster dipole-dipole model [eq. (8)] and the experimental data. In table 2, the Co and R, values calculated from the best fit of the data, for various acceptor concentrations in PR:CV and AO:RB systems, are presented. The average R, values obtained by spectral integration and the fluorescence decay data for
Residuals
Intcnsiy (counts)
Intensity (counts)
I
0
.!\
4
8 Tii
12
(ns)
Fig. 4. Fluorescence decay curves of a&dine orange (1 x 10T5 M) containing 1.3 x 10e3 M of rhodamine B, in which the points represent the experimental data and the solid line is the fitted curve using: left, a single exponential function (TV = 2.7 ns, xf = 2.1, DW = 0.94), and right, F&ster kinetics [eq. (8), (a = 0.296 ns-‘, b = 0.297 ns-‘/2, ~3 = 0.95, DW = 2.13)].
197
E.-P. Niu et al. / Energy transfer between fluorescent &es Table 2 Critical molar concentrations from analysis of fluorescence
(G) and critical transfer distances decay profiles using eqs. (8), (11)
PR : CV in methanol lo4 x C,, (M) 1.0 1.9 2.8 3.6 4.4
0.01290 0.02463 0.04331 0.04886 0.06390
Mean values
at various acceptor concentrations
(C,,). Values determined
: RB in ethanol
A0
b (ns-“2)
both D-A pairs The R, values quite well. This kinetic model is
(Rc)
Ro (A)
lo3 x Co (M)
lo3 x C, (M)
b (ns-‘/*)
Ro (A)
10’ x C,, (M)
39.6 39.7 42.1 40.3 41.2
7.2 7.2 6.0 6.9 6.4
0.67 1.3 1.9 2.4 2.9
0.14516 0.29757 0.43916 0.53394 0.63799
45.2 46.0 46.2 45.6 45.9
4.8 4.6 4.5 4.7 4.6
40.6( f 1.5)
6.7( f 0.7)
45.8( kO.6)
4.6( k 0.3)
are given in table 3 (see below). obtained by each method agree is further evidence that Fiirster’s valid in both alcohol systems.
4.3. Energy transfer processes in Nafion membrane
The R, values of the D-A pairs in Nafion calculated from the spectral integral data are found to be larger than in solution systems. This mainly results from the higher fluorescence quantum .yield of the donors in Nafion membrane when compared with the solution case.
In fig. 5 the fluorescence excitation spectra of the acceptors in the absence and presence of the donors are shown. Since a component of the excitation spectrum of the acceptor corresponds to the absorption spectrum of the donor, it is clear that energy transfer is occurring between the dyes within Nafion membranes. Since the D-A pairs were incorporated in a very thin membrane (- 0.018 cm), reabsorption and self-absorption effects would be negligible, The efficiency of energy transfer (E) in Nafion can be measured by determining the relative quantum yields of donors in the absence and presence of an acceptor [eq. (a)].
Intensity (arbitraryunits) Intensity (arbitraryunits)
@+a)
(i)
absorption maximum of
A
absorption maximum of pr (donor) at 450 run
480 Wavelength (run) Fig. 5. Fluorescence
excitation
Wavelength (run)
spectra of acceptors in the absence (A) and presence (D + A) of the donors. Left, excitation cresyl violet in Nafion; right, excitation spectra of rhodamine B in Nafton.
spectra of
E. -P. Niu et al. / Energy transfer betweenfluorescent @es
198 Table 3 D:A
PR:CV AO:RB
Solvent
J (cm6 mol-‘)
10’ x c, (M)
E =)
ss
FD b,
SS
FD b,
FD =)
6.0 4.5
6.7
0.39
4.2 3.1
4.6
3
(A)
MeOH Nafion
1.7x10-‘3 2.6 x lo-l3
42.7 47.2
40.6
EtOH Nafion
4.2x10-I3 3.7x10-13
48.0 53.4
45.8
r (A) 1-
had /+d
0.62
43.5
0.90
37.0
0.41
‘) The concentration of donor is - 1 x 10e3 M and acceptor is -2x10-3MforCVand1.5x10-3MforRB. b, From table 2. ‘) Using eq. (10). SS From steady-state spectral data. FD From fluorescence decay analysis.
The distance r between acceptors and donors can be calculated from E (table 3). At the concentrations studied, and assuming the dyes are homogeneously distributed in the membranes, the average separation between donor and acceptor would be 80-85 A. The much shorter distances between donor and acceptor determined from energy transfer data suggest that these molecules are non-uniformly distributed in Nafion membranes. As indicated by the spectral properties mentioned above, the cationic dyes are probably bound with the sulfonated head groups within the hydrophilic cluster region and are not incorporated in the hydrophobic regions of the membrane. Because the ionic clusters in Nafion are approximately 30-50 A in diameter [4], the D-A distances of 43.5 and, 37.0 A (for PR:CV and AORB, respectively) suggest that incorporation of the donor and acceptor molecules into the same cluster may be possible. Using the mean values of the “critical concentration” (table 2) and the same acceptor concentration as in Nafion membrane, the energy transfer efficiencies in alcohol systems were calculated for comparison, using eq. (10) (cf. table 3). It can be seen that they are much less than in Nafion systems. This result is due to the different spectroscopic and photophysical properties of the dyes in the two environments, combined with the non-uniform distribution of the dyes in Nafion.
5. Conclusion The results presented above show that incorporated cationic dye molecules are heterogeneously
distributed in Nafion membranes and are most probably anchored within hydrophilic cluster regions of Nafion. For a given concentration of the donor: acceptor pair, the energy transfer efficiency in Nafion is higher than in alcohols. This is attributable to the non-uniform distribution and modified photophysical properties of the dyes in Nafion. The results also suggest that the micellelike polymer may be a useful medium for optimizing energy transfer processes.
Acknowledgement This work was supported by a University of Melbourne/CSIRO Collaborative Research Grant.
References 111Th. Forster, Z. Naturf. 4A (1949) 321. 121J.R Lakowicz, Principles of Fhtorescence Spectroscopy (Plenum, New York, 1983). [31 K. Allinger and A. Blumen, J. Chem. Phys. 72 (1980) 4608. [41 D.P. Millar, R.J. Robbins and A.H. Zewail, J. Chem. Phys. 75 (1981) 3649. 151 S.J. Sondheimer, N.J. Bunce and C.A. Fyfe, JMS-REV. Macromol. Chem. Phys. C26 (1986) 353. [61 E.-P. Niu, K.P. Ghiggino, A.W.-H. Mau and W.H.F. Sasse, J. Lumin. 40 (1988) 563. 171 P.C. Lee and D. Meisel, Photochem. Photobiol. 41 (1985) 21. [81 J. Ferguson and A.W.-H. Mau, Aust. J. Chem. 26 (1973) 1617.
E.-P. Niu et al. / Energy transfer between jluorescent dyes [9] A. Kellmam [lo] [ll] [12] [13]
and Y. Lion, Photcchem. Photobiol. 29 (1978) 217. E. Blatt, A. Launikonis, A.W.-H. Mau and W.H.F. Sasse, Aust. J. Chem. 40 (1987) 1. K.P. Ghiggino, S.W. Bigger, T.A. Smith, P.F. Skilton, and K.L. Tan, ACS Symp. Ser. Vol. 358 (1987) Ch. 28, p. 369. D.V. O’Connor and D. Phillips, Time-Correlated Single Photon Counting (Academic, New York, 1983). P.C. Lee and D. Meisel, J. Am. Chem. Sot. 102 (1980) 5477.
199
[14] M.N. Szentirmay, N.E. Prieto and C.R. Martin, J. Phys. Chem. 89 (1985) 3017. [15] D. Magde, J.H. Bramon, T.L. Cremers and J. Ohnsted, III, J. Phys. Chem. 83 (1979) 6%. [la] D.B. James and W.R. Ware, Chem. Phys. Lett. 120 (1985) 455. [17] M.J. Snare, F.E. Treloar, K.P. Ghiggino and P.J. Thistlethwaite, J. Photochem. 18 (1982) 335.
191
46 (1990) 191-199
ENERGY TRANSFER BETWEEN AND IN NAFION MEMBRANES
FLUORESCENT
DYES IN ALCOHOLS
E.-P. NIU a, K.P. GHIGGINO a,1, T.A. SMITH a and A.W.-H. MAU b a Department of Physical Chemistry, University of Melbourne, Parkville, Victoria, 3052, Australia b CSIRO
Division of Chemicals and Polymers, Private Bag IO, Clayton,
Victoria, 3168, Australia
Received 10 July 1989 Accepted 1 November 1989
Singlet state energy transfer between proflavine (donor) and cresyl violet (acceptor) and between acridine orange (donor) and rhodamine B (acceptor) in alcohols and in Nafion (a perfluorosulfonated polymer membrane) has been investigated by steady-state and time-resolved fluorescence spectroscopy. In solution, the fluorescence decay profiles of the donor in the presence of acceptors are non-exponential but follow kinetics predicted from Wrster’s dipole-dipole model for energy transfer. The critical transfer distances obtained from the time-resolved experiments are in good agreement with the values calculated from spectral overlap data in solution. Higher energy transfer efficiencies than expected are observed for the dyes in Nafion membrane. ‘Ihe results suggest that dye molecules are heterogeneously distributed in the micelle-like polymer.
1. Introduction
Singlet state energy transfer in the condensed phase has been a subject of interest in various chemical, physical and biological subdisciplines. Non-radiative transfer of the excited state energy from a donor (D) to an acceptor (A) is primarily a result of dipole-dipole interaction. According to Forster’s model [l], the rate of energy transfer depends on a number of factors including: (i) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, (ii) the relative orientation of the donor and acceptor transition dipoles, and (iii) the distance between these molecules. Non-radiative energy transfer provides a wealth of information concerning the properties and location of a donoracceptor pair, resulting in the widespread application of such measurements. In systems where the distance between donor and acceptor molecules does not change during the lifetime of the donor, the transfer of energy can be described by Fiirster kinetics [l], and
’ Author for correspondence.
Forster’s model has been extended in order to describe the energy transfer that takes place in solution where the distance between the donor and acceptor varies as a result of Brownian motion during the lifetime of the excited donor [2,3]. Time-resolved fluorescence spectroscopy (TRFS) studies are particularly useful for studying such processes, since the fluorescence decay of the donor may be analyzed to determine the energy transfer parameters directly. Nanosecond and picosecond TRFS studies of singlet energy transfer have found that the Forster model is valid over a wide time scale and acceptor concentration range [41. Excitation energy transfer in dye mixtures has been utilized to achieve better dye-laser performance at the desired wavelength. In this paper we report experimental work on several fluorescent dyes in solution and in Nafion, a perfluorosulfonated polymer membrane which is believed to exhibit an inverted micelle-like structure containing both hydrophobic and aqueous cluster regions (fig. 1) [5]. Non-polar aromatic molecules and dyes can be taken up from solutions into Nafion, where they exhibit novel photochemical and nhatonhvcin~l
192
E.-P. Niu et al. / Energy transfer between fluorescent &es
quantum yield of donor emission in the absence of quenching, n is the refractive index of the medium, N is Avogadro’s number, rd is the excited donor lifetime in the absence of acceptor, and J is the overlap integral which expresses the degree of spectral overlap between the donor emission and the acceptor absorption, i.e. J=
=%(J+a(V)
J0
dv
v4
.
~-40.4
d-1OA Fig. 1. Proposed channel and aqueous cluster networks for water-swollen Nafion. 8: SO;. D = 40 A; d = 10 A.
behaviour [6,7]. The purpose of this paper is to report the results of experimental investigations of the mechanism of energy transfer between fluorescent dyes in solution and in Nafion. In addition, the polymer was investigated as a potential useful medium for promoting energy transfer processes. The donor: acceptor systems chosen for study were proflavine (PR): cresyl violet (CV), and acridine orange (AO): rhodamine B (RB) in both alcohol and Nafion membrane. The data show that the FSrster model for energy transfer is valid in these systems. The energy transfer efficiencies are higher in Nafion than in alcohol solutions, which can be attributed to the spectral properties of the dye molecules in the different media and their heterogeneous distributions in the polymer membrane.
In eq. 2, &(Y) is the donor fluorescence intensity at v (normalized to unit area on a wavenumber scale) and Ed is the extinction coefficient of the acceptor at v. R, is the Fijrster “critical transfer distance” at which the transfer rate is equal to the decay rate of the donor in the absence of the acceptor (kT = T;~) and can be derived from eq. (1) (3) R, can thus be calculated from the known spectral properties of D and A. Using this definition of R, and eq. (l), the rate of energy transfer can be simply given by
(4) The energy transfer efficiency, E, is the fraction of photons absorbed by the donor, which are transferred to the acceptor, and is given by
2. Theoretical background By considering the weak coupling of the electronic and vibronic states of donor and acceptor by a dipole-dipole interaction, Forster [l] showed that the rate of energy transfer from a specific donor to a specific acceptor (kr), separated by a fixed distance (r), may be described by eq. (1)
The transfer efficiency is frequently calculated from the relative fluorescence yield in the presence (@da,) and absence of acceptor (@da>,or from the lifetimes under these respective conditions ( rda and
7d)
E=l-(Gdahd’d),
E=l-
(7d,/Td>a
(6)
Substituting eq. (4) into eq. (5) yields the transfer efficiency as a function of distance where ~~ is a factor describing the relative orientation in space of the transition dipoles of D and A (K’ = f assuming a random orientation), +d is the
ES_
R6, R6,+r6'
E.-P. Niu et al. / Energy transfer between jluorescent &es
Measurement of the rate of energy transfer permits the distance between the donor and acceptor to be calculated. These expressions [eq. (1) and eqs. (3-7)] are only applicable to D-A pairs which are separated by a fixed distance but this condition is generally not found for a mixture of D and A in solution. In the latter case more complex expressions, which are normally derived by averaging the transfer rate over the assumed spatial distribution of D-A pairs, are required. The energy transfer rate will decrease with increasing time after excitation, and the donor fluorescence decays non-exponentially according to eq. (8) [4] -k = p0 exp[ -at
-
g:a312n,Ri
- W2],
(8)
where p (t ) is the ensemble averaged donor decay probability, n, is the acceptor number density (molecules cme3) and g is a numerical factor given by g = ( ;(K2))1’2.
(9)
The average orientation factor (tc’) depends on how the orientations are averaged. In this work, we assume g = 1 for all cases [4]. The energy transfer efficiency for the solution system is
EcnV2(g)
exp(+2[I-erf(E)]V
(IO)
where C is the molar concentration of the acceptor, and C-, is the “critical” molar concentration of the acceptor, given by 01)
Co = (2nzR;) and erf(m) = 2/\/;;/r exp(-x2)
dx.
3. Experimental section 3.1. Materials Cresyl violet and rhodamine B were laser grade dyes (Exciton Chemical, Ohio, USA). The purity of proflavine and acridine orange was ascertained
193
by their absorption and fluorescence spectra [8,9]. Concentrations of dye solutions were determined spectrophotometrically. Spectroscopic grade ethanol, methanol and triply distilled water were used throughout these experiments. Nafion (117) membrane (with thickness of 0.018 cm) was kindly donated by the Polymer Division of DuPont, Delaware, USA. 3.2. Procedure for preparation samples
of Nafon
membrane
Prior to use, the Nafion membranes were cleaned by immersion in boiling concentrated nitric acid and in boiling water (2 x 30 minutes each) [lo]. Sodium-form membranes of Nafion were prepared by stirring the pretreated Nafion in concentrated NaOH solution for at least 1 day. Excess base was then removed by stirring the membrane in several portions of pure water. All membrane samples were equilibrated with the desired solution overnight. The amounts of dye taken up by the membranes were determined by spectrophotometric analysis of the dye remaining in solution. Concentrations of the dyes in Nafion were calculated based on the total membrane volume (- 0.054 cm3 for each sample). 3.3. Instrumentation Absorption spectra relative to a solvent reference at room temperature were obtained using a Hitachi, 150-20 spectrophotometer. The molar extinction coefficient (c) of the acceptors (BB and CV) in membranes were obtained by measuring the optical density of those samples which contained known concentrations of the dyes. Fluorescence spectra and corrected fluorescence quantum yields were obtained at room temperature with a Perkin-Elmer MPF-44A spectrofluorometer by using a resolution of 3 run. Fluorescence decay profiles were recorded by the time-correlated single-photon counting technique by using as an excitation source a synchronously mode-locked and cavity-dumped dye laser (Spectra Physics 171 argon ion laser and Spectra Physics 375 dye laser using Bhodamine 6G). Further details of the instrumentation can be found elsewhere [ll]. Fluo-
194
E.-P. Niu et al. / Energy transfer between fluorescent &es
rescence decay data were analyzed on a VAX 11/780 (Digital) computer by using non-linear least-squares iterative reconvolution programs which were developed in our laboratory. Goodness-of-fit to the fluorescence decay data can be judged by inspection of the weighted residuals, autocorrelation function of the weighted residuals, reduced &i--squared (xf) value and the DurbinWatson parameter (DW) [12].
4. Results and discussion 4. I.
Fluorescence spectral properties of ees
Many studies have shown the ability of Nafion to concentrate polycyclic aromatic hydrocarbons when swollen by water or alcohol [13,14]. The solubility of such molecules in Nafion is surprisingly high compared with an aqueous solution. In this work, four laser-dye molecules - AO, PR, CV and RB - have been successfully incorporated into Nafion membranes by the method described above (see section 3.2). Concentrations of 2 X lop3 M can be readily obtained. Table 1 lists the absorption and fluorescence spectral properties of dyes in water-swollen Nafion (H,O-Nafion) membrane together with those in solution for comparison.
The spectral properties of PR, A0 and RB were found to be strongly pH dependent. We have found that the absorption and emission spectra of CV are also concentration dependent. This may reflect the concentration dependence of the acidbase equilibrium of CV in solution as noted for other dyes [8]. In table 1, the steady-state spectral data of the base form for AO, PR and RB in alcohol solutions are presented. The concentrations of AO, PR, RB and CV were approximately lop5 M in aqueous solutions and 10e4 M in Nafion membranes. The spectral properties of these four dyes in water-swollen Nafion are similar to those in aqueous solutions, although they all show some slight spectral shift. We have also found that their spectra were changed by changing the swelling agent from water to alcohols suggesting that the most probable location for these positively charged chromophores is in the solventaccessible cluster region. Our results suggest that, in H,O-Nafion, dye molecules are most likely in their acid form. The corrected fluorescence quantum yields of the dyes in solution and in Nafion membrane were obtained relative to RB and A0 in ethanol (base form, @aa = 0.71, +Ao = 0.17) [8]. The fluorescence quantum yields are generally higher in H,O-Nafion than in water. It has been reported by Szentirmay et al. [14] that when cationic luminescent probes are taken up from aque-
Table 1 Spectral properties of dyes in different solvents Dye
Solvent
Aabs (rw
Proflavine
MeOH
Cresyl violet
Hz0 Nafron MeOH
A&dine orange
Hz0 Nafion EtOH
Rhodamine B
Hz0 Nafion EtOH H,O
a) Average decay lifetime. b, Ref. (151. ‘) Ref. 181.
405 445 448 593 584 582 430 492 493 542 553 553
(X104) (cm-’ M-l)
c
5.0
6.8 1.2 3.4
11 9.8
x flu
h”
7fI” (ns)
0.19 0.27 0.28 0.54 b, 0.18 0.56 0.17 =) 0.11 0.41 0.71=) 0.34 0.96
4.62
(nm) 500 515 499 623 621 626 525 528 515 570 582 514
4.21 ‘) 3.08 3.79 a) 3.62 3.53 a) 3.20 4.31 a)
E.-P. Niu et al. / Energy transfer between fluorescent dyes
ous solution into Nafion membrane, binding of the dye ions to the anionic sites (within the water cluster phase) on polymer chains occurs. These authors found that in some cases . electrostatic th emission and binding results in a blue shift Qf-.?T an increase in emission quantum yield of these incorporated molecules which they attributed to the interactions of the bound molecules with the polymer chains. The same spectral changes were observed in our experiments. These spectral observations also suggest that the dye molecules are primarily bound to the sulfonated head groups in membrane clusters, as mentioned above. The fluorescence decay profiles of the dyes in Nafion were all non-single exponential but could be adequately fitted by a two-exponential function. It is likely that the dye molecules are in a range of environments resulting in the heterogeneous decay behavior. The average lifetimes, as defined in ref. [16], of the dyes in Nafion are listed in table 1. It is interesting to note that the quantum yield of RB, a widely used laser dye, is 2.8 times higher in Nafion than in H,O and 1.4 times higher than in ethanol. Previous studies [17] have indicated that microviscosity and polarity are important in determining the fluorescence quantum yield from RB. Further spectroscopic studies, particularly of the acid-base spectral properties of these dye molecules in Nafion membrane, will be presented elsewhere.
480
520
560
195
The considerable overlap between the donor fluorescence and acceptor absorption spectrum is illustrated in fig. 2. The spectral overlap integral, J [eq. (2)], was evaluated by numerical integration of the donor fluorescence and acceptor absorption spectra shown in fig. 2. The values of R, calculated from J and other known spectral parameters [eq. (3)] are listed in table 3 (see below). It was assumed that K* in eq. (3) has a value of : corresponding to a random orientation in a homogeneous solvent, although in the heterogeneous environment of the membrane, deviations from this value may apply. Because of the non-single exponential decay of the donors in Nafion membrane, it is difficult to determine R, from the decay data accurately. In this case, only the steady-state spectral data were used to determine the energy transfer parameters. 4.2. Energy transfer processes in alcohol systems The fluorescence decays of donors in the presence of the acceptor at different concentrations were measured for both PR: CV and AORB in alcohol. Donor decays in the absence of an acceptor were exponential with lifetimes of 4.62 ns (PR in methanol) and 3.62 ns (A0 in ethanol). The xf values of the single-exponential fits were about 1.0-1.2, and the Durbin-Watson parameters were larger than 1.8 which indicates an acceptable fit
480
520
560
Fig. 2. The donor fluorescence and acceptor absorption spectra in the spectral overlap region for: left, proflavine: cresyl violet in Nafion, and, right, acridine orange: rhodamine B in Nafion.
E.-P. Niu et al. / Energy transfer between fluorescent dyes
196
represents the one-component
P(Q’Po
Time
(ns)
Fig. 3. Fluorescence decay curves of acridine orange (1 X 10m5 M) in ethanol containing: (a) 0, (b) 1.3X10m3 M and (c) 2.4 x lo- 3 M of rhodamine B.
[12]. In the presence of acceptors the donor fluorescence decay deviated from mono-exponential behaviour. The fluorescence decay curves of the excited donors are shown as a function of acceptor concentrations (fig. 3), displaying the qualitative features expected for dipole-dipole energy transfer. Figure 4 shows the fluorescence decay of A0 in the presence of RB. In fig. 4(a) the solid line
exP
fit using eq. (13)
(2 1*
03)
From the very high values of xf (xf = 2.1), the non-random residuals and low Durbin-Watson parameter (DW = 0.94), it is clear that a single exponential function is not suitable. The curves decay faster and become more non-exponential as the acceptor concentration increases. Equation (8) was used to fit all the fluorescence decay curves of the donors in the presence of acceptors in both solution systems. The quality of the fits were excellent over the entire range of acceptor concentration in each system, as indicated by the improved values of the reduced &i-squared and Durbin-Watson parameters (x: = 0.945, DW = 2.13). As an example, fig. 4(b) shows the fit of the decay curve of A0 in the presence of 1.3 x 10e3 M RB. It can be seen that there is very good agreement between the Fiirster dipole-dipole model [eq. (8)] and the experimental data. In table 2, the Co and R, values calculated from the best fit of the data, for various acceptor concentrations in PR:CV and AO:RB systems, are presented. The average R, values obtained by spectral integration and the fluorescence decay data for
Residuals
Intcnsiy (counts)
Intensity (counts)
I
0
.!\
4
8 Tii
12
(ns)
Fig. 4. Fluorescence decay curves of a&dine orange (1 x 10T5 M) containing 1.3 x 10e3 M of rhodamine B, in which the points represent the experimental data and the solid line is the fitted curve using: left, a single exponential function (TV = 2.7 ns, xf = 2.1, DW = 0.94), and right, F&ster kinetics [eq. (8), (a = 0.296 ns-‘, b = 0.297 ns-‘/2, ~3 = 0.95, DW = 2.13)].
197
E.-P. Niu et al. / Energy transfer between fluorescent &es Table 2 Critical molar concentrations from analysis of fluorescence
(G) and critical transfer distances decay profiles using eqs. (8), (11)
PR : CV in methanol lo4 x C,, (M) 1.0 1.9 2.8 3.6 4.4
0.01290 0.02463 0.04331 0.04886 0.06390
Mean values
at various acceptor concentrations
(C,,). Values determined
: RB in ethanol
A0
b (ns-“2)
both D-A pairs The R, values quite well. This kinetic model is
(Rc)
Ro (A)
lo3 x Co (M)
lo3 x C, (M)
b (ns-‘/*)
Ro (A)
10’ x C,, (M)
39.6 39.7 42.1 40.3 41.2
7.2 7.2 6.0 6.9 6.4
0.67 1.3 1.9 2.4 2.9
0.14516 0.29757 0.43916 0.53394 0.63799
45.2 46.0 46.2 45.6 45.9
4.8 4.6 4.5 4.7 4.6
40.6( f 1.5)
6.7( f 0.7)
45.8( kO.6)
4.6( k 0.3)
are given in table 3 (see below). obtained by each method agree is further evidence that Fiirster’s valid in both alcohol systems.
4.3. Energy transfer processes in Nafion membrane
The R, values of the D-A pairs in Nafion calculated from the spectral integral data are found to be larger than in solution systems. This mainly results from the higher fluorescence quantum .yield of the donors in Nafion membrane when compared with the solution case.
In fig. 5 the fluorescence excitation spectra of the acceptors in the absence and presence of the donors are shown. Since a component of the excitation spectrum of the acceptor corresponds to the absorption spectrum of the donor, it is clear that energy transfer is occurring between the dyes within Nafion membranes. Since the D-A pairs were incorporated in a very thin membrane (- 0.018 cm), reabsorption and self-absorption effects would be negligible, The efficiency of energy transfer (E) in Nafion can be measured by determining the relative quantum yields of donors in the absence and presence of an acceptor [eq. (a)].
Intensity (arbitraryunits) Intensity (arbitraryunits)
@+a)
(i)
absorption maximum of
A
absorption maximum of pr (donor) at 450 run
480 Wavelength (run) Fig. 5. Fluorescence
excitation
Wavelength (run)
spectra of acceptors in the absence (A) and presence (D + A) of the donors. Left, excitation cresyl violet in Nafion; right, excitation spectra of rhodamine B in Nafton.
spectra of
E. -P. Niu et al. / Energy transfer betweenfluorescent @es
198 Table 3 D:A
PR:CV AO:RB
Solvent
J (cm6 mol-‘)
10’ x c, (M)
E =)
ss
FD b,
SS
FD b,
FD =)
6.0 4.5
6.7
0.39
4.2 3.1
4.6
3
(A)
MeOH Nafion
1.7x10-‘3 2.6 x lo-l3
42.7 47.2
40.6
EtOH Nafion
4.2x10-I3 3.7x10-13
48.0 53.4
45.8
r (A) 1-
had /+d
0.62
43.5
0.90
37.0
0.41
‘) The concentration of donor is - 1 x 10e3 M and acceptor is -2x10-3MforCVand1.5x10-3MforRB. b, From table 2. ‘) Using eq. (10). SS From steady-state spectral data. FD From fluorescence decay analysis.
The distance r between acceptors and donors can be calculated from E (table 3). At the concentrations studied, and assuming the dyes are homogeneously distributed in the membranes, the average separation between donor and acceptor would be 80-85 A. The much shorter distances between donor and acceptor determined from energy transfer data suggest that these molecules are non-uniformly distributed in Nafion membranes. As indicated by the spectral properties mentioned above, the cationic dyes are probably bound with the sulfonated head groups within the hydrophilic cluster region and are not incorporated in the hydrophobic regions of the membrane. Because the ionic clusters in Nafion are approximately 30-50 A in diameter [4], the D-A distances of 43.5 and, 37.0 A (for PR:CV and AORB, respectively) suggest that incorporation of the donor and acceptor molecules into the same cluster may be possible. Using the mean values of the “critical concentration” (table 2) and the same acceptor concentration as in Nafion membrane, the energy transfer efficiencies in alcohol systems were calculated for comparison, using eq. (10) (cf. table 3). It can be seen that they are much less than in Nafion systems. This result is due to the different spectroscopic and photophysical properties of the dyes in the two environments, combined with the non-uniform distribution of the dyes in Nafion.
5. Conclusion The results presented above show that incorporated cationic dye molecules are heterogeneously
distributed in Nafion membranes and are most probably anchored within hydrophilic cluster regions of Nafion. For a given concentration of the donor: acceptor pair, the energy transfer efficiency in Nafion is higher than in alcohols. This is attributable to the non-uniform distribution and modified photophysical properties of the dyes in Nafion. The results also suggest that the micellelike polymer may be a useful medium for optimizing energy transfer processes.
Acknowledgement This work was supported by a University of Melbourne/CSIRO Collaborative Research Grant.
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