J. Phys. Chem. 1993,97, 1701-1706
1701
Pyrene Excimer Formation in Individual Oil Droplets Dispersed in Gelatin Matrices: Space- and Time-Resolved Fluorescence Spectroscopy Kiyoharu Nakatani,' Hiroaki Misawa, Keiji Sasaki, Noboru Kitamura,' and Hiroshi Masuhnra' Microphotoconversion Project? ERA TO,Research Development Corporation of Japan, 15 Morimoto-cho, Shimogamo, Sakyo- ku, Kyoto 606, Japan Received: July 17, 1992; I n Final Form: October 23, 1992
Pyrene excimer formation in individual liquid paraffin droplets dispersed in aqueous gelatin matrices was studied by three-dimensional space- and time-resolved fluorescence spectroscopy. The pyrene excimer formation efficiency in the droplets was constant irrespective of the droplet size. The parameters of the excimer formation were determined for each droplet and compared with those observed in bulk pyrene/liquid paraffin solution. The results were discussed on the basis of the depth profiles and the dynamics of pyrene fluorescence in individual liquid paraffin droplets. A distribution equilibrium of pyrene between the oil and the aqueous gelatin was also considered.
1. Introduction
2. Experimental Section
Time-resolved spectroscopic studies are an essential basis to elucidate molecular mechanisms of chemical reactions. Indeed, improvement of time resolution in emission and absorption spectroscopy from microsecond in the 19609 to femtosecond in the late 1980s has provided great advances in mechanistic understandings of photoinduced chemical reactions such as electron transfer, proton transfer, isomerization,ionic dissociation, and so forth.'-' Besides such important roles of lasers in spectroscopy, their applicationto spatially-resolved spectroscopy with high time resolution has been scarcely explored. Recently, wedevelopeda space- and time-resolved fluorescence spectroscopic system, in which a picosecond time-correlated single-photoncountingtechnique was combined with a confocal-laser-scanning microscope.' The new spectrpscopic system was applied to study photochemical and photophysical dynamics in minute volumes such as polymer latex particl&s,4oildroplets? and microcapsules6J dispersed in solution. Among the systems studied, in particular, the chemistry in individual microcapsules was quite unique, interesting, and very important since an effiency of pyrene excimer formation was different between the c a p s ~ l e s .This ~ was ascribed to the concentration distribution of pyrene in the capsules. It is worth noting that such conclusions can be derived only by spectroscopic measurements on individual particles. In the present work, we extended the work to pyrene excimer formation in oil-in-water emulsions (OWES). OWES are of particular importanceas photoinduced electrontransfer reactions across theoil/water interface, which have been extensivelystudied in referenceto chemicalconversion of light energy.* In industrial fields, furthermbte, OWES have been commonly used as color photographic systems.9 Photochemical studies oh OWES are therefore important for understanding electron transfer and the distribution equilibrium of a solute(s) across the interface. We report here on space- and time-resolved fluordcace spectroscopic studfd of excimer formation and the distribution equiiibrium of pyrene in individual liquid paraffin droplets stabilized by surfactants and gelatin. The results are compared with those on individual pyrene/toluene microcapsules and discussed on the factors controllingthepyrene excimer formation in minutevolume. We also investigated a distribution equilibrium of pyrene across the liquid paraffin/gelatin interface.
Materials .nd Sample Preparations. All the chemicals used in this study except liquid paraffin were purchased from Nacali Tesque Inc. Pyrene (GR grade) was recrystallized twice from ethanol after column chromatography on silica gelln-hexane. Methyl viologen (MV2+;GR grade, chloride salt) was recrystallized from ethanol. Liquid paraffin (Uvasol, Merck), sodium dodecyl sulfate (SDS;SP grade), and gelatin (GR grade) were used without further purification. Water was purified by deionization and distillation (Yamato Autostill WG25). Liquid paraffin (1.17g) containing an appropriate amount of pyrene was mixed with 7.99 g of water and an aqueous solution of gelatin (12.5 wt 96, 4.68 g) and SDS ( 5 wt 96, 1.17 g) at 50 OC. The resulting emulsion was further stirred by a homogenizer at 104 rpm for 5 min.I0 The emulsion (0.10g) with the initial pyrene concentration in liquid paraffin ([Pylo) of 8.80 X 10-3 or 2.64 X M was redispersed in aqueous gelatin ( 5 wt 5%,9.90 g) at 50 OC (abbreviated as EO or El, respectively). An El emulsion containing M V2+(0.1 mol/kg) in the aqueous gelatin phase was also prepared (E2)to study the distribution equilibrium of pyrene between the oil and the aqueous gelatin. The diameter of the droplets (4in the emulsions was normally smaller than -25 pmas estimated by an optical microscope. It is worth noting that, although the glass transition temperature of gelatin is dependent on the volume composition of water and gelatin, Brownian motion of the droplets is completely frozen at room temperature under the present conditions of 5 wt 5% gelatin in water. Each emulsion was placed between a slide glass and a quartz cover glass and was sealed with Teflon films, followed by coating with epoxy resin. For fluorescence measurements, deaeration of the sample is preferable. However, degassing of the sample solutions during all the procedures mentioned above was difficult, so we performed the experimentsin air. According to fluorescence lifetime ( T ) measurementsin bulk liquid paraffin solution, the fluorescenceis quenched by oxygen to -605% under aerated condition; s(under air)/T(under Ar) = 151 4 3 7 4 ns = -0.4 for the solution with [Py]= 6.6 X 10-4 M. Spce- a d Time-Resolved Fluorescence Spectroscopy. Fluorescence characteristics of pyrene in an individual droplet were measured by a space- and time-resolved fluorescencespectroscopic system consisting of a confocal-laser-scanning microscope (Zeiss, UMSP-50) and a timecorrelated single-photon-counting system. The experimental setup has been reported in detail el sew her^.^ Briefly, the second harmonic of a cavity-dumped dye laser (Coherent, a 702-1 dye laser and a 7220 cavity dumper), synchronously pumped by a CW mode-locked Nd3+:YLF laser
To whom all correspondence should be addressed. All the correspondences after Sept 1993 should be seat to the permanent address of H. Masuhara at Department of Applied Physics, Osaka University, Suita 565, Japan.
' Five-years term project: Oa 1988Sept 1993.
0022-365419312097-1701SO4.OOlO
0 1993 American Chemical Society
Nakatani et al.
1702 The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 3.75 , X
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Wavelengthlnm Figure 1. Fluorescence spectrum of pyrene in a single liquid paraffin droplet. (a) In El, d = 14 pm; (b) in E2, d = 15 pm. The fluoresence intensities were normalized at 384 nm. (Quantronix, 4217ML), was used as the excitation light source (333 nm, 2 ps, 0.95-3.8 MHz). The excitation laser beam was introduced to the microscope and focused (spot size 51 pm) into the sample solution through an objective lens (X 100, NA = 1.25) of the microscope. The fluorescence was collected by the same objective lens and focused onto the exit pinhole (diameter = 80 pm) of the microscope. Since the system is essentially based on a confocal system with respect to excitation- and fluorescencemonitoring optics, the present experimental setup provides both lateral (XUaxes; -0.5 pm) and longitudinal ( 2 axis; -2 pm) spatial resolution, in addition to time and spectral resolution of -2 ps and 5 nm," respectively. Practically, the spatial resolutions for fluorescence spectral and decay measurements were adjusted to 0.5 pm (Xu)X 2 pm (Z)and 4 pm (Xu)X 32 pm (Z), respectively, depending upon the fluorescenceintensity. Under such conditions,both thedroplet and the gelatin phase were probed, although the excitation laser beam was irradiated to the center of each droplet. Since no emission was detected from the gelatin phase, the fluorescence observed was ascribed to that from pyrene in the individual droplets. All fluorescence measurements were performed at ambient temperature (19-22 "C). 3. Results and Discussion
Pyrene Excimer Formation in Oil-in-Gelatin Emulsions. A typical example of the fluorescence spectrum of pyrene from a single liquid paraffin droplet in El (d = 14 pm) is shown in Figure 1 . Although the vibrational structure of pyrene monomer fluorescence (370-420 nm) is not well resolved owing to the low spectral resolution,Il the spectrum clearly demonstrates an efficient pyrene excimer formation as characterized by broad and structureless fluorescence around 475 nm. To observe the spectrum, the excitation laser beam is focused into a 5 1-pm spot so that the laser intensity is expected to be very high in the focal spot. However, the fluorescence spectral band shape of pyrene did not depend on the variation in the excitation intensity by 1 order of magnitude. Although a high excitation intensity sometimes causes a change in monomer-to-excimer fluorescence intensity ratio thrbugh singlet-singletannihilation,l2such an effect was negligible under the present conditions. The fluorescence intensity ratio of the monomer (I,,, at 384 nm) to the excimer (I, at 475 nm) can be therefore used as a measure for the excimer formation efficiency in the individual pyrene/liquid paraffin droplets. In order to characterize the individual droplets, furthermore, wemeasured the fluorescencedepth (Zaxis) profileof thedroplet. When pyrene is distributed homogeneously in the droplet, the fluorescence intensity profile should obey Lambert-Beer's law.4 Indeed, both I,,,and I, in the logarithmic scale decrease linearly with the depth of the droplet as shown in Figure 2. Furthermore,
10
15
Depthlpm Figure 2. Fluorescence depth profiles of pyrene in a single El liquid paraffin droplet. Monitored at 394 nm (A) and 475 nm ( 0 ) .
5
10
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20
Diameterlpm Figure 3. Dependence of Ie/I,,, on the droplet diameter in EO (+) and E l (0).
since the slopes for I,,,and I, agree with each other, it can be concluded that pyrene is distributed homogeneouslyin the droplet as far as spatial resolution of the spectroscopicsystemis concerned (- 2 pm). According to Lambert-Beer's law, the slope of the profile should correspond to [Py]c33~,where [Py]and €333 are the concentration of pyrene in the droplet and the molar extinction coefficient of pyrene at 333 nm, respectively. €333 was assumed to be equal to that determined for a thin liquid paraffin film of pyrene (2.64 X 10-2 M) under the same experimental conditions: e333 = 2.4 X lo4 M-l cm-1. e333 also agreed with that observed for the same solution by a conventional absorption spectrometer without a microscope. The slope of the profile and 6333 gave [Py] in the droplet to be 1.3 X 10-2 M, which was much lower than [PylO= 2.64 X 10-2 M (discussed later). For pyrene/toluene microcapsules, we previously reported that [Py] could be determined by comparing I,/Im in each capsule with that in homogeneous pyrene/toluene solution.6 The fluorescence depth profile measurements shown above are more direct and reliable to determine [Py]in individual droplets, since the depth profile analysis is essentially based on Lambert-Beer's law. Even if the absolute value of €333 is not known, a relative concentration of pyrene can be determined from the fluorescence depth profile measurements. This is extremely useful to study the chemistry in minute volume. We determined I,/I,,, for the individual droplets in EO and E l as summarized in Figure 3. Figure 3 clearly demonstrates that Ie/I,,, is almost independent of the droplet size and constant at 1 or -0.4 for El or EO, respectively, in the diameter range of 5-20 pm. This is quite contrasting to the results for the pyrene/ toluene microcapsules, where &/I,,, is scattered with the capsule size.6 Scattering of IJI,,, with the capsule size was attributed to concentration distributions of pyrene between the capsules, which was suggested to be originated from mixing processes of water
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The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1703
Pyrene Excimer Formation in Oil Droplets
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equilibrium concentration of pyrene in water partitioned with a liquid paraffin solution of pyrene ([Py] = 2.64 X 10-2 M) is 4 X lo-* M: P = C o i ~ / C=~7 tX~lo5. In the presenceof SDS (3.9 X le3%),thecorrespondingPvaluedecreases to 2 X 10s. Gelatin consists of various proteins and amino acids9 so that the presence of gelatin in water will further induce a partition of pyrene into the aqueous phase through hydrophobic interaction. If one takes such a circumstance in the emulsion into account, P = Coi~/Cgel = 1.3 X lo3 may be a reasonable consequence. Fluorescence D ~ M ~ of C Pyrene S Excimer Formlrtion. Fluorescence rise and decay profiles monitored for pyrene monomer (384 nm) and excimer (500 nm) in the individual droplets are shown in Figure 4. The SIN ratios of the data were good in spite of spectroscopic measurements on a micrometer single droplet. The profiles in Figure 4 were analyzed by eqs 1 and 2, according i M ( t )= A, exp(-t/TJ
+ A, exp(-t/r,)
(1)
iD(t)= A , exp(-t/T,)
- A, exp(-t/T,)
(2)
Timehs 4, m I
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0 .
4
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-41
0
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a
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Timehs Figure 4. Fluorescence decay of the monomer (a) and the decay/rise of the excimer (b) in a single EO (A; d = 10 pm) or El ( B d = 10 pm) liquid paraffin droplet.
and a toluene solution of pyrene. Namely, polymerization of the capsule resin wall (melamine resin) around pyrene/toluene droplets is supposed to proceed before reaching a distribution equilibriumof pyrene between the water and the toluene solutions so that the pyrene concentrationis different between the capsules. In the present system, on the other hand, since the droplets are surrounded by aqueous solution without a polymeric resin wall, distributions of pyrene, paraffin, and/or water between the water and oil phases will be sufficiently equilibrated during sample preparation procedures. This will be a possible reason for the results in Figure 3. Indeed, although the solubility of pyrene in pure water is very poor, pyrene is soluble in an aqueous solution of SDS and gelatin. In the present case, furthermore, the weight percent of liquid paraffin in the emulsion is very low (7.8 X so that pyrene is likely to partition to the aqueous phase during mixing processes. The lower pyrene concentration in the droplets ([Py]= 1.3 X 10-2 M for E l ) relative to [Py]0 = 2.64 X M proves this. The distribution coefficient, P = c o l l / c ~ e l 9where Coil and CseI are the weight molar concentrations of pyrene in the droplet and the gelatin, respectively, is thus calculated to be 1.3 X 103. The
to the Birks kinetics model.13 i M ( r ) and iD(t)are the time response of the monomer and the excimer fluorescence, respectively. 7 , (i = 1-4) is the time constant, and A, (i = 1-4, A, > 0) is the preexponential factor relevant to T,. In the present case, decay of the monomer fluorescence was analyzed by fixing ~2 to T, because the contribution of the former component was very small compared to that of 71. The T, and A, values determined for the data in Figure 4 are summarized in Table I. T I agreed well with 73, and A2 was extremely small as expected. Such unusual pyrene excimer formation dynamics is not characteristic to the micrometer droplets in the gelatin but was observed even in homogeneous bulk liquid paraffin solutions of pyrene with [Py] * 0.88 X 10-2, 1.32 X 10-2, or 2.64 X 10-2 M, abbreviated as SO, S1, or S2, respectively. Although both i M ( t ) and iD(t)for SO, S1, and S2 were fitted by double-exponential functions, T Z did not agree with T ~ the ; results are included in Table I. The bulk solution'data indicate that the contribution of the 7 2 component to iM(t) is very small: A2 = 0.07-0.09. In such a case, exact analysis of iM(t) is, in general, very difficult, so disagreement between 72 and T , will be due to the uncertainty in determining T Z . It is worth noting that the T~ values observed for the droplets are always longer than those for the bulksolutions. As an example, although IJI,,, is almost the same in EO and SO, T I in the former is longer than that in the latter. In a dilute pyrene/liquid paraffin solution([Py] = 6.6 X lo-*M),themonomerfluorcscencelifetime under air (rM(air)) or Argas atmosphere (TM(&)) is 15 1 or 374 ns, respectively. In the gelatin matrices, the effects of 0 2 on the pyrene fluorescence are considerably smaller than those in homogeneoussolution. One explanation is to assume that effective [02] in gelatin is lower than that in pure water. It has been known that gelatin (mixture of water-soluble proteins) contaminates in general small amounts of reducing agents such as inorganic salts, aldehydes, and so forth, and these chemicals in gelatin will react with 0 2 . 9 Therefore, [O,] in gelatin could be lower than that in pure water. The solubility of 02 in liquid paraffin is higher than that in water, and liquid paraffin and aqueousgelatin are vigorously stirred at 50 OC for the preparation of the samples. The actual [02] in the droplets dispersed in the gelatin matrices is thus lower than that in bulk liquid paraffin. This will explain the relatively long T , in droplets as compared with those in the bulk. Further analyses were made based on the Birks kinetics model')
Nakatani et al.
1704 The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 TABLE I: Fluomance Time Comtnnb for Pyrene Excimer Formation sample [ P ~ ] / 1 0 -M~ +]Ins rdns &/AI co.01 EO 0.44 25 1 45.4" 0.03 El 1.3 161 43.8' E2
0.39 0.88 1.32 2.64
so SI
s2
243 134 123 100
43.7" 25.0 39.8 18.5
rdns 248 160 238 136 124 104
co.01
0.07 0.07 0.09
nlns
A4/A3
Iellln
45.4 43.8 43.7 34.5 34.9 33.6
0.91 0.94 0.91 0.90 0.89 0.91
0.43 1 .O
0.36 0.40 0.60 1.2
These values were fixed to 14. TABLE II: Rate Parameters for Pyrene Excimer Formation in Paraffin ~
~~
droplets solutions M
M + M
where DM and ~ M are D the rate constants of excimer (ID*) formation and dissociation of ID*to excited-state (lM*) and ground-state pyrene (M), respectively. ~ F M k, G M , and k q are ~ the radiative, nonradiative, and oxygen-quenching rate constants of 'M*, respectively, and ~ F D ~, G D and , k q represent ~ those of ID*.According to the scheme, i M ( t ) and i D ( t ) can be expressed respectively13 as
where
AI,^ = '/2{x+Y r ( ( Y - x ) *+ ~ ~ M D ~ D M [ M ] ) ' " ]
c = (X-X,)/(X,
x = k,M
-x)
+ k F M + kqMIo21 + k D M I M l
Y = kcD + k F D + k M D + kq,[O21 iM(t) and i D ( t ) should be now characterized by the common rate parameters of XI and X2. In the present case, however, the 7 2 values are less reliable compared with other 7 ,values as discussed above. Therefore, we performed analyseson i M ( r ) and i D ( t ) under theassumptionsof l/XI = ( r l 73)/2and 1 1 x 2 = 74. Thevalidity of the assumptions is discussed later. k D M , ~ M D and , k~ ~ G +D ~ F +D kqD[O2] can be determined on the basis of [MIdependenceson XI + A2 and X1X2 if k~ = kcM + ~ F +M kqM[02] is known. In the bulk solution, k~ corresponds to T M - ~(air) = 6.62 X 106 s-l, as described above. For the liquid paraffin droplets in the gelatin, the contribution of kqM[O2] is small, so we used the data of TM-'(Ar) for kM. Although the number of the data for variation in [MIare limited, in particular for the droplet data, we estimated these rate parameters as listed in Table 11. In cyclohexane, k D M , ~ M Dand , kD have been reported to be 6.7 X lo9 M-1 s-l. 6.5 X lo6 s-1. and 1.55 X lo7 s-I, respecti~e1y.l~Since liquid paraffin is highly viscous (7 100 CPat 20 "C),smaller DM and ~ M values D in both droplets and solutions as compared with those in cyclohexane ( I ) = 0.650 CP at 20 "C)I4arequite understandable. kD is supposed to be almost independent of solvent viscosity, so the present results are in good agreement with k~ in cyclohexane if one takes the variation of kqD[o]2 with the systems into account. DM determined for the bulk liquid paraffin solution is larger than the diffusion-limited rate constant in this solvent, as
+
-
~~
3.5 1.7
~~
~~
I .8 2.4
3.8 3.8
calculated by k d l f f = 8RT/30007 0.65 X lo* M-' s-' (at 20 'c, 7 = 100 cP). The discrepancy between DM and kdlff is much larger for the droplets. Since &MD and k~ for both the droplet and the bulk solution data almost coincide with each other, the larger DM in the droplets relative to that in the bulk solution is meaningful. One possible reason for this is a decrease in 7,which is caused by partition of water and/or SDS to the liquid paraffin. Indeed, the El droplets showed a larger Ie/Im (-1.0) than did the S1 solution (-0.6) even at a similar [Py] of 1.3 X 10-2 M, which supports the above explanation. Another idea to explain DM > kdlff is to assume partial ground-state dimer formation of pyrene and subsequent excimer formation upon excitation. If this is the case, ~ D contains M a rapid component for the excimer fluorescence fr kdlm. This is consistent with A4/A3 < 1.0. The discussion described above is based on the assumptions of l/hl = ( T I + rd/2 and l/X2 = ~ 4 which , are now worth checking their validity. T I and ~3 agreed with each other within experimental error for all the systems (Table I), so l/XI = ( T I + ~ 3 ) / 2 will be reasonable. On the other hand, the assumption of l/X2 = ~4 is not common. However, the rate parameters obtained (Table 11) were quite reasonable as discussed above in detail. To confirm thevalidityofthis assumption, wecalculated A2/A1 (=C, eq 3) based on the rate parameters (Table I). For S2, C was calculated to be 0.070, which is in good agreement with the observed A ~ / A value I of 0.09. Similarly, C was estimated to be 0.065 for El. The Cvalue clearly indicates a small contribution of the TZ component to the overall i M ( t ) for the droplet data, which renders the failure of a double-exponentialfit to iM(t). All the results are consistent with each other, and the present results are well explained along the single context described above. Fluoresc" Analysis of Pyrene Distribution Equilibrium. In order to study thedistribution equilibrium of pyrene between the oil and the aqueous solution, fluorescence quenching of pyrene by MV2+was examined on E2. The fluorescencespectrum from the droplet in E2 is included in Figure 1, where the intensity is normalized to that of El at 384 nm. In the presence of MV2+, the total fluorescence intensity and I,/&, decreased considerably as compared with those in the absence of M V + . Phenomenologically, the pyrene fluorescence is quenched by MV2+. Ic/Im values in E2 droplets are summarized in Figure 5. The values were almost constant around 0.36, independent of the droplet diameter. Figure 6 shows the pyrene fluorescence depth profiles of the E2 droplet monitored at both the monomer and the excimer bands. Fluorescence quenching of pyrene by M V + is expected to proceed at the liquid paraffinlgelatin boundary, since MV2+is concentratedon thesurfaceofthedroplet owing toelectrostaticattraction between MV2+and the sulfate group of SDS,which is solubilized with the dodecyl group being directed to the oil phase. If fluorescence quenching occurs in a wider region than spatial resolution of the spectroscopic system (2 rm), I, or I,,, in the
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The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1705
Pyrene Excimer Formation in Oil Droplets
0*45 0.40
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L
0.30 5 0.25
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Diameterlpm Figure 5. Dependence of I e / I m on the droplet diameter in E2. 11
0
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~~
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Depthlpm Figure 6. Fluorescence depth profiles of pyrene in a single E2 liquid paraffin droplet. Monitored at 394 nm (0)and 475 nm (*).
periphery of the droplet should decrease as compared with that in the central part. However, the fluorescence depth profiles in Figure 6 are analogous to those in the absence of MV2+ (Figure 2), except for the slopes of the profiles along the depth direction. Furthermore, although the fluorescence intensity is different between Z,, and Z,, the profile of Zm is analogous to that of I,. The results indicate that the decreases in the total fluorescence intensity and Ie/Zmin the presence of MV2+ are not ascribable to fluorescence quenching by MV2+. The important point is that the slopes of the fluorescencedepth profiles in Figure 6 are one-third of those in Figure 2. We conducted analogous experiments for E l by adding NaCl (0.1 mol/&) instead of MV2+. However, theslopeof thedepth profile was completely the same with that obtained for the E l droplet, so ionic strength effects cannot explain the present results. If any absorption overlaps with pyrene absorption at the excitation wavelength, the slope of the depth profile should increase so that this possibility can also be neglected. The moderate slopes of the profiles in Figure 6 should, therefore, be explained in terms of a decrease in either [Py]or (333 in the droplet. To test such an idea, we studied the effects of MV2+ on the partition of pyrene for the two-phase system of liquid paraffin ([Pyla = 2.64 X M, 5 g) and water ( 5 g). Addition of MV2+ (0.1 M) to the water phase leads to the appearance of a new absorption band at 339 nm in the water phase (absorbance = -0.3 for a 10-mm optical path), which has been assigned as a ground-state nonfluorescent complex (1 :1) between pyrene and MV2+. Indeed,the association constant between pyrene and MV2+ in water has been reported to be I 1 M-I,l6 though a new absorption reported for the pyrene/ MV2+ CT complex in methanol" was not observed in the liquid paraffinphase. These results indicate that the slopes of the profiles in Figure 6 are responsible for a decrease in [Py]in the droplet. [Py]in the E2 droplet was estimated to be 3.9 X M from the depth profiles. This value gives P = Coil/Cgel= 2.3 X lo2, which is - 5 times smaller than that in the absence of MV2+ (P = 1.3 X 103). Partition of pyrene between the liquid paraffin and
0
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Timelns Figure 7. Fluorescence decay of the monomer (a) and decay/rise of the excimer (b) in a single E2 liquid paraffin droplet (d = 10 pm).
gelatin is very much facilitated by the nonfluorescent pyrene/ MV2+ complex formation in the gelatin phase. Figure 7 showed fluorescence rise and decay curves of pyrene in the E2 droplet. The rise and decay curves of the monomer and excimer fluorescence were best fitted by double-exponential functionssimilar to the results in Figure 4. The Tivaluesobtained are included in Table I. Table I indicates that the T~ values for E2 are almost in good agreement with those for EO: T I = 243251 ns, T~ = 238-248 ns, and T~ = 43.7-45.4 ns. According to the preceding analyses and discussion, the pyrene concentrations in the EO and E2 droplets are almost identical with each other ([Py] = (0.39-0.44) X lo-* M) irrespective of [Pylo. The rate parameters for the excimer formation and Ze/Z,,, in each droplet are therefore governed by [Py]. The results are also in good accordance with no fluorescence quenching of pyrene by MV2+ across the liquid paraffin/gelatin interface. For a study on bimolecular reactions in emulsion systems such as the present case, distribution of the solute between two phases is one of the important factors to be considered. However, determination of the distribution coefficient or the concentration of a solute in droplets and the surrounding phase is in general very difficult. Even if the spectrum of the solute in oil is distinguishablefrom that in water, spectroscopiccharacterization of the solute in individual oil dropletsis impossible by conventional spatially-unresolved spectroscopy. In the present study, the concentration of pyrene in each droplet was determined on the basis of the fluorescence depth profile measurements. This was an essential basis for analyses of both the excimer formation and the distribution of pyrene between the liquid paraffin and the surrounding gelatin phases. Nonetheless, although absorption
1706 The Journal of Physical Chemistry, Vol. 97, No. 8, 1993
spectral changes of pyrene by addition of MV2+were observed by spatially-unresolved absorption measurements for the bulk solution, we failed to find them with absorption spectroscopy on the individual droplets owing to light scattering and strong absorption by MVZ+ and gelatin. Further development of threedimensionalspace- and time-resolvedfluorescenceand absorption spectroscopy permits more exact analysis of bimolecular reaction mechanismsand solute partition in complicated emulsionsystems.
4. Concluding Remarks Three-dimensional space- and time-resolved fluorescence spectroscopy was shown to have a high potential to investigate photochemical and photophysical processes occurring in minute volume. The pyrene excimer formation in individual pyrene/ liquid paraffin droplets dispersed in gelatin matrices was analyzed on the basis of fluorescencedepth profiling as well as fluorescence dynamics in the droplet. A study of the chemistry in OWES is in general performed by spatially-unresolved methods, so the characteristic properties of each droplet and/or the distribution of the solute(s) between a droplet and the surrounding phase have never been discussed. In this point, the present work clearly demonstrated the importance of space- and time-resolved measurements to study both the microscopic and macroscopic aspects on the chemistry and physics in OWEs. Although the present study is limited to fluorescence spectroscopy, we have already developed a space- and time-resolved absorption spectroscopic system,'* and actually, we have started to investigate singletsinglet and triplet-triplet absorption spectra of a dye in individual oil droplets.I9 We are convincedthat such approaches will engage further advances in the research on the chemistry/physics in minute volume as well as on OWEs. References and Nota (1) Mathematical and Physical Science. Application of Picosecond Spectroscopy to Chemistry; Eisenthal, K. B., Ed.; NATO AS1 Series C; D. Reidel: Dordrecht, 1984, Vol. 127.
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