In Situ Fluorescence Imaging and Time-Resolved Total Internal

Jul 27, 2001 - Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan. Langmuir , 2001, 17 (17), pp 53...
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Langmuir 2001, 17, 5337-5342

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In Situ Fluorescence Imaging and Time-Resolved Total Internal Reflection Fluorometry of Palladium(II)-Tetrapyridylporphine Complex Assembled at the Toluene-Water Interface Naozumi Fujiwara, Satoshi Tsukahara, and Hitoshi Watarai* Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received February 19, 2001. In Final Form: May 29, 2001 Assemblies of palladium(II)-5,10,15,20-tetra(4-pyridyl)porphine (tpyp) complex were formed spontaneously at the toluene-water interface, when a tpyp toluene solution was contacted with a PdCl2 aqueous solution under an acidic condition. The interfacial assemblies of tpyp were formed with Pd(II) more effectively than with other divalent metal ions, Ni(II), Cu(II), and Zn(II). It was suggested that Pd(II) of the complex was bound to the nitrogen(s) of the pyridyl groups of tpyp and not to the pyrrole nitrogen. In situ fluorescence microscopy elucidated the formation of two kinds of complex assemblies: assembly 1 (AS1) was generated in a lower tpyp concentration, and assembly 2 (AS2) was generated in a higher tpyp concentration. The fluorescence excitation and emission spectrum of interfacial AS2 showed a red-shift of 7-11 nm in the maximum wavelength relative to that of tpyp in toluene, suggesting a weak π-stacking interaction between tpyp molecules in the assembly AS2. Time-resolved total internal reflection fluorometry determined the fluorescence lifetime of AS1 and AS2 as 0.15 ( 0.05 and 1.1 ( 0.1 ns, respectively. The average stoichiometric composition of Pd/tpyp was suggested to be 3:1 and 1:1 for AS1 and AS2, respectively, by equilibrium analysis, but each tpyp of AS1 was considered to be bound to four Pd(II) with the fluorescence quenching effect by Pd(II). From these results, we concluded that two Pd(II) were shared between two tpyp molecules and that the Pd(II) bridged the two tpyp molecules of AS1.

Introduction Specific phenomena at liquid-liquid interfaces have attracted the interest of researchers in various fields,1,2 such as phase-transfer catalysis,3 colloidal chemistry, solvent extraction,4 and the ion or electron-transfer process.1,5,6 In particular, the role of interfacial complexation in the solvent extraction kinetics has been extensively studied by means of interfacial tension measurement7 and high-speed stirring method.3,4,8 Recently, various spectroscopic methods including second harmonic generation (SHG) spectroscopy,9,10 centrifugal liquid membrane method,11 attenuated total internal reflection (ATR) spectroscopy,12,13 and total internal reflection fluorometry (TIRF)5,14 have been developed so as to measure the liquidliquid interface directly. These methods, however, could usually afford only average spectral information for adsorbed species at the interface. A combination of microscopy and these interfacial spectroscopies is a promising strategy for the direct (1) Liquid-Liquid Interfaces. Theory and Methods; Volkov, A. G., Deamer, D. W., Eds.; CRC Press: Boca Raton, FL, 1996 and references therein. (2) Nelson, A. Langmuir 1996, 12, 2058. (3) Onoe, Y.; Watarai, H. Anal. Sci. 1998, 14, 237. (4) Ohashi, A.; Tsukahara, S.; Watarai, H. Anal. Chim. Acta 1998, 364, 53. (5) Fujiwara, M.; Tsukahara, S.; Watarai, H. Phys. Chem. Chem. Phys. 1999, 1, 2949. (6) Dryfe, R. A. W.; Ding, Z.; Wellington, R. G.; Brevet, P. F.; Kuznetzov, A. M.; Girault, H. H. J. Phys. Chem. A 1997, 101, 2519. (7) Shioya, T.; Tsukahara, S.; Teramae, N. Chem. Lett. 1996, 469. (8) Watarai, H.; Satoh, K. Langmuir 1994, 10, 3913. (9) Higgins, D. A.; Corn, R. M. J. Phys. Chem. 1993, 97, 489. (10) Tohda, K.; Umezawa, Y. Bunseki Kagaku 1999, 47, 1027. (11) Nagatani, H.; Watarai, H. Anal. Chem. 1998, 70, 2860. (12) Sperline, R. P.; Freiser, H. Langmuir 1990, 6, 344. (13) Tsukahara, S.; Watarai, H. Chem. Lett. 1999, 89. (14) Dryfe, R. A. W.; Ding, Z.; Wellington, R. G.; Brevet, P. F.; Kuznetzov, A. M.; Girault, H. H. J. Phys. Chem. A 1997, 101, 2591.

observation and measurement of interfacial compounds at liquid-liquid systems, although it has been often utilized for gas-liquid and solid-liquid interfaces.15-17 Recently, scanning tunneling microscopy (STM) and atomic force microscopy (AFM) have been applied extensively to the studies of surfaces. However, there has been only one report on the observation of liquid-liquid interfaces by AFM18 as far as we know. In the present study, in situ fluorescence microscopy was applied to the study of assemblies of palladium(II)-5,10,15,20-tetra(4pyridyl)porphine (tpyp) complex at the toluene-water interface. In addition, the structure of the interfacial assemblies was discussed with fluorescence lifetimes of tpyp and quenching effects by Pd(II) of the assemblies measured by time-resolved TIRF. Experimental Section Chemicals. 5,10,15,20-Tetra(4-pyridyl)-21H,23H-porphine (tpyp; purity, 97%; Aldrich) and PdCl2 (purity, 99.99%; Wako Pure Chemicals) were used as purchased. Sodium chloride, sodium perchlorate, perchloric acid, and sodium hydroxide were of analytical reagent grade, which were used to adjust the chloride ion concentration, pH, and ionic strength (I) of aqueous phases. Water was distilled and deionized with a Milli-Q system (Milli-Q SP. TOC., Millipore). Tpyp was dissolved in toluene, and its initial concentration was 3.1 × 10-8 to 1.4 × 10-6 M. The initial concentration of PdCl2 in the aqueous phase was set to 1.0 × 10-5 M if not stated. All the aqueous solutions of PdCl2 contained 1.0 × 10-3 M chloride (15) Yoneyama, M.; Fujii, A.; Maeda, S.; Murayama, T. J. Phys. Chem. 1992, 96, 8982. (16) Donner, D.; Bo¨ttcher, C.; Messerschmidt, C.; Siggel, U.; Fuhrhop, J. H. Langmuir 1999, 15, 5029. (17) Taniguchi, M.; Ueno, N.; Okamoto, K.; Karthaus, O.; Shimomura, M.; Yamagishi, A. Langmuir 1999, 15, 7700. (18) Hartley, P. G.; Grieser, F.; Mulvaney, P.; Stevens, G. W. Langmuir 1999, 15, 7282.

10.1021/la010259a CCC: $20.00 © 2001 American Chemical Society Published on Web 07/27/2001

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Fujiwara et al. Table 1. Comparison of Reactivity of Tpyp with Several Metal Ions at the Toluene-Water Interfacea

Figure 1. Schematic illustration of a drum-shaped optical cell for the measurement of time-resolved fluorescence decay at the toluene-water interface under the total internal reflection configuration. ion at pH 2.93 and I ) 0.1 M, in which Pd(II) existed as PdCl(OH) dominantly.19 Except for the experiments for the mole ratio method, the amount of PdCl2 in the aqueous solution was in large excess of that of tpyp in the toluene solution. Fluorescence Microscopy. Assemblies of Pd(II)-tpyp complex formed at the toluene-water interface were observed with a fluorescence microscope (BX60, Olympus) and a cooled CCD camera (ImagePoint, Photometrics). An aliquot (5.5 mL) of 1.0 × 10-5 M Pd(II) of aqueous solution (1.0 × 10-3 M Cl-; pH, 2.93; I ) 0.1 M NaClO4) was introduced in an open cell, and then an equal volume of 3.1 × 10-8 to 3.2 × 10-7 M tpyp toluene solution was quietly added on the aqueous phase (interfacial area, 12.6 cm2). After only the aqueous phase was gently stirred with a magnetic stirrer for 5 h without disturbing the interface, the microscopic images of the interface were obtained by soaking an objective lens (10×) in the toluene phase directly. Excitation and detection wavelengths of the microscope were set to 420 and 470-800 nm, respectively, because tpyp has an absorption maximum at 418 nm and two emission maxima at 656 and 718 nm in toluene. Steady-State TIRF. Steady-state TIRF spectra for assemblies of Pd(II)-tpyp complex at the interface were measured with a spectrofluorometer (650-40, Hitachi) by the same method as reported previously.20 A standard quartz optical cell (1 cm × 1 cm) with two quartz rectangular prisms (1 cm × 1 cm × 1 cm) was used for the measurements. An aliquot (0.56 mL) of 1.0 × 10-5 M Pd(II) aqueous solution (1.0 × 10-3 M Cl-; pH, 2.93; I ) 0.1 M NaClO4) was introduced to the cell, and then 0.65 mL of 4.0 × 10-8 or 2.0 × 10-7 M tpyp toluene solution was quietly added on the aqueous phase. After only the aqueous phase was mildly stirred with a magnetic stirrer overnight without disturbing the interface, steady-state TIRF spectra were measured with an excitation wavelength of 430 nm at an incident angle of 74°, which was larger than the critical angle (θc ) 63°) for the toluenewater interface. The fluorescence emission spectra of the interface were recorded in the range of 500-800 nm. Time-Resolved TIRF. Figure 1 shows the optical arrangement including a TIRF cell used.21,22 The toluene-water interface was prepared in the drum-shaped cell. An aliquot (4.0 mL) of 1.0 × 10-5 M Pd(II) aqueous solution (1.0 × 10-3 M Cl-; pH, 2.93; I ) 0.1 M NaClO4) was introduced to the cell, and then 3.5 mL of 4.0 × 10-8 to 2.0 × 10-7 M tpyp toluene solution was quietly added on the aqueous phase. Only the aqueous phase was gently stirred with a magnetic stirrer overnight without disturbing the interface. In this situation, the interfacial area was 5.3 cm2. A mode-locked Ti:sapphire laser (Tsunami, Spectra Physics; wavelength, 800 nm; pulse width, 120 fs; repetition rate, 82 MHz; power, 600 mW) was pumped by a cw Nd:YVO4 laser (Millennia, Spectra Physics; wavelength, 532 nm; power, 5.0 W). The pulsed light was led to a frequency doubler (model 3980, Spectra Physics) (19) Baes, C. F., Jr.; Mesmer, R. E. The Hydrolysis of Cations; John Wiley & Sons: New York, 1974. (20) Watarai, H.; Funaki, F. Langmuir 1996, 12, 6717. (21) Tsukahara, S.; Yamada, Y.; Hinoue, T.; Watarai, H. Bunseki Kagaku 1998, 47, 945. (22) Tsukahara, S.; Yamada, Y.; Watarai, H. Langmuir 2000, 16, 6787.

metal ion

[tpyp]ib/ mol cm-2

reactivityc/%

Ni(II) Cu(II) Zn(II) Pd(II)

0 6.3 × 10-10 9.1 × 10-11 0 9.0 × 10-10

0 70 10 0 99

log βd

fluorescence

1.90 2.49 1.00 8.4

(not adsorbed) quenched quenched (not adsorbed) observed

a The initial concentration of tpyp in the toluene phase was 1.4 × 10-6 M, and that of metal ion in the aqueous phase was 1.0 × 10-4 M. The pH of the aqueous phase is 2.93, and the volumes of the organic and aqueous phases were 0.65 and 0.56 mL, respectively. b Total interfacial concentration of tpyp adsorbed with metal ion. c The reactivity was defined as 100 × [tpyp] /[tpyp] i init, where [tpyp]init means the hypothetical interfacial concentration, provided that all the tpyp in the toluene phase was adsorbed at the interface. d Logarithmic stability constant for metal(II)-pyridine 1:1 complex in an aqueous phase at 25 °C, I ) 0.1 M (refs 23 and 24).

to produce a second harmonic laser light (wavelength, 400 nm; power, 15 mW). The second harmonic light was irradiated to the interface for excitation of tpyp at an incident angle of 70°, which was larger than the critical angle. The fluorescence was collected from the bottom of the cell by a condensed lens and introduced to a streakscope (C4334, Hamamatsu Photonics) through an optical fiber. Mole Ratio Method for Pd(II)-Tpyp Complex. The composition of the Pd(II)-tpyp complex formed at the interface at 25 °C was determined by the mole ratio method at a higher concentration range. A standard quartz optical cell (1 cm × 1 cm) was used, and the volumes of the toluene and aqueous phases were 0.65 and 0.56 mL, respectively. The initial concentration of tpyp in the toluene phase was fixed to 1.4 × 10-6 M, and that of PdCl2 in the aqueous phase was varied in the range of 1.0 × 10-7 to 1.0 × 10-5 M. After the Pd(II)-tpyp complexation at the interface reached its equilibrium, absorbance of tpyp remaining in the toluene phase was measured in the cell directly with a UV-visible spectrophotometer (UVIDEC-430A, JASCO). The interfacial concentration of Pd(II)-tpyp complex was obtained from the difference in absorbance of tpyp in the toluene phase before and after the complexation. The complex was soluble neither in toluene nor in aqueous phases.

Results and Discussion We carried out preliminary experiments for adsorption of tpyp alone at the toluene-water interface as a function of pH. In the pH range of 1.0-1.8, protonated tpyp at both the pyridyl and pyrrole nitrogens was distributed dominantly in the aqueous phase. In the pH range of 1.8-2.9, protonated tpyp at the pyridyl nitrogens was dominantly adsorbed at the toluene-water interface. When the pH was higher than 2.9, all tpyp existed in the toluene phase. In the following experiments, the pH of the aqueous phase was fixed to 2.93 to prevent the adsorption of tpyp by itself at the interface and to hinder the formation of metal ion hydroxide. Formation of Interfacial Complex. The reactivity of tpyp to form complexes at the interface was examined for some divalent metal ions such as Ni(II), Cu(II), Zn(II), and Pd(II) under the same conditions. An aliquot (0.56 mL) of aqueous phase containing 1.0 × 10-4 M metal ion and 1.0 × 10-3 M Cl- (pH, 2.93; I ) 0.1 M NaClO4) was introduced into the 1.0 cm × 1.0 cm quartz cell, and 0.65 mL of tpyp toluene solution (1.4 × 10-6 M) was added on the aqueous phase. After stirring only the aqueous phase for 5 h mildly, the concentration of tpyp remaining in the toluene phase was directly determined by spectrophotometry. The percentage of tpyp adsorbed with the metal ion was determined from the remaining tpyp concentration, as listed in Table 1. In the absence of metal ions, all tpyp remained in the toluene phase as mentioned above.

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The reactivity of tpyp with the metal ions increased with the increase in the stability constant for the formation of metal-pyridine complex,23,24 suggesting that the metal ions were bound to the nitrogen(s) of the pyridyl group(s) of tpyp. Pd(II) showed the highest reactivity with tpyp at the interface, and in this case almost all tpyp in the toluene phase was adsorbed at the interface. Tpyp formed a yellowish complex with Pd(II) at the interface, and the interfacial complex emitted fluorescence. Pd(II) has a quenching effect for fluorophore due to the heavy atom effect,25 and this effect increases as the distance between Pd(II) and the fluorophore becomes shorter. It is known that a porphyrin complex of Pd(II), in which Pd(II) enters into the center of the porphyrin ring, can barely emit fluorescence under hydrophilic environments26-28 and that the stability constant of Pd(II) with pyridine is very high.29,30 Therefore, Pd(II) is considered to be bound to the nitrogen(s) of the pyridyl group(s) of tpyp. In Situ Fluorescence Microscopic Images. The fluorescence microscopic images of the Pd(II)-tpyp complex formed at the interface were successfully obtained and shown in Figure 2a-d, where the initial concentrations of tpyp in the toluene phase were 3.1 × 10-8, 4.7 × 10-8, 6.2 × 10-8, and 3.2 × 10-7 M, respectively, and the PdCl2 concentration in the aqueous phase was 1.0 × 10-5 M in all cases. The specific interfacial area, which was defined as Si/Vo where Si and Vo were the interfacial area and the volume of the organic phase, respectively, was 2.3 cm-1. The total interfacial concentration of tpyp bound to Pd(II) was calculated from the initial concentration of tpyp and the specific interfacial area by assuming that all tpyp in the toluene phase formed complexes with Pd(II) at the interface, because Pd(II) was very reactive with tpyp as shown in Table 1 and the amount of PdCl2 was in large excess of that of tpyp. Then, the total interfacial concen-

trations of tpyp in Figure 2a-d were calculated as 1.3 × 10-11, 2.0 × 10-11, 2.7 × 10-11, and 1.4 × 10-10 mol cm-2, respectively. In Figure 2a, which was the case of the lowest concentration of tpyp, there were formed relatively large and smooth domains (100 µm to 1 mm in size) from which fluorescence was emitted uniformly. These domains were abbreviated as assembly 1 (AS1). In Figure 2b, the interface was saturated with AS1, and in addition, bright and small domains (10-20 µm in size) appeared in the AS1 domains. The brighter domain was abbreviated as assembly 2 (AS2). The fraction of AS2 domains increased with the increase in the tpyp concentration (Figure 2c), and finally most of the interface was covered with AS2 (Figure 2d). Both AS1 and AS2 domains floated at the interface without changing their form during the measurements, indicating that AS1 and AS2 have rigid structures from the macroscopic viewpoint. The fluorescence microscopic images displayed that the interface was saturated with AS1 at the interfacial tpyp concentration of 1.7 × 10-11 mol cm-2. Under this condition, the interfacial area occupied by one tpyp molecule was calculated to be 9.8 nm2. When the porphyrin ring of tpyp lies at the interface horizontally, the area occupied by one tpyp molecule is estimated to be 1.2 nm2 by a CACheMOPAC computer program on Macintosh.31 Therefore, AS1 was considered to be a monolayer possessing many mesoscopic apertures. In the case of Figure 2d, the interfacial area occupied by one tpyp molecule was calculated to be 1.2 nm2, being comparable to the value obtained by the program. However, Figure 2d displays a heterogeneous interface, implying that tpyp of AS2 does not lie horizontally but leans by some orientation angle against the interfacial plane. The fluorescence microscopic images of metal complex assemblies formed at the liquid-liquid interface are novel results, according to our knowledge. Steady-State TIRF Spectra. The steady-state TIRF spectra of the interfacial complexes were shown in Figure 3. All the tpyp molecules in the toluene phase were adsorbed at the interface at the equilibrium. The shape and maximum of the TIRF spectrum of AS1 (Figure 3a) were similar to those of tpyp in toluene (Figure 3c), though the fluorescence intensity of AS1 was very weak. This implies the isolation of tpyp molecules of AS1. When the interfacial tpyp concentration increased from 2.6 × 10-11 mol cm-2 (Figure 3a) to 1.3 × 10-10 mol cm-2 (Figure 3b), the maximum wavelength of the emission spectrum was shifted from 657 to 668 nm and the fluorescence intensity increased. The excitation maximum was also shifted from 429 to 436 nm (not shown). These effects are attributable to the increase in the amount of AS2. It is known that the shifts of the fluorescence excitation and emission maximum of porphyrins are caused by the intermolecular interaction in their aggregates,32-36 for example, J- and H-aggregates. A smaller red-shift of 7-11 nm than others33-36 proposes the aggregation of tpyp of AS2 by weaker interaction. Time-Resolved TIRF. Examples of fluorescence decay curves for Pd(II)-tpyp complexes were shown in

(23) Banerjea, D.; Kaden, T.; Sigel, H. Inorg. Chem. 1981, 20, 2586. (24) Arena, G.; Cucinotta, V. Inorg. Chim. Acta 1981, 52, 275. (25) Muswald, C.; Hartwich, G.; Po¨llinger-Dammer, F.; Lossau, H.; Scheer, H.; Michel-Beyerle, M. E. J. Phys. Chem. B 1998, 102, 8336. (26) Igarashi, S. Bunseki Kagaku 1997, 46, 1. (27) Schmehl, R. H.; Whitten, D. G. J. Phys. Chem. 1981, 85, 3473. (28) Roza-Fema´ndez, M.; Valencia-Gonza´lez, M. J.; Dı´az-Garcia, M. E. Anal. Chem. 1997, 69, 2406. (29) Anderegg, G.; Wanner, H. Inorg. Chim. Acta 1986, 113, 101. (30) Ettorre, R. Inorg. Chim. Acta 1984, 91, 167.

(31) Molecular orbitals were calculated by PM3, MOPAC version 94.10 in CAChe, version 3.7, CAChe Scientific, 1994. (32) Saitoh, Y.; Watarai, H. Bull. Chem. Soc. Jpn. 1997, 70, 351. (33) Maiti, N. C.; Mazumdar, S.; Periasamy, N. J. Phys. Chem. B 1998, 102, 1528. (34) Akins, D. L.; O ¨ zc¸ elik, S.; Zhu, H.-R.; Guo, C. J. Phys. Chem. 1996, 100, 14390. (35) Maiti, N. C.; Ravikanth, M.; Mazumdar, S.; Periasamy, N. J. Phys. Chem. 1995, 99, 17192. (36) Barber, D. C.; Freitag-Beeston, R. A.; Whitten, D. G. J. Phys. Chem. 1991, 95, 4074.

Figure 2. Fluorescence microscopic images of the Pd(II)-tpyp complex at the toluene-water interface. The images were taken with a 10× objective lens at various total interfacial tpyp concentrations: (a) 1.3 × 10-11, (b) 2.0 × 10-11, (c) 2.7 × 10-11, and (d) 1.4 × 10-10 mol cm-2. Aqueous phase: 1.0 × 10-5 M Pd(II); 1.0 × 10-3 M Cl-; pH, 2.93; I ) 0.1 M NaClO4.

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Figure 3. (a and b)The steady-state fluorescence emission spectra of the Pd(II)-tpyp complex at the toluene-water interface under the total internal reflection condition. Initial concentrations in the toluene phase were (a) 4.0 × 10-8 or (b) 2.0 × 10-7 M tpyp. Aqueous phase: 1.0 × 10-5 M Pd(II); 1.0 × 10-3 M Cl-; pH, 2.93; I ) 0.1 M NaClO4. Total interfacial tpyp concentrations at equilibrium were (a) 2.6 × 10-11 mol cm-2 (solid line) and (b) 1.3 × 10-10 mol cm-2 (broken line). (c) The fluorescence emission spectrum of tpyp in toluene (dotted line). The intensities at the maximum wavelength (a, 656 nm; b, 657 nm; c, 668 nm) of the spectra were normalized to 1.0. The excitation wavelength was 430 nm.

Figure 4. The decay curves of the fluorescence integrated in the range of 504-796 nm for the Pd(II)-tpyp complex at the toluene-water interface under the total internal reflection condition. The total interfacial tpyp concentrations were (b) 2.6 × 10-11 and (O) 8.0 × 10-10 mol cm-2. The solid curves are the best fitted curves of eq 1.

Figure 4 at the total interfacial tpyp concentrations of 2.6 × 10-11 and 8.0 × 10-10 mol cm-2, where AS1 and AS2 were intermingled. For all the other cases, the decay curves were successfully fitted to a double-exponential function with two fluorescence lifetimes, τ1 and τ2, as

I(t) ) I1(0) exp(-t/τ1) + I2(0) exp(-t/τ2)

(1)

where I(t) is the observed fluorescence intensity at the time t and I(0) is the initial fluorescence intensity. The two lifetimes indicate that there are two kinds of species at the interface, and this is consistent with the results of fluorescence images. The I1(0) and I2(0) values are expected to be proportional to the concentrations of the two interfacial species, provided that the corresponding lifetimes are constant. The values of τ1 and τ2 were obtained as 0.15 ( 0.05 and 1.1 ( 0.1 ns, respectively. The ratio of I1(0)/I2(0) decreased with the increase in the interfacial tpyp concentration, indicating that the species 1 and 2 corresponded to AS1 and AS2, respectively. This idea can also be supported by Figure 4; the participation of species 1 in I(t) is larger at the lower interfacial concentration of tpyp, while the participation of species 2 is larger at the higher concentration. As shown in Table 1, tpyp is not adsorbed at the interface in the absence of Pd(II), and thus at least one Pd(II) must be bound to tpyp of AS1 and AS2. This assumption is consistent with the facts that Pd(II) is a quencher for fluorophores and that the τ values of tpyp of AS1 and AS2 are much less than that of tpyp in toluene, 8.1 ns. The shorter τ of tpyp of AS1 than that of AS2 suggests that more Pd(II) are bound to tpyp of AS1 than AS2. Composition of Interfacial Complex. Figure 5 shows the result of the mole ratio method: the plot of the equilibrium interfacial tpyp concentration against the

Figure 5. The mole ratio relationship between the initial concentration of PdCl2 and the total interfacial tpyp concentration, [tpyp]i, at equilibrium. The point of intersection of the two lines is [PdCl2] ) 1.4 × 10-6 M and [tpyp]i ) 8.3 × 10-10 mol cm-2, which corresponds to the formation of the 1:1 complex at the interface.

initial concentration of PdCl2. At the interfacial concentrations higher than 1.7 × 10-11 mol cm-2, the dominant interfacial species is AS2. The point of intersection of two straight lines in Figure 5 implies that 8.3 × 10-10 mol of tpyp was consumed by 7.8 × 10-10 mol of Pd(II) to produce the interfacial Pd(II)-tpyp complex, AS2. Therefore, the mole ratio of Pd(II)/tpyp of AS2 was determined to be 1:1. It was difficult to determine the composition of the interfacial complexes at a much lower interfacial concentration range. Therefore, the following method was adopted to estimate the composition of AS1 in the present

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study. The following equilibrium was assumed for the formation of AS2 from AS1 at the interface: K

PdntpypAS1 + (n - 1)tpypo y\z (Pdtpyp)n,AS2

(2)

where the subscript o refers to the organic phase, n is the stoichiometric coefficient of Pd(II) in AS1 or the average aggregation number of Pd(II)-tpyp complex in the AS2 assembly, and K is the equilibrium constant defined by the equation

K)

[(Pdtpyp)n]AS2 [Pdntpyp]AS1[tpyp]n-1 o

(3)

The wavelength range integrated in the TIRF measurements (504-796 nm) covers the whole emission range of tpyp of AS1 and AS2 (see Figure 3), and hence the detection efficiencies for both AS1 and AS2 can be assumed to be nearly equal. Therefore, the ratio of I1(0)/I2(0) is equalized to the ratio of the equilibrium interfacial concentration of tpyp of AS1 to that of AS2, and I1(0)/I2(0) is expressed as

I1(0) I2(0)

≈

[Pdntpyp]AS1 n[(Pdtpyp)n]AS2

Figure 6. The interfacial concentration of PdntpypAS1 (b) and (Pdtpyp)n,AS2 (O) against the free tpyp concentration in the toluene phase in the range of the total interfacial tpyp concentration of 2.6 × 10-11 to 1.3 × 10-10 mol cm-2, where AS1 and AS2 were intermingled. The broken line shows the average value (1.7 × 10-11 mol cm-2) of [Pdntpyp]AS1, and the solid curve is the fitting curve of eq 6 with n ) 3.

(4)

The values of I1(0) and I2(0) were obtained from the timeresolved TIRF measurement in the tpyp concentration range where AS1 and AS2 were intermingled, and at the same time the values of the tpyp concentration remaining in the toluene phase, [tpyp]o, were measured by the steadystate fluorescence measurement of the phase. Since the initial tpyp in the toluene phase was converted to AS1 or AS2 or remained in the toluene phase, the following relationship held:

([Pdntpyp]AS1 + n[(Pdtpyp)n]AS2)Si/Vo + [tpyp]o ) [tpyp]init (5) From eqs 4 and 5 and the observed I1(0)/I2(0) and [tpyp]o, [Pdntpyp]AS1 and [(Pdtpyp]n]AS2 were obtained for various initial tpyp concentrations, [tpyp]init. Equation 3 was changed to

n[(Pdtpyp)n]AS2 ) nK[Pdntpyp]AS1[tpyp]n-1 o

(6)

Figure 6 shows the plots of [Pdntpyp]AS1 and n[(Pdtpyp)n]AS2 against [tpyp]o at the equilibrium. In this concentration range, [Pdntpyp]AS1 was almost a constant of 1.7 × 10-11 mol cm2, which well agreed with the value obtained in the previous section of microscopic images. By fixing the [Pdntpyp]AS1 to 1.7 × 10-11 mol cm2, n and log K were obtained as 2.9 ( 0.4 and 18 ( 3, respectively, by the nonlinear least-squares fitting for the points in Figure 6 into eq 6. When n was fixed to an integer of 3, the log K value (K in M-2) was given as 18.65 ( 0.03. The calculated line by eq 6 with the obtained K value was superimposed in Figure 6, which well reproduced the observed points. This result suggested that the assumed models of AS1 and AS2 were valid and that the composition of AS1 was Pd3tpyp. The large stability constant of log K ) 18.65 meant that the reaction could proceed spontaneously. The binding of three Pd(II) to one molecule of tpyp of AS1 can explain the observed significant quenching and the short fluorescence lifetime of tpyp of AS1 by the heavy atom effect of Pd(II).25 When one Pd(II) of AS1 bridges two tpyp molecules in the assembly, the quenching effect

Figure 7. Linear relationship between the reciprocal lifetime (τ) of tpyp in toluene, those of tpyp of AS1 and AS2, and the number of Pd(II) bound to tpyp. The composition of AS1 was suggested to be Pd3tpyp (O), but the linearity is better when the number of Pd(II) is four per one tpyp (b).

will be further enhanced. The linear relationship between the reciprocal fluorescence lifetime (τ-1) and the quencher concentration has been widely known, and it has also been reported that the fluorescence of Eu(III) compounds is quenched through the OH vibration of water molecules in the first coordination sphere of Eu(III) and that τ-1 is proportional to the number of the water molecules.37 Therefore, we examined the relationship between τ-1 of tpyp and the Pd(II) number as shown in Figure 7. The composition of AS1 was suggested to be Pd3tpyp (open circle in the figure), but the linearity is better when the Pd(II) number is enlarged to four (closed circles). This fact may imply that the number of the effective Pd(II) (37) Horrocks, W. D.; Sudnick, D. R. J. Am. Chem. Soc. 1979, 101, 334.

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Figure 8. Possible structures of (a) AS1 and (b) AS2 units and (c) a schematic illustration of AS1 and AS2 at the interface.

participating in the fluorescence quenching is four instead of the three that is recommended from the composition of Pd3tpyp. There are a few studies on the fluorescence lifetime of aggregates of porphyrins, and thus a little information on the relationship between aggregate structure and fluorescence properties has been obtained so far. Both the excitation maximum at the Soret band and the emission maximum for J-aggregates of porphyrins are red-shifted, and the τ value is shortened.33-36 The mechanism of fluorescence quenching for aggregates is complicated, but a strong intermolecular interaction tends to result in a larger shift of the maximum wavelength and a shorter τ for J-aggregates. For instance, red-shifts of 42 and 51 nm were observed for J-aggregates of diprotonated tetraphenylporphinetetrasulfonate in aqueous solutions, and much shorter τ values of 0.1 and 0.3 ns, respectively, were obtained (3.5-3.9 ns for monomer).33,34 A J-aggregate of a picket-fence porphyrin possessed a red-shift of about 15 nm at the Soret band and showed a shorter τ of 5.24 ns (13 ns for monomer) under some condition.36 In the present case of tpyp of AS2, the red-shifts of the excitation maximum at the Soret band and emission maximum may indicate the J-type aggregate, but the shifts of 7-11 nm are smaller than those of the other J-aggregates of porphyrins. This fact suggests that the aggregation causes a smaller quenching effect and supports the above idea that the dominant quenching process of tpyp of AS2 is not the path of aggregates but through Pd(II). Possible Structures of AS1 and AS2. In situ fluorescence images clarified that AS1 was a monolayered and rather homogeneous assembly and that many apertures existed in the layer from the mesoscopic viewpoint. In addition, the composition ratio of Pd(II)/tpyp of AS1 was found to be 3:1, but the larger quenching effect suggests that tpyp may be bound to four Pd(II). On the basis of these results, we can propose the structure of AS1 as shown in Figure 8a,c. The unit of the 3:1 complex forms assemblies with apertures at the interface. Two diagonal pyridyl groups of the tpyp molecule are probably bound to Pd(II), and hence tpyp molecules are bridged by Pd(II)Cl(OH) to form an assembly. The other two pyridyl nitrogens may be occupied by Pd(II)Cl(OH). In situ fluorescence imaging and steady-state TIRF elucidated that after the interface was covered with AS1, the assembly AS2 was formed from the assembly AS1 and tpyp in the toluene phase. The large formation

constant for the conversion from AS1 to AS2 suggested the spontaneous process of the self-assembly. In addition, the composition ratio of Pd(II)/tpyp in AS2 was found to be 1:1, and the aggregation number of the 1:1 unit complex is 3 on average. On the basis of these results, we postulated the structure of AS2 as drawn in Figure 8b,c. One pyridyl group of tpyp of AS2 was bound to Pd(II) and three molecules of the 1:1 complex aggregates by a weak π-stacking interaction between the tpyp molecules. The steady-state fluorescence intensity of a fluorophore was almost proportional to its concentration and fluorescence lifetime. The weaker fluorescence intensity of AS1 as compared to AS2 seen in the microscopic images can be explained by the lower interfacial concentration of tpyp (Figure 6) and the greater number of quencher Pd(II) bound to tpyp of AS1 (Figure 7). Conclusions We found the specific complexation between Pd(II) and tpyp and the aggregation of the complex at the toluenewater interface. In situ fluorescence microscopy of the liquid-liquid interface indicated the existence of two kinds of assemblies (AS1 and AS2) and suggested the presence of mesoscopic apertures in AS1. The steady-state fluorescence spectrum revealed a π-stacking between tpyp molecules of AS2. Time-resolved TIRF suggested that the difference in lifetime between AS1 (0.15 ns) and AS2 (1.1 ns) was caused by the difference in the composition ratio between them: tpyp of AS1 and AS2 was bound to three and one Pd(II), respectively, where Pd(II) acted as a fluorescence quencher. The average percentage of Pd(II) remaining in the aqueous phase was 0.7% (data number, 6) in the Pd(II) concentration range of 1.0 × 10-7-1.0 × 10-6 M with an excess amount of tpyp (1.4 × 10-6 M, see Figure 5). This means that tpyp has a high ability to accumulate Pd(II) by self-assembly formation at the interface. In view of analytical chemistry, the present reaction scheme can be developed to a new interfacial concentration method of Pd(II) with higher reactivity than the other metal(II) ions as shown in Table 1. Acknowledgment. This work was supported by a Grant-in-Aid for Scientific Research (B) and (A) from the Ministry of Education, Science, Sports and Culture, Japan (No. 10440222 and No. 12304045) and from the Kao Foundation for Arts and Science. LA010259A