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Fluorescence Lifetime of 5-(4-Carboxyphenyl)-10,15,20-tritolylporphyrinin a Mixed Langmuir-Blodgett Film with Dioleoylphosphatidylcholine. A Proposed Standard? Harold A. Dick and James R. Bolton* Photochemistry Unit, Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N 6 A 5B7
Gilles Picard, Gagtan Munger, and Roger M. Leblanc" Centre de recherche en photobiophysique, Universitg du QuGbec 6 Trois-RiviBres, Trois-RiviGres, Qugbec, Canada C9A 5H7 Received April 28, 1987. In Final Form: July 28, 1987 Time-resolved fluorescence lifetime measurements of 5-(4-carboxyphenyl)-lO,l5,20-tritolylporphyrin (TI'Pa) with dioleoylphosphatidylcholiie(DOPC) in mixed Langmuil-Blodgett (LB) films on quartz slides were performed at two different laboratories. TTPa in the mixed LB film exhibited a simpler and longer decay profile than did a pure "Pa monolayer. At a DOPC/TTPa molar ratio of 501, the decay consisted primarily of one lifetime of 10.7 f 0.2 ns. This mixed LB f i i is being offered as a standard for time-resolved fluorescence lifetime measurements of LB films. Simplification and lengthening of the lifetime were attributed to reduction of TTPa aggregate formation in the film. This effect is also seen in fluorescence and absorption spectra. The fluorescence lifetime of the standard system at the air-water interface was also measured and found to be essentially the same as that of the LB film.
Introduction The fluorescence lifetime of fluorophors in LangmuirBlodgett (LB) films can provide important information concerning the intermolecular photophysics and organization of molecules within the film. A number of such lifetime measurements have been made for various systems.' Experience has shown that these lifetimes can be difficult to reproduce. Compound, solvent, and subphase purity and physical parameters such as compression speed, subphase temperature, aqd surface pressure play an important role in determining the final condition of the monolayer film. Subtle changes in aggregation may produce large variations in the results. The problem is further compounded by the diversity of measuring equipment and data reduction techniques. This paper offers a monolayer system, extensively tested at two laboratories, to act as a standard for future monolayer fluorescence work. The system consists of a single mixed monolayer (50:l molar ratio) of dioleoylphosphatidylcholine (DOPC) and 5-(4-carboxyphenyl)10,15,20-tritolylporphyrin (TTPa) deposited from a Langmuir trough onto quartz slides. Mixing of the TTPa with DOPC reduces the probability of fluorescence quenching by aggregate formation. Quenching is also minimized by the small overlap between the fluorescence and absorption profiles of the monomeric and aggregate pigment species, respectively.2 The LB films have a shelf life of at least 1week in light and air a t 20 OC. They are also stable under an irradiance of 200 W m-2 a t 295 nm for the duration of the fluorescence measurement. Finally, the T T P a is easily synthesized and purified, and the DOPC is commercially available in high purity. At present three fluorescence lifetime measurement techniques are popular: phase shift fluorometry,3 flash photolysis employing a pulse laser and a streak camera: and time-correlated single photon ~ o u n t i n g . Of ~ the three techniques, the last has come into widest use and is the Contribution No. 379, Photochemistry Unit, Department of Chemistry, The University of Western Ontario.
method of choice for this paper.
Experimental Section Experiments were conducted in parallel both at the University of Western Ontario (UWO) and at the Universitg du Qugbec ti Trois-RiviBres (UQTR). The following is a description of the technique used at UWO. Mixed monolayers of TTPa and DOPC were deposited onto quartz slides from an air-water interface by the LangmuirBlodgett method! The DOPC/TTPa molar ratio was varied between 1OO:l and 0:l. The subphase, unbuffered water, was purified as follows. House distilled water was passed through scale elimination, deionization, organic removal columns (D8921, D8922 SYBRON/Barnstead), distilled in a quartz bidistillation apparatus (Bi4, Englehart, Amersil Quartz Division), and then stored in Pyrex volumetric flash fitted with inverted ground glass stoppers. The conductivity of the water was -lo4 Q-' m-l. The LB trough was fabricated from Teflon. The surface area of the air-water interface was adjusted by a movable barrier riding along the top of the sides of the trough. Surface pressure was measured by a movable Teflon float attached to a torsion balance coupled to a linear transducer. The apparatus was cleaned periodically by soaking in a solution of the detergent Decon (BDH Chemicals) followed by thorough rinsing with filtered water. It was also rinsed before every deposition with absolute ethanol followed by Fisher spectral grade methanol. The quartz slides (Corning)were cleaned by soaking for 3 days in concentrated chromic acid. They were then given 15 rinses (1)(a) Picard, G.; Munger, G.; Leblanc, R. M.; LeSage, R.; Sharma,
;.)I Siemiarczuk,A.; Bolton, J. R. Chem. Phys. Lett. 1986,129,41-47.(b) Agrawal, M.L.; Chauvet, J. P.; Patterson, L. K. J . Phys. Chem. 1985,89, 2979. (2)Turro, N. Modern Molecular Photochemistry; Benjamin/Cummings: London, 1978;p 303. (3)Cundall, R. B. In Time-Resolued Fluorescence Spectroscopy in Biochemistry and Biology; Cundall, R. B., Dale, R. E., Eds.; Plenum: New York, 1983;pp 59-83. (4)(a) Shapiro, S.L.; Kollman, V. H.; Campillo, A. J. FEBS Lett. 1975, 54,358-362. Beddard, G.S.;Porter, G.; Tredwell, C. J. Nature (London) 1976,258,166-168. (5)Ware, W. R. In Tine-Resolved Fluorescence Spectroscopy in Biochemistry and Biology; Cundall, R. B., Dale, R. E., Eds.; Plenum: New York, 1983;pp 23-57. (6)Blodgett, K. B.; Langmuir, I. Phys. Reu. 1937,51, 964-982.
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134 Langmuir, Vol. 4, No. 1, 1988 with Barnstead filtered water and sonicated for 15 min in 0.01 M NaOH (Fisher). This was followed by 10 rinses with filtered water, 5 rinses with triply distilled water, and sonication for 15 min in the fmal rinse. The slides were then dried with prepurified nitrogen. The TTPa was synthesized by the method of Little et al.' The monoacid compound (TTPa) was separated on a 2.5 cm X 50 cm silica gel column with 1% methanol in chloroform as an eluant. The TTPa fraction produced a single spot on a TLC slide with 5% methanol in chloroform. Its identity was confirmed by mass spectroscopy,UV-vis absorption, and fluorescence spectroscopy. The extinction coefficients in chloroform were found to be as follows: Amax. (nm) [e (M-' cm-')I 646.5 [4.75 X lo3], 590 [5.27 X 1031, 552 [8.96X lo3], 516.5 [1.73 X lo'], 420 [4.49 X 1051. Since the absorption of TTPa in chloroform follows Beer's law to the solubility limit, its concentration can be determined by colorimetry. The DOPC obtained from Sigma was weighed on a microbalance before being dissolved in chloroform. The two solutions were mixed in the desired proportion by using Pressure-Lok syringes (Precision Sampling Corp.). Fisher spectral grade chloroform was used as solvent throughout. The solution (lo4 M)was deposited on the subphase (20 i 2) "C with a syringe, with a minimum of 3 s between drops. Compression was begun 1 min after the end of deposition at a rate of 5.5 X 10'' A2rnin". The film was compressed to a surface pressure of 20 mN m-l at which point the slide was dipped in and out of the water at a rate of 2.0 cm min-'. During transfer of the monolayer the surface pressure was maintained at 20 mN m-' by continual reduction of the surface area. Only slides exhibiting a deposition ratio of 1.0 0.1 were accepted. The technique followed at UQTR differs slightly at points from the above. These differences are deemed to be inconsequential since identical results were obtained between the two labs. For a detailed description of the UQTR technique, see Munger et al.8 Fluorescence measurements were performed within the range 24 h to 1 week after deposition. Both laboratories used PRA International Model 3000 fluorescence lifetime single photon counting equipment. During measurement the monolayers were exposed to room air. Differing modifications of the equipment between the two laboratories are as follows. The UWO laboratory generated excitation light by a cavitydumped dye laser (590 Coherent) synchronously pumped by a mode-locked argon ion laser (Cr-8 Coherent). Rhodamine 6G was used as the lasing dye. The laser light (15-ps pulse width, 590 nm) was focused through a linear polarizer onto the sample with an intensity of los photons/flash at a repetition rate of 1 MHz. Sample fluorescence was focused through a 620-nm cut-off filter and a monochromator tuned to 650 nm. Data were analyzed by a statistical reconvolution program designed at UW0.9 The UQTR laboratory generated excitation liiht by a nitrogen flash lamp (4-11s pulse width, le photons/flash, 30-lrHz repetition rate). The light passed through a monochromatorset atb420nm. Fluorescence was collected through a 630-nm cut-off filter by an ellipsoidal mirror focused on the photomultiplier tube. Data were analyzed by a PRA statistical reconvolution program. Absorption spectra were measured on a HewletbPackard8450A diode array spectrophotometer. Fluorescence spectra were taken on a Spex Fluorolog 2 spectrofluorometer. In addition to measurements of the LB film, the UQTR group repeated the fluorescence lifetime measurement for the standard mixture at the air-water interface (Le., before transferring to the quartz slide). For this measurement the sample compartment of the PRA 3000 was modified to contain an LB trough identical with that described in ref 8. The excitation light was passed through a 100-nm band-pass fiiter (Oriel) centered at 440 nm and focused directly onto the water surface. Two interwoven fiberoptic bundles placed perpendicular to the excitation beam transferred the fluorescenceemission through a 10-nm band-pass interferentialfiter (660nm, Ditric Optics) to the ellipsoidal mirror focused on the detection photomultiplier. (7) Little, R. G.; Anton, J. A.; Loach, P. A.; Ibers, J. A. J.Heterocycl. Chem. 1975,12, 343-349. (8)Munger, G.; Lorrain, L.; Gagn6, G.; Leblanc, R. M. Rev. Sci. Instrum. 1987,58, 285-288. (9)James, D.R.;Ware, W. R. Chem. Phys. Lett. 1986, 126, 7-11.
Dick et al.
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Figure 1. Surface pressurearea isotherm of a 501 DOPC/l"Pa mixed monolayer. Subphase, unbuffered water at 20 2 "C.
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Figure 2. Surface pressure-area isotherm of a pure TTPa monolayer. Subphase, unbuffered water at 20 f 2 "C.
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Wavelength (nm)
Figure 3. Optical absorption spectra of TTPa in methylene chloride (lo4 M) (- -), in a LB film of TTPa alone (- - -), and in a 50:l DOPC/TTPa mixed film (-).
Results and Discussion The surface pressure-area isotherms of a DOPC/TTPa monolayer and of a pure TTPa monolayer are shown in Figures 1and 2, respectively. The DOPC/TTPa isotherm is essentially identical with that for pure DOPC. The observed molecular area at 20 m N m-' surface pressure and 20 "C subphase temperature is 72 (fl) A2 molecule-' for the mixture, 71 (fl) A2 molecule-' for pure DOPC, and 79 ( f l ) A2 molecule-' for pure TTPa. It has been shown that the DOPC monolayer is in a quasi-liquid state at this surface pressure, which is favorable for homogeneous mixing of the diluted pigment.'O In addition, the mo(10)Ducharme, D.; Salesse, C.; Leblanc, R. M. Thin Solid Films 1985, 132, 83-90.
Langmuir, Vol. 4, No. I , 1988 135
Standard for Lifetime Measurements of LB films
Table 1. Fluorescence Lifetime Measurements for Monolayer Films of TTPa ______~
system
0
501 DOPC/T"Pa mixed LB film pure TTPa LB film 501 DOPC/TTPa mixed monolayer at air-water interfaceb TTPa in CH2C1;'
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Figure 4. Fluorescence excitation spectra of 'ITPa in methylene chloride (10" M) (- -), in an LB film of TTPa alone (- - -), and in a 50.1 DOPC/TTPa mixed film (-); A,, = 650 nm. lecular area of the mixture agrees with the weighted addition of the molecular areas of the two components, indicating an ideal mixture, i.e., no specific association. Figure 3 compares the absorption spectrum of a film of TTPa alone and a film of the " P a / D O P C mixture with that of T T P a in methylene chloride (lo4 M). The redshifted aggregate peak earlier reported for the TTPa film" is entirely absent from the spectrum of the mixed film, suggesting the predominance of the same monomeric form as found in dilute solution. The fluorescence excitation and emission spectra in Figures 4 and 5 support this hypothesis in a similar fashion. In both c u e s the spectra of the mixed film agree closely with the solution spectra and differ from the spectrum of the T T P a monolayer shown here and reported by Bardwe1l.l' The entire excitation spectrum for each of the three cases is shown in Figure 6. The intensity of the 422-nm shoulder in the pure TTPa monolayer spectrum varies from sample to sample as this feature is highly sensitive to the subphase pH." Spectral variations of this magnitude are not observed for the mixed film. A predominance of monomeric species in the monolayer is desirable since any aggregate formed may act as a fluorescence quenching complex. In the case of the TTPa film at least two aggregate species are speculated to exist.12 Fluorophors find themselves in a nonuniform quenching environment, and the resulting decay profile consists of three closely spaced short-lifetime components (see Table I). Fluorophors in the mixed monolayer find themselves in an environment primarily made up of nonquenching DOPC molecules rarely encountering another fluorophor. This results in a simplification of the decay to two com(11)(a) Bardwell, J. A,; Bolton, J. R. Photochem. Photobiol. 1984,39, 735-748. (b) Bardwell, J. A. Ph.D. Thesis, The University of Western Ontario, 1983,pp 63-78; Dissert. Abstr. B 1983,#(lo), 3091. (12)Bardwell, J. A. Ph.D. Thesis,The University of Western Ontario, 1983,pp 81-83.
690 725 Wavelength (nm)
7 IO
Figure 5. Fluorescence emission spectra of TTPa in methylene chloride (10" M) (--), in an LB film of TTPa alone (-- -), and in a 501 DOPC/TTPa mixed film (-); A,, = 420 nm. $1C
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Figure 6. Fluorescence excitation spectra of TTPa in methylene chloride (10" M)(--), in an LB film of TTPa alone (---), and in a 501 DOPC/TTPa mixed film (-); A,, = 650 nm. ponents, the longer of which predominates. The long component is likely due to monomeric species in the film since its lifetime is similar to that found in dilute solution. The mean value of the long component was found to be 10.7 ns with a standard deviation of 0.2 ns. Since we have found this lifetime to be highly reproducible both within and between our two laboratories, we propose this system as a standard for future testing of time-resolved fluorescence of monolayer systems. Agreement between the two laboratories was found to be within 0.05 ns. The standard deviation in 24 measurements on the same sample was 0.2 ns. These measurements spanned the period of 1 week with no noticeable trend in the results indicating the chemical stability of the system with respect to the photophysics. The standard deviation in the measurement of eight different samples was 0.1 ns. At a 501 DOPC/TTPa molar ratio the change in the long component value as a function of mixing ratio was 1.25 X loe2 ns/ratio point. Hence, an error of 16% in the mixing ratio is acceptable before a change in the long component value is seen.
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Langmuir 1988,4 , 136-140
The short component is probably due to an aggregate species by analogy to the pure TTPa film. This species makes up only 7% of the fluorescent population and thus does not noticeably red-shift the fluorescence and absorption spectra. While a good fit may be obtained with a two-component decay for measurements involving up to 20 OOO counts, the fit becomes increasingly inadequate a t higher counts. It is suspected that the decay is, in fact, made up of a distribution of 1ifeth1es.l~ The distribution is currently being analyzed by using a program for measurements at 200 OOO counts and will be described in a forthcoming paper. The TTPa monolayer was found to be stable a t least over a period of 2 weeks in air a t room temperature. Futhermore, no bleaching of the pigment was seen under irradiation by either light source. (13)(a) James, D.R.; Liu, Y.4.; de Mayo, P.; Ware, W. R. Chem. Phys. Lett. 1985, 120, 460-465. (b) James, D.R.; Ware, W. R. Chem. Phys. Lett. 1986, 126,7-11. (14)Siemiarczuk, A.; McIntosh, A. R.; Ho, T.-F.; Stillman, M. J.; Roach, K. R.; Weedon, A. C.; Bolton, J. R.; Connolly,J. S. J. Am. Chem. SOC.1983,105, 7224-7230.
The results of measurements of the same mixture at the air-water interface are also included in Table I. The photophysical situation appears to be close to that in the LB film. This is an important observation since it shows that the transfer of the monolayer film from the air-water interface to the quartz slide has little effect on the nature of the film.
Conclusions It was found that TTPa aggregation in LB films could be reduced by dilution in DOPC. The resulting monomeric TTPa fluoresced with a reproducible lifetime of 10.7 f 0.2 ns, which we offer as a standard. The reduced aggregation was also apparent in the absorption and fluorescence spectra as a blue shift away from the earlier reported aggregate spectrum toward that of TTPa in methylene chloride. The authors will make available, on request, samples of T T P a and/or certified LB films of the standard system. Registry No. TTPa, 93082-03-2;DOPC, 10015-85-7;quartz, 14808-60-7.
Monomer-Micellar Equilibrium of Fluorocarbon and Hydrocarbon Surfactant Solutions by Ultrafiltration Tsuyoshi Asakawa,*t Kazuhiro Johten, Shigeyoshi Miyagishi,*t and Morie Nishida*t Department of Industrial Chemistry, Faculty of Technology, Kanazawa University, Kanazawa 920, Japan Received February 20, 1987. I n Final Form: August 3, 1987 An ultrafiltration method was used to study fluorocarbon and hydrocarbon surfactant mixtures such as lithium perfluorooctanesulfonate (LiF0S)-lithium alkyl sulfate, having a different chain length of 10 (LiDeS),12 (LiDS), or 14 (LiTS), and sodium perfluorooctanoate (SPFO)-sodiumdodecyl sulfate (SDS). The method was useful to reveal the monomer-micelle equilibrium beyond the mixture cmc. The azeotropic point was observed close to the maximum of the mixture cmc curve for the LiFOS-LiTS and LiFOS-LiDS systems, respectively. This result was interpreted by the occurrence of micelle demixing.
Introduction Recently, we have used a group contribution model to predict critical micelle concentrations of binary surfactant mixed systems.' The method was also applied to quantitative investigation of the mixing of fluorocarbon and hydrocarbon surfactants. The immiscibility has been worthy of remark in the viewpoint of technical interest such as oil repellent and fire-extinguishing properties.2 The coexistence of two kinds of mixed micelles was predicted by Mukerjee et al.3*4 The micelle demixing is quite plausible, but it has not been proven. A more direct experimental method is needed to resolve the issue. Several experimental data for this subject were presently available.'-lo The monomer compositions were also predicted, but they have not been determined by a direct experimental method. If the demixing of micelles would occur, the concentrations of monomers must be constant according to the pseudo-phase-separation approximation of micellization under the constant temperature and pressure Department of Chemistry and Chemical Engineering, Faculty of Technology, Kanazawa University, 2-40-20Kodatsuno, Kanazawa 920,Japan.
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conditions. The monomer concentrations were not verified because of the experimental difficulty in direct measurement of monomer concentrations. Thus, the monomer needs to be separated from the micellar solution by an appropriate method. An ultrafiltration is effective at removing dissolved high molecular weight organics from water.11-14 Surfactant monomer passes through an ultrafiltration membrane with (1)Asakawa, T.; Johten, K.; Miyagishi, S.; Nishida, M. Langmuir 1985, 1, 347. (2)Shinoda, K.;Nomura, T. J. Phys. Chem. 1980, 84, 365. (3)Mukerjee, P.;Mysels, K. J. ACS Symp. Ser. 1975, 9, 239. (4)Mukerjee, P.;Yang,A. Y. S. J. Phys. Chem. 1976,80, 1388. (5)Mysels, K.J. J. Colloid Interface Sci. 1978, 66, 331. 1982,59, 573. (6)Mukerjee, P.J. Am. Oil Chem. SOC. (7) Funasaki, N.; Hada, S. J. Phys. Chem. 1980, 84, 736. (8)Funasaki, N.;Hada, S. J. Phys. Chem. 1983, 87, 342. (9) Carlfors, J.; Stilbs, P. J. Phys. Chem. 1984, 88, 4410.
(10)Asakawa, T.;Miyagbhi, S.; Nishida, M. J. Colloid Interface Sci.
1985, 104, 279.
(11)Osborne-Lee, I. W.; Schechter, R. S.; Wade, W. H. J. Colloid interface Sci. 1983, 94, 179. (12)Osborne-Lee, I. W.: Schechter, R. S.; Wade, W. H.; Baraket, Y.
J. Colloid Interface Sci. 1986, 108, 60. (13)Scott, H. J. Phys. Chem. 1964, 68, 3612. (14)Warr, G. G.;Grieser, F.; Healy, T. W. J. Phys. Chem. 1983, 87, 1220.
0 1988 American Chemical Society
