Influence of Molecular Structure on the Aggregating Properties of

dichloro-9-ethylthiacarbocyanine (THIATS) and its cationic analogue 3,3′-diethyl-5 ...... ATS/DODAB film a homogeneous layer of closely packed fluor...
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Langmuir 2000, 16, 9518-9526

Influence of Molecular Structure on the Aggregating Properties of Thiacarbocyanine Dyes Adsorbed to Langmuir Films at the Air-Water Interface Nadia Vranken, Mark Van der Auweraer,* and Frans C. De Schryver Laboratory for Molecular Dynamics and Spectroscopy, Department of Chemistry, KU Leuven, Celestijnenlaan 200 F, B-3001 Heverlee (Leuven), Belgium

Hugo Lavoie, Philippe Be´langer, and Christian Salesse GREIB, De´ partement de Chimie-Biologie, Universite´ du Que´ bec a` Trois-Rivie` res, Trois-Rivie` res, Que´ bec, Canada, G9A 5H7 Received June 27, 2000. In Final Form: September 4, 2000

The influence of substituents on the adsorption and aggregation of the anionic 3,3′-disulfopropyl-5,5′dichloro-9-ethylthiacarbocyanine (THIATS) and its cationic analogue 3,3′-diethyl-5,5′-dichloro-9-ethylthiacarbocyanine (TDC) was investigated in Langmuir films at the air-water interface. Dioctadecyldimethylammonium bromide (DODAB) and octadecylammonium chloride (ODACl) were used to adsorb THIATS. Arachidic acid (AA) and dihexadecyl phosphate (DHP) were used to adsorb TDC. J-aggregate formation has been observed for the four different combinations of dyes and surfactants. The absorption and emission spectra of THIATS at the air-water interface revealed one narrow intense J-band which was not influenced by the chemical structure of the amphiphiles or by the concentration of THIATS in the subphase. Fluorescence microscopy experiments suggest that, depending on the subphase dye concentration, aggregation of dye molecules leads either to a layer of J-aggregates distributed homogeneously on the scale of a few micrometers or to the observation of individual domains of J-aggregates. For TDC adsorbed to an AA film, the absorption and emission spectra revealed the formation of two J-bands that coexist at the interface. The absorption spectra of TDC adsorbed onto a DHP film showed only one J-band, whose maximum gradually shifted from an initial value of 646 nm to 637 nm and then to 652 nm. The flexible alkyl spacer chain between the localized negative charges of the sulfate groups and the nitrogen atoms of the benzthiazole units allows a packing of the THIATS molecules that is less dependent on the charge distribution in the oppositely charged lipid film than for TDC. Fluorescence micrographs of THIATS/DODAB and TDC/AA films reveal different spatial features. They confirm the predominant influence of the lipid on the TDC/AA film morphology, as concluded from the spectral data. The presented study offers substantial information that was still lacking in this field of research: the time-dependence adsorption of the dyes onto charged monolayers as well as an investigation of the influence of the substitution pattern and the role of the packing of the amphiphiles on the formation of a specific type of aggregate.

Introduction Upon increasing the concentration of an aqueous solution of several cyanine dyes, J-aggregate formation has been observed, resulting in a red shift and a narrowing of their absorption band.1,2 Due to strong intermolecular van der Waals attractive forces and hydrophobic interactions, two-dimensional aggregate formation3-5 can occur under specific experimental conditions. The formation of J-aggregates of cyanine dyes adsorbed to silver halides prompted the interest in this phenomenon for the design of photographic materials.6-8 The Langmuir-Blodgett (LB) technique has also been shown to yield large two* To whom correspondence should be addressed. E-mail: [email protected]. Fax: 016/32 79 90. (1) Jelley, E. E. Nature 1936, 138, 1009. (2) Jelley, E. E. Nature 1937, 139, 631. (3) Kobayashi, T. J-aggregates; World Scientific Publishing Co. Pte. Ltd.: 1996. (4) Kuhn, H.; Czikkely, V.; Fo¨rsterling, H. D. Chem. Phys. Lett. 1970, 6 (1), 11. (5) Kuhn, H.; Czikkely, V.; Fo¨rsterling, H. D. Chem. Phys. Lett. 1970, 6 (1), 207. (6) Wo¨rz, O.; Scheibe, G. Z. Naturforch. 1969, 14b, 381. (7) Maskasky, J. E. Langmuir 1991, 7, 407. (8) The Theory of the Photographic Processs, 4th ed.; James, T. H., Ed.; Macmillan: New York, 1977.

dimensional aggregates of cyanine dyes9-12 and could thus potentially be used for this purpose. Two different approaches have been used to prepare these aggregates. When the cospreading method is used, amphiphilic dye molecules with long alkyl chains are mixed with unsubstituted fatty acids to form monolayers at the air-water interface.9 An alternative method consists of the adsorption of charged, water-soluble dyes to a lipid monolayer bearing an opposite charge.10-12 When a spread film of amphiphiles is compressed, a charged surface is created at the air-water interface which attracts oppositely charged ions and eventually results in the adsorption of a monolayer of dye molecules dissolved in the underlying subphase. This concept can lead to several applications if the preparation of such films is well characterized. However, previous studies were mainly dealing with deposited LB films, which can reveal different spectral properties compared to those of the original Langmuir films at the air-water interface before deposition.13 These (9) Kuhn, H.; Mo¨bius, D. Angew. Chem. 1988, 92, 5035. (10) Saito, K.; Ikegami, K.; Kuroda, S. I.; Tabe, Y.; Sugi, M. J. Appl. Phys. 1992, 71, 1401. (11) Reich, C.; Pandolfe, W. D.; Bird, G. R. Photogr. Sci. Eng. 1973, 17, 335. (12) Lehmann, U. Thin Solid Films 1988, 160, 257. (13) Dutta, A. K.; Salesse, C. Langmuir 1997, 13, 5401.

10.1021/la000896l CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000

Thiacarbocyanine Dyes Adsorbed to Langmuir Films

Figure 1. Chemical structure of the thiacarbocyanine dyes THIATS (1) and TDC (2) and the amphiphiles DODAB (3), ODACl (4), DHP (5), and AA (6).

studies were lacking information on (1) the kinetics of the adsorption of the dyes onto charged monolayers, (2) the dependence of the formation of a specific type of aggregate on the packing requirements and substitution pattern of the dye, and (3) the dependence of the dye adsorption and aggregation on the lipid film. In this contribution we have investigated the timedependent adsorption of dyes onto charged amphiphilic molecules. Moreover, we have studied the influence of the 3,3′-substitution of a thiacarbocyanine dye on the formation of J-aggregates by in situ absorption and fluorescence spectroscopy at the air-water interface. In addition, the influence of the amphiphilic compound on the aggregate formation has also been evaluated by comparing the spectroscopic properties of the dye molecules in the presence of an amphiphile with either one or two alkyl chains. Finally, fluorescence microscopy experiments were performed to correlate film morphology with the spectral properties of these films. Experimental Section The thiacarbocyanine dyes 3,3′-disulfopropyl-5,5′-dichloro-9ethylthiacarbocyanine (THIATS) (1, Figure 1) and 3,3′-diethyl5,5′-dichloro-9-ethylthiacarbocyanine (TDC) (2, Figure 1) were gifts from Agfa. The amphiphilic compounds dioctadecyldimethylammonium bromide (DODAB) (3, Figure 1), octadecylammonium chloride (ODACl) (4, Figure 1), dihexadecyl phosphate (DHP) (5, Figure 1), and arachidic acid (AA) (6, Figure 1) were purchased from Aldrich. The organic solvents used were of spectroscopic quality. A home-built LB trough was first filled with pure Millipore filtered water. Water was obtained by running tap water first through filters to remove the organic compounds. Subsequently, the water is pumped into a NANO pure-filter system, yielding water with a resistivity above 18 MΩ‚cm-1, a pH of 5.5, and a surface tension of 71 mN/m. The temperature of the room was thermostatically kept at 20 °C, and the air humidity was maintained at 50%. The design of the instrument for the in situ spectroscopic measurements at the air-water interface has been described in detail elsewhere.14 A round Teflon mask (with a diameter of 6 cm and a height of 8 mm), with a door (with a width of 2 cm and a height of 5 mm) was placed in the trough at the position where the spectra are measured. The films of the different amphiphiles are compressed until a surface pressure of 30 mN/m is reached. The surface (14) Gallant, J.; Lavoie, H.; Tessier, A.; Munger, G.; Leblanc, R. M.; Salesse, C. Langmuir 1998, 14, 3954.

Langmuir, Vol. 16, No. 24, 2000 9519 pressure is measured with a Langmuir balance. After closing the door of the mask, a given amount of a concentrated dye solution in pure water was injected into the subphase inside the mask until the desired dye concentration (10-6 or 10-7 M) was reached. A spectrum was taken every 10 or 15 min to follow the changes in the absorption and emission spectra of the dye. Not all spectra are shown in the figures for the sake of clarity. Absorption and fluorescence spectra are recorded in transmission and in reflection mode, respectively.14 The fluorescence polarization has been determined by placing a polarizer just after the excitation monochromator and one just before the emission monochromator. The direction of the polarizers can be changed manually. In the polarization experiments, the two polarizers were either put perpendicular (s-polarization) or parallel (ppolarization) to the plane of incident light. A fluorescence microscope was coupled to a home-built Langmuir trough. The trough is made of aluminum covered with a sheet of adhesive precoated Teflon (Johnston Industrial Plastics). The width of the trough is 121 mm, and the monolayer can be compressed from a length of 767 mm down to 19.9 mm. The surface pressure (π) is detected with a Wilhelmy plate system using a filter paper.15 All trough operations are computer controlled using the same software as for the in situ spectroscopic experiments. The epifluorescence microscope used in this study is similar to that described by Meller16,17 and has been described elsewhere.18 The filter set used for the observation of J-aggregates (“J-filter set”) consisted of an excitation filter at 610 nm with a bandwidth of 20 nm, an emission filter at 670 nm with a bandwidth of 40 nm, and a dichroic mirror at 630 nm (Omega Optical, Brattleboro, VT). The other filter set used for the observation of the monomers (“M-filter set”) was a Nikon G-2A set, which included an excitation filter at 535 nm with a bandwidth of 50 nm, a 590 nm high-pass emission filter, and a 580 nm dichroic mirror.

Results and Discussion To find out whether the Langmuir films of the amphiphilic compounds were influenced by the dye molecules in the subphase, isotherms of the amphiphiles, measured in the presence and absence of dye molecules in the subphase, were compared. At a surface pressure of 30 mN/m the area per amphiphile molecule was the same in the absence and presence of the dye, suggesting that, contrary to the results obtained by Kirstein and Mo¨hwald,19 the adsorption of the dye molecules does not modify the density of the lipid layer. The molecular area of both AA and ODACl at 30 mN/m amounts to 20 ( 0.5 Å2/molecule, whereas that of DHP and DODAB amounts to 40 and 60 ( 1 Å2/molecule, respectively. Aggregate Formation of THIATS Adsorbed onto a DODAB Film. The adsorption and aggregation of THIATS onto a compressed layer of DODAB were studied at a 10-6 and a 10-7 M dye concentration in the subphase. J-aggregation was observed in both cases. The amount of dye necessary to obtain a monolayer coverage in the mask was calculated from the area of the mask and the area expected to be occupied by one adsorbed dye molecule. Taking into account the depth of the mask in the subphase (6 mm), a concentration of 2.5 × 10-7 M over the total volume inside the mask was found to be necessary to obtain a full monolayer coverage at the air-water interface inside the mask. In this calculation, the dimensions of a THIATS molecule are taken to be 20 × 10 × 5 Å3 and the dye molecules are assumed to adsorb edge-on.20 These dimensions, calculated from the molecular structure, are in good (15) Albrecht, O. Thin Solid Films 1983, 99, 227. (16) Meller, P. Rev. Sci. Instrum. 1988, 59, 2225. (17) Meller, P. J. Microsc. (Oxford) 1989, 156, 241. (18) Maloney, K. M.; Grandbois, M.; Grainer, D. W.; Salesse, C.; Lewis, K. A.; Roberts, M. F. Biochim. Biophys. Acta 1995, 1235, 395. (19) Kirstein, S.; Mo¨hwald, H.; Shimomura, M. Chem. Phys. Lett. 1989, 154 (4), 303. (20) Saijo, H.; Shiojiri, M. J. Imag. Sci., Technol. 1996, 40 (2), 111.

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Vranken et al. Table 1. Averaged Spectral Data of the Different Dye/ Lipid Complexes dye/lipid complexes

FW2/3M(M) (cm-1)

FW2/3M(J) (cm-1)

Neff

THIATS/DODAB (10-6 M) THIATS/DODAB (10-7 M) THIATS/ODACl (10-6 M) TDC/AAa (10-6 M) TDC/AAb (10-6 M)

1000 ( 20 1070 ( 40 1060 ( 30 980 ( 10 1000 ( 15

TDC/DHP (10-6 M) TDC/DHP (10-7 M)

990 ( 10 1090 ( 50

230 ( 10 250 ( 20 280 ( 20 190 ( 10 250 ( 40 (J1) 260 ( 10 (J2) 290 ( 70 400 ( 100

19 ( 1 19 ( 3 14 ( 2 26 ( 5 18 ( 5 (J1) 15 ( 1 (J2) 14 ( 4 8(4

a

Spectra not shown. b Spectra shown in Figure 5a.

length of 19 ( 1 molecules was calculated using eq 1.

Neff )

Figure 2. (a) Absorption spectra of a layer of THIATS/DODAB at the air-water interface with an initial dye concentration in the mask of 10-6 M; spectra taken after 5 min (0), 35 min (4), 65 min (O), and 95 min (3). The arrows at the M-band and J-band, pointing downward and upward, indicate a decrease and increase, respectively, of the absorbance of the corresponding band, after longer periods of time. (b) Emission spectra taken of the THIATS/DODAB layer at the air-water interface with an initial dye concentration in the mask of 10-6 M; excitation was performed at 620 nm; spectra taken after 5 min (0), 30 min (4), 50 min (O), and 60 min (3).

agreement with the dimensions obtained from X-ray diffraction measurements performed on a single crystal of TDC, where the length and width of the molecule were found to be 20 and 4 Å, respectively.21 Absorption Spectra. Figure 2a shows the absorption spectra of a layer of THIATS/DODAB at the air-water interface. The absorption spectra consist of a monomer (M) band with a maximum at 553 nm and a J-aggregate (J) band with a maximum at 657 nm. It can be seen that the J-band increases at the expense of the M-band, indicating that an increased number of J-aggregates are formed as a function of time. The full bandwidth at twothirds of the maximum (FW2/3M) for the J-aggregates is 230 ( 10 cm-1, which is considerably smaller than the value of 1000 ( 20 cm-1 (Table 1) found for the monomers. The bandwidths were taken at two-thirds of the maximum of the absorption band because a vibrational band, which is no longer present at 2/3 of the maximum, broadens the M-absorption band. Assuming that the bandwidth scales with the effective coherence length (Neff),22 a coherence (21) Asanuma, H.; Ogawa, K.; Fukunaga, H.; Tani, T.; Tanaka, J. Proceedings ICPS ′98 International Congress on Imaging Science; University of Antwerp (UIA): Belgium, 1998; Vol. 1, p 178. (22) Knapp, E. W. Chem. Phys. 1984, 85, 73.

(

)

FW2/3M(M) FW2/3M(J)

2

(1)

From the experimental setup, it is not clear whether all monomer dye molecules are adsorbed to the Langmuir film. It is possible that part of the residual monomer absorbance after 95 min is due to the presence of a certain amount of dye monomers in the bulk subphase, which is, however, not the case for the aggregates.23 Indeed, the large difference between the maximum of the J-aggregate band in solution (625 nm)24-26 and the one in Figure 2a (657 nm) argues in favor of the presence of only adsorbed J-aggregates under our experimental conditions. In the calculation of the effective coherence length, it is assumed that the monomer is only present as a single isomer (the presence of several isomers will cause band broadening not related to increased electron phonon coupling27). If several isomers of the monomer would be present, part of the bandwidth reduction could be due to preferential adsorption of one isomer rather than to an increase of the coherence length. The spectral data for the measurements at concentrations of 10-6 and 10-7 M are listed in Table 1. The features of the spectra are almost identical for both dye concentrations. The absorption spectrum obtained after 18.5 h (not shown) indicates that there are almost no nonaggregated dye molecules left in the mask after this long period of time. This absorption spectrum was separated into a M-band and a J-band ranging from 400 to 600 nm and from 600 to 750 nm, respectively. The ratio between the M-band and the J-band was calculated by dividing the integrated absorbance from the M-band by the integrated absorbance from the J-band, thus yielding a value of 0.08. This observation, that almost no monomer absorption is left after such a long period of time, is an additional indication that as soon as a dye molecule adsorbs, it becomes part of the J-aggregate. Emission Spectra. Figure 2b shows the emission spectra of the THIATS/DODAB complexes at a 10-6 M dye concentration. It can be seen that the intensity of the J-band increases with time. In all spectra, the emission maximum was situated at 658 nm, indicating a negligible Stokes shift for the J-aggregates. The FW2/3M of the emission band was found to be 230 ( 20 cm-1. A maximum (23) Van der Auweraer, M.; Vranken, N.; Grim, K.; Hungerford, G.; De Schryver, F. C.; Vitukhnovskhy, A.; Varnavsky, O. Proceedings Excon ′96: 2nd International Conference on Excitonic Processes in Condensed Matter; Bad Schandau: Germany, 1996; p 178. (24) Hada, H.; Honda, C.; Tanemura, H. Photogr. Sci., Eng. 1977, 21 (2), 83. (25) Honda, C.; Hada, H. Photogr. Sci., Eng. 1977, 21 (2), 91. (26) Kemnitz, K.; Yoshihara, K.; Tani, T. J. Phys. Chem. 1990, 94, 3099. (27) Steiger, R.; Kitzing, R.; Hagen, R.; Stoeckli-Evans, H. J. Photogr. Sci. 1974, 22, 151.

Thiacarbocyanine Dyes Adsorbed to Langmuir Films

emission intensity is reached after 60 min of dye adsorption (spectra taken after longer periods of time are not shown in Figure 2b). No monomer emission could be seen during the time window of the experiment. Even when the excitation occurred in the monomer band, at 555 nm, only emission from the aggregates could be observed. Even if some light is absorbed by monomers at or close to the interface, a combination of the very small fluorescence quantum yield of monomers of THIATS in aqueous solution (10-3) and energy transfer from monomer dye molecules to aggregated dye molecules will make it very difficult to observe any monomer fluorescence.28 Qualitative polarization measurements were performed on these films at the air-water interface to determine the orientation of the dye molecules with respect to the interface. Since emission spectra with a “horizontal” polarization (s-polarization) both at the excitation and the emission monochromator showed a higher fluorescence intensity than emission spectra with a “vertical” polarization (p-polarization) at the excitation and emission monochromators, one can conclude that the dye molecules are preferentially oriented with their long axes, with coinciding transition dipole moments, parallel to the air-water interface.29 Aggregate Formation of THIATS Adsorbed onto an ODACl Film. To investigate the influence of the structure of the amphiphile on the adsorption and aggregation of THIATS at the air-water interface, the experiments were repeated with a film of ODACl, an amphiphile with one long alkyl chain whereas DODAB contains two long alkyl chains (compare compounds 3 and 4, Figure 1). For this system the aggregation of THIATS was also followed as a function of time for a 10-6 and a 10-7 M dye concentration. Because the Langmuir film of ODACl was somewhat less stable, as suggested by the drop in surface pressure observed when the compression is stopped, the mask was kept open during the adsorption process. In this way, the surface pressure could be kept constant at 30 mN/m. However, this procedure could in principle allow diffusion of the dye molecules out of the mask. For the 10-7 M dye concentration, very little aggregate formation was detected at first, and after 20 min, the J-band disappeared completely (spectra not shown). A possible explanation for the disappearance of the J-band is a charge density that is smaller than expected for totally protonated ODACl molecules at the interface. This would lead to a reduced adsorption of dye molecules and a less extensive cooperativity. On the basis of the solution pKa of 4.7530 of ODACl, the ratio [NH4+]/[NH3] would amount to 103 at a pH of 5.5, so that this effect at a first glance should in this case be negligible. However, one should take into account that the electrostatic potential at the interface and the lower local dielectric constant of the lipid layer can decrease the pKa considerably.31 In contrast to the case of the 10-7 M solution, aggregate formation was observed for a 10-6 M solution of THIATS. Absorption Spectra. The absorption spectra of the 10-6 M solution of THIATS adsorbed as a function of time onto an ODACl film are very similar to the ones obtained for the THIATS/DODAB film (compare Figures 2a and 3a). Aggregate formation also occurs within an adsorption time of 5 min (Figure 3a). The maxima of the absorption (28) Noukakis, D.; Van der Auweraer, M.; Toppet, S.; De Schryver, F. C. J. Phys. Chem. 1995, 99, 11860. (29) Ferre´, Y.; Larive´, H.; Vincent, E. J. Soc. Photogr. Sci. Eng. 1974, 18, 457. (30) Lide, D. R. Handbook of Chemistry and Physics, 73nd ed.; CRC Press Inc.: Boca Raton, FL, 1992-1993. (31) Fromherz, P.; Arden, W. J. Am. Chem. Soc. 1980, 102, 6211.

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Figure 3. (a) Absorption spectra of a THIATS/ODACl layer at the air-water interface with an initial dye concentration of 10-6 M; spectra taken after 5 min (0), 20 min (4), 50 min (O), and 80 min (3). The arrows at the M-band and J-band, pointing downward and upward, indicate a decrease and increase, respectively, of the absorbance of the corresponding band, after longer periods of time. (b) Emission spectra taken of the THIATS/ODACl layer at the air-water interface with an initial dye concentration of 10-6 M; excitation was performed at 620 nm; spectra taken after 5 min (0), 30 min (4), 60 min (O), and 90 min (3).

bands for the monomer and the aggregate are at 553 and 654 nm, respectively, and the corresponding FW2/3M values of the bands are 1060 ( 30 and 280 ( 20 cm-1 (Table 1). The effective coherence length is calculated to be 14 ( 2. Repeating the experiment several times yields data that are identical except for the intensity of the shoulder in the J-absorption band located around 630 nm. This shoulder is however never pronounced. The slight difference in the absorption maximum of the J-aggregate using ODACl instead of DODAB may result from a somewhat different packing of the aggregate, since the packing conditions for ODACl (charge density, area per amphiphile head) differ from those of DODAB. In addition, H-bonding may also occur between ODACl molecules while this is impossible for DODAB. This may lead to a more rigid film than for DODAB. Those differences may also explain the slightly larger bandwidth at two-thirds of the maximum and thus the smaller effective coherence length observed with these dye/lipid complexes compared to the THIATS/DODAB complexes (Table 1). However, the spectra of the J-aggregates of THIATS adsorbed onto ODACl resemble better those formed onto a DODAB film or an AgBr crystal than those formed in an aqueous solution.23-26 Emission Spectra. The emission spectra (Figure 3b) obtained for the 10-6 M THIATS subphase underneath

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an ODACl film at different adsorption times are also very similar to those obtained for the THIATS/DODAB film. The emission maximum is situated at 656 nm, and the FW2/3M is 220 ( 20 cm-1. As for the THIATS/DODAB film, the Stokes shift is negligible. The films were excited at 620 nm, where only the aggregates absorb, but excitation at 555 nm in the monomer band yielded identical emission spectra. This suggests that even if some light would be absorbed by the monomers at 555 nm, complete transfer of excitation energy occurs from the monomers to the adsorbed aggregates. Fluorescence Microscopy of a Film of Adsorbed THIATS at the Air-Water Interface. Fluorescence micrographs were taken from a film of THIATS/DODAB. These experiments were not repeated for a film of THIATS/ ODACl, since the spectral properties of both films are very similar. The absorption spectra, using the 10-7 M dye-containing subphase, indicated that J-aggregates could be observed after only 15 min of adsorption time. In contrast, formation of very mobile (a dynamic movement of the domains can be observed), circular domains of J-aggregates with a diameter smaller than 5 µm could be observed with the fluorescence microscope using the J-filter set only 90 min after the onset of adsorption (micrograph not shown). This difference in time regime could originate from the limited resolution of the microscope. Indeed, a reliable detection of fluorescent structures is only possible when large enough domains are present. This corresponds, under our experimental conditions, with the micrometer range. Smaller domains could be present much earlier, but they cannot be observed with the microscope. Domains, with a diameter of around 15 µm, were also occasionally observed. The absorption spectra, using the 10-7 M dye-containing subphase, indicated that after one night of adsorption all monomer absorption had disappeared and only absorption of J-aggregates was left. The fluorescence microscopy experiments for this dye concentration were therefore also performed over this period of time, to find out how the fluorescence characteristics and thus the morphology of the film evolve. Micrographs 4a and 4b were taken after 5.5 and 20 h of adsorption time, respectively. Although many circular, individual fluorescent domains with sizes varying between 5 and 15 µm, already observed after 90 min of adsorption time, remain present, approximately half the surface of the film is occupied by an inhomogeneous fluorescent area after 5.5 h of adsorption time (micrograph 4a). This was accompanied by an increased rigidity of the film, since the film revealed a decreased mobility (these inhomogeneous fluorescent areas show no movements of the circular domains). After 20 h of adsorption time, the surface occupied by the inhomogeneous fluorescent area is largely increased at the expense of the individual domains (micrograph 4b). It can also be seen that a rather large area of the film is devoid of fluorescence. Since we have observed by absorption spectroscopy that monomers are no longer present after this adsorption time, this suggests that the 10-7 M dye concentration is not quite sufficient to cover the whole film surface. A concentration of 10-6 M of dye in the subphase is more than sufficient to provide a monolayer coverage of the Langmuir film. In contrast to the 10-7 M experiment, we were able to detect fluorescence (using the J-filter set) from this film already after 35 min (micrograph 4c), which corresponds with the time dependence of the spectral data. Micrographs 4c and 4d were taken respectively after 35 and 50 min of dye adsorption. The film in micrograph 4c

Vranken et al.

consists of a large number of closely packed fluorescing domains with a diameter of 1-2 µm as well as some inhomogeneous fluorescent areas. While the fluorescence characteristics do not change with longer adsorption times, the size and distribution of the domains shown in micrograph 4d are more homogeneous than those in micrograph 4c. In this case, the film is clearly more rigid than that for the 10-7 M dye concentration (micrographs 4a and 4b), as bleaching can be observed (the bleaching area in micrograph 4d is observed within a few seconds). Despite the fact that we worked with the same amphiphile (DODAB) and with the same dye concentration in the subphase (10-6 M), we obtained totally different fluorescence characteristics for our film at the air-water interface than Kirstein and Mo¨hwald.19,32 This is most probably due to two factors. First, they used a structurally different thiacarbocyanine dye, that forms a different type of aggregates (a Herringbone aggregate) and reveals an H-band as well as a J-band in its absorption spectrum. Furthermore, this J-band is situated at 620 nm, whereas the J-aggregates of THIATS absorb and emit around 655 nm. Second, their experiment consists of a sequence of compressions and relaxations of the DODAB layer, which had a pronounced influence on the film morphology. In the present work, the DODAB layer was first compressed to a surface pressure of 30 mN/m; the dye was then injected in the subphase to reach a concentration of 10-6 M. When DODAB was spread and compressed on a subphase containing 10-6 M THIATS, the same spectral data were obtained. However, under those conditions, the fluorescence microscopy images show larger microcrystals of the dye. A more extensive polarization study was performed for those films which will be discussed elsewhere. Aggregate Formation of TDC Adsorbed onto an AA Film. To study the TDC/AA complexes, 10-6 and 10-7 M dye concentrations were used. Since the surface pressure of the film of AA decreased slowly, we left the mask open to keep the surface pressure constant by adjusting the barrier position. For a 10-7 M dye concentration, in analogy to the THIATS/ODACl complexes, hardly any aggregate formation could be seen at the beginning of the experiment and the small aggregate band even disappeared after some time due to diffusion of the dye out of the mask. Hence, only the spectra obtained for the 10-6 M solution are discussed in more detail. Absorption Spectra. The first time-dependent measurements of the absorption spectra of a 10-6 M dye concentration of TDC adsorbed onto an AA film showed a monomer absorption band with a maximum at 551 nm and a bandwidth (FW2/3M) of 980 ( 10 cm-1 (Table 1). The J-aggregate band in this spectrum was initially characterized by a maximum at 651 nm while the FW2/ 3M value for this J-band was 190 ( 10 cm-1 (Table 1). This suggests an effective coherence length of 26 ( 5 molecules. Furthermore, we observed the formation of a shoulder on the blue side of the J-band (spectra not shown). When the experiment was repeated, the shoulder became more intense and even developed into a distinct band with a maximum at 634 nm. This can be seen in Figure 5a, where the observed shoulder is already very pronounced after 20 min and eventually becomes a straightforward band. Under those conditions, the FW2/3M of this second band amounts to 260 ( 10 cm-1. For the J1-band absorbing at 652 nm, the FW2/3M amounts to 250 ( 40 cm-1 (Table 1). This leads to an effective coherence length of 18 ( 5 (32) Kirstein, S.; Bliznyuk, V.; Mo¨hwald, H. Physica A 1993, 200, 759.

Thiacarbocyanine Dyes Adsorbed to Langmuir Films

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Figure 4. Fluorescence micrographs of THIATS adsorbed from a 10-7 M (a, 5.5 h adsorption time; b, 20 h adsorption time) and a 10-6 M (c, 35 min adsorption time; d, 50 min adsorption time) dye solution to a compressed DODAB layer to 30 mN/m. Imaging was done using the J-filter set. The scale bar is 25 µm.

for the J1-band, the J-band at the longest wavelength, and 15 ( 1 for the J2-band, the J-band at the shortest wavelength. Emission Spectra. Emission spectra obtained upon excitation of these films at 600 nm are shown in Figure 5b. Almost immediately after injection of the dye, a narrow fluorescence band can be observed with a maximum at 652 nm and a FW2/3M of 230 ( 20 cm-1. After approximately 1 h, a shoulder which was attributed to emission of the J2-aggregates appears at 637 nm. At longer periods of time, this shoulder becomes more pronounced and develops into a separate band. The emission of the J1-band does not disappear. The presence of a predominant emission of the J2-aggregate suggests that, despite a good spectral overlap and an important absorption of the J1aggregates, no efficient energy transfer from the J2- to the J1-aggregates occurs. This result strongly suggests that both types of aggregates form separate and electronically uncoupled domains of aggregates with a size exceeding at least the Fo¨rster distance (2-10 nm). The polarization of the fluorescence of the TDC/AA complexes indicates that the dye molecules are oriented parallel to the air-water interface. In contrast to the results obtained with THIATS, we observe with TDC the coexistence of two types of J-

aggregates: one, denoted J1, which absorbs and emits at a longer wavelength while the other one, J2, absorbs and emits at a shorter wavelength. Since the conversion of the J1- into the J2-aggregate can be observed in the absorption as well as in the fluorescence spectra, we can conclude that the J1-aggregate of TDC is a metastable species. This conversion has already been observed for other thiacarbocyanine dyes for which the layer or the substratum to which the dyes are respectively adsorbed or deposited has an influence on the aggregate packing.26 The different behavior of THIATS and TDC could be rationalized by the following argument. The positive charge of TDC is delocalized over the core of the dye molecule; on the other hand, the negative charges, that are the driving force for the THIATS adsorption, are highly localized and situated at the end of the side chains of the molecule. Aggregate Formation of TDC Adsorbed onto a DHP Film. The amphiphile DHP was used for a comparison with the experiments of the THIATS/DODAB complexes, as well as for a determination of the effect of the lower charge density of DHP with respect to AA, on the adsorption of TDC. DHP forms a stable Langmuir film which allows us to close the mask (as in the case of the THIATS/DODAB film). The adsorption and aggrega-

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Figure 5. (a) Absorption spectra of the TDC/AA layer at the air-water interface with an initial dye concentration of 10-6 M, taken under the same experimental conditions as for a THIATS/DODAB layer; spectra taken after 20 min (0), 40 min (4), 80 min (O), and 100 min (3). The arrows at the M-band and J1-band, pointing downward, and the arrow at the J2-band, pointing upward, indicate a decrease and increase, respectively, of the absorbance of the corresponding bands. (b) Emission spectra of the TDC/AA layer at the air-water interface with an initial dye concentration of 10-6 M; excitation was performed at 600 nm; spectra taken after 5 min (0), 20 min (4), 50 min (O), 80 min (3), and 125 min (]).

tion from a 10-6 M and a 10-7 M dye concentration inside the mask were studied. Absorption Spectra. Figure 6a presents the absorption spectra obtained for a 10-6 M dye concentration in the mask. Only one J-band is observed that shifts from 641 to 634 nm after longer periods of time. The FW2/3M of the band was equal to 290 ( 70 cm-1 (Table 1). As the monomer band has a maximum at 552 nm and a FW2/3M of 990 ( 10 cm-1, an effective coherence length of 14 ( 4 (Table 1) could be calculated using eq 1. The FW2/3M of the J-band for the 10-7 M dye concentration was found to be wider: a value of 400 ( 100 cm-1, yielding a smaller effective coherence length of 8 ( 4 (Table 1) (spectra not shown). For this system, aggregation only occurred after an adsorption time of approximately 1 h. The observed shift of the absorption maximum for the 10-6 M dye concentration, however, was also detected for the 10-7 M dye concentration (Table 1) (spectra not shown). Emission Spectra. Since the absorption spectra of the 10-6 and 10-7 M TDC/DHP complexes were almost identical, only the emission spectra obtained for the 10-6 M TDC/DHP complexes upon excitation at 600 nm (Figure 6b) will be discussed. After 5 min of adsorption, the emission maximum is situated at 646 nm (Table 2). This

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Figure 6. (a) Absorption spectra of the TDC/DHP layer at the air-water interface with an initial dye concentration in the mask of 10-6 M; spectra taken after 5 min (0), 20 min (4), 50 min (O), and 80 min (3). The arrows at the M-band and J-band, pointing downward and upward, indicate a decrease and increase, respectively, of the absorbance of the corresponding band, after longer periods of time. (b) Emission spectra of the TDC/DHP layer at the air-water interface with an initial concentration of 10-6 M; excitation was performed at 600 nm; spectra taken after 5 min (0), 20 min (4), 35 min (O), 50 min (3), 65 min (9), 80 min (2), 95 min (b), and 125 min (1).

maximum shifts to the blue and reaches its most blueshifted value of 636 nm after 35 min. A shoulder that becomes more pronounced as a function of time appears on the red side of the band and develops, after 2 h, into a band emitting at a maximum of 652 nm. Although during the first 35 min the absorption and emission spectra evolve in the same way, the counterpart of the red shift of the emission toward the J1-band occurring at longer periods of time was never observed in the absorption spectra. The spectral data for the TDC/DHP complexes differ from those of both the THIATS/DODAB and TDC/AA complexes. The difference with the THIATS/DODAB and THIATS/ODACl complexes can be attributed to the different positions of the charges of the dye molecules, yielding a different adsorption energy or a different adsorption geometry. In contrast, the differences between the TDC/AA and the TDC/DHP films are probably related to a different surface charge density. Since DHP and AA bear respectively two and one long alkyl chain, it is easy to understand that, for DHP, the number of charges per unit area is only half that of AA (we have measured a molecular area of 20 and 40 Å2/molecule for AA and DHP, respectively). This may result in a different packing of the dye molecules in the aggregates. For the absorption spectra, we observed that there is no distinct J1-band present in the TDC/DHP film. However, it may be possible

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Figure 7. Fluorescence micrographs of a layer of TDC adsorbed from a 10-6 M dye solution to a compressed AA layer to 30 mN/m (a, 5 min adsorption time; b, 50 min adsorption time). Micrograph a was taken using the M-filter set; micrograph b was taken using the J-filter set. The scale bar is 15 µm. Table 2. Time Dependence of the Emission Maximum of a TDC/DHP Layer at the Air-Water Interface with an Initial Dye Concentration of 10-6 M adsorption time (min) 5 20 35 50 65 80 95 125 emission max (nm) 646 640 636 637 648 650 651 652

that this band is initially hidden underneath the pronounced J2-band. When the values of FW2/3M of the absorption bands are compared, we observe a narrowing of the J-band at longer periods of time which may confirm this hypothesis (Table 1). Although, after the eventual equilibrium of the dye adsorption, the J2-aggregates are predominant in the absorption spectrum, the emission is, however, to a large extent due to J1-aggregates. This emission can be due to a direct excitation of a small fraction of J1-aggregates. In this case, the fluorescence quantum yield of these J1-aggregates is an order of magnitude larger than that of the J2-aggregates. Another possible explanation is that both types of aggregates have a similar fluorescence efficiency while excitation transfer occurs from the J2-aggregates to the J1-aggregates. This indicates that, in contrast to TDC adsorbed on AA, the average distance to the J1-aggregates is of the same order of magnitude as the distance for Fo¨rster transfer (generally between 2 and 10 nm).33-35 The different behavior of TDC adsorbed on AA compared to DHP is also observed in fluorescence microscopy. While for TDC adsorbed on DHP very efficient photobleaching excluded the recording of fluorescence micrographs, we had no problem in recording fluorescence micrographs for TDC adsorbed on AA (see next section). Although in both cases TDC adsorbs with the core of the molecule, over which the charge is delocalized, the difference in charge density for the Langmuir films of AA and DHP can lead to a different free energy for the packing of J1- and J2-aggregates. Fluorescence Microscopy of a Film of Adsorbed TDC at the Air-Water Interface. Fluorescence micrographs were obtained for TDC absorbed to a Langmuir film of AA. When it was attempted to repeat the experiments for TDC adsorbed to a Langmuir film of DHP, the (33) Nakashima, N.; Yoshihara, K.; Willig, F. J. Chem. Phys. 1980, 73, 3553. (34) Laguitton-Pasquier, H.; Van der Auweraer, M.; De Schryver, F. C. Langmuir 1998, 14, 5172. (35) Kilså Jensen, K.; Albinsson, B.; Van der Auweraer, M.; Vuorimaa, E.; Lemmetyinen, H. J. Phys. Chem. B 1999, 103, 8514.

fluorescence bleached too fast to allow one to record micrographs. Fluorescence micrographs of TDC/AA were obtained from a film prepared the same way as described before for THIATS/DODAB, with a 10-6 M TDC solution in the mask. Micrograph 7a was taken 5 min after the onset of adsorption, using the M-filter set. A weak and apparently homogeneous fluorescence can be detected. Furthermore, nonfluorescent, dark domains with an irregular shape can be observed together with a certain number of very intense fluorescent domains. Changing the M-filter set for the J-filter set to image the TDC/AA film does not lead to other fluorescence characteristics of the film, except for a pronounced bleaching of the homogeneous fluorescence. When we use the M-filter set, the absorbed energy has to be transferred to the J-aggregates, whereas in the case of the J-filter set the excitation energy is directly absorbed by the J-aggregates, and thus bleaching occurs much faster. The intense fluorescent domains, however, do not seem to bleach (see micrograph 7b, taken after 50 min of adsorption time). The dark domains might be due to collapsed AA material that prevents fluorescence of the aggregates. This collapse could result from injecting the dye in the subphase, through the compressed layer of AA, inside the mask, since under normal conditions the collapse pressure of an AA film is much higher than 30 mN/m.36 Actually, the observed features are very similar to those of collapsed material that was observed when AFM-experiments were performed on deposited films of AA (unpublished results). Only the dimensions are of course much larger in this case. An interesting feature is the very intense fluorescent domains with a diameter of 10-15 µm. A possible explanation might be that these intense fluorescent domains are made up of 3D aggregates with a larger absorbance or fluorescence quantum yield. In that case, the intense fluorescence should be observed only after longer periods of times, as it takes more time to grow 3D-aggregates. This possible explanation is thus unlikely, since micrograph 7a has been taken after only 5 min of adsorption time. Another possibility is that the intense fluorescing domains are made up of a different type of J-aggregate, since both the absorption and emission (36) Insoluble Monolayers at the Liquid-Gas Interfaces; Gaines, J. L., Jr., Ed.; Wiley & Sons: 1966.

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spectra revealed the coexistence of two types of Jaggregates. Correlating this assumption with the spectral data, the intense fluorescence should be that of the J2type aggregate, emitting at 634 nm, and the background fluorescence should be that of the J1-type aggregate, emitting at 652 nm. This does not contradict the fact that the J-filter set is optimized for fluorescence at 650 nm, since differences in quantum yield can overrule differences in filter sensitivity. Comparing the observations from fluorescence microscopy with the spectral properties of the TDC/AA film indicates that this bleaching assists the conversion from the J1-type aggregate (at 650 nm) to the J2-type aggregate (at 634 nm) observed in the spatially unresolved fluorescence spectra. Conclusions We have shown that THIATS forms J-aggregates at the air-water interface after adsorption to an oppositely charged Langmuir film of an ammonium salt bearing either one or two octadecyl chains. The large difference in charge density between these amphiphiles does not lead to a significant difference in the spectroscopic properties of the J-aggregates. Their absorption spectra revealed a narrow, intense new band that was shifted to the red for more than 100 nm with respect to the monomer band. In the case of the DODAB film, no influence of the dye concentration underneath the charged Langmuir film with respect to the aggregate formation could be detected. The emission spectra revealed a mirror image fluorescence band with no Stokes shift. Polarized fluorescence experiments showed that the dye molecules are aligned with their long axes parallel to the air-water interface, which is consistent with a brickstone packing of the molecules at the interface.4 The charged SO3- groups of THIATS determine the packing of this dye molecule. Because of the C3H6 “spacer” chain, the dye molecules can pack optimally in a two-dimensional brickstone packing, adopting an all-trans conformation. It is that packing of the dye molecules that leads to the large bathochromic shift. This type of aggregate formation (J1) presumes a well-ordered packing of the dye molecules in the monolayer.27 Excitation of the monomer band does not change the fluorescence properties of this film, indicating an efficient energy transfer from the monomer dye molecules to the aggregated ones. Fluorescence microcopy experiments suggest that at a sufficient dye concentration (10-6 M) the aggregation of the dye molecules can lead to a homogeneous layer of closely packed domains. From the timedependent character of the experiments, we can conclude that the number of dye molecules that adsorb onto the compressed layer of amphiphiles from the subphase increases with time. When the dye concentration in the subphase was lower than necessary for a monolayer

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coverage, it was even possible, after a long period of time, to adsorb all dye monomers from the subphase. The dye TDC also forms J-aggregates at the air-water interface but reveals a different behavior. Using AA as amphiphile, both the absorption spectra and the emission spectra reveal two kinds of J-aggregates. The J1-aggregate absorbing around 650 nm is formed initially and is converted into a second type of J-aggregate (J2) absorbing 20 nm at shorter wavelengths. The two types of Jaggregates coexist in the absorption spectrum as well as in the emission spectrum, indicating that the fluorescence of the J2-aggregate is not quenched by the J1-aggregates. This suggests that they are distant from each other by at least 10 to 20 nm. Using DHP as amphiphile, the absorption spectra revealed only one J-band that shifts slightly to the blue with time. From observations of band narrowing with time, we can conclude that there might possibly be an underlying “band” present of the J1-type of aggregates. The emission spectra first go through a blue shift, and after a period of coexistence, the fluorescence shifts again to the red, confirming the persistence of the J1-aggregate type that was seen for the TDC/AA complexes. As the dye adsorption is determined by Coulomb interactions between the charge of TDC that is delocalized over the core of the chromophore and the charges of the lipid layer, the packing of the dye molecules will be determined more extensively by the lipid film than in the case of THIATS. Since a TDC/AA film revealed an aggregation behavior different from that of a THIATS/ DODAB film, the different film morphologies observed with fluorescence microscopy were expected. For a THIATS/DODAB film a homogeneous layer of closely packed fluorescent domains could be observed under specific experimental conditions, whereas for a TDC/AA film other characteristics, namely a weak homogeneous fluorescence, coexisting with intense fluorescent domains, were observed. Acknowledgment. M.V.d.A. is a “Onderzoeksdirecteur” of the F.WO. (Fonds voor Wetenschappelijk Onderzoek Vlaanderen). N.V. thanks the “Vlaams instituut voor de bevordering van het wetenschappelijk en technologisch onderzoek (IWT)”. The authors gratefully acknowledge the continuing support from DWTC (Belgium) through grant IUAP-IV-11, the F.W.O.-Vlaanderen, the Nationale Loterij, and the Flemish-Que´bec cooperation. The authors are grateful to Agfa N.V. for the gifts of the dyes. The authors are indebted to the Natural Sciences and Engineering Research Council of Canada, the “Fonds pour la formation de Chercheurs et l′Aide a` la Recherche” (FCAR), and FRSQ for financial support. LA000896L