Langmuir 2009, 25, 1651-1658
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Suspensions of Organic Microcrystals Produced in the Presence of Polymers: Diversity of UV/Vis Absorption and Fluorescence Properties According to the Preparation Conditions Mouhammad Abyan,† Dominique de Caro,‡ and Suzanne Fery-Forgues*,† Laboratoire des Interactions Mole´culaires Re´actiVite´ Chimique et Photochimique, UMR CNRS 5623, UniVersite´ Paul Sabatier, 31062 Toulouse cedex 9, France, and Laboratoire de Chimie de Coordination, UPR CNRS 8241, 205 route de Narbonne, 31077 Toulouse cedex 4, France ReceiVed July 4, 2008. ReVised Manuscript ReceiVed NoVember 21, 2008 Free-standing microcrystals of an organic fluorescent dye, specifically, 4-n-octylamino-7-nitrobenz-2-oxa-1,3diazole, were prepared using a solvent-exchange process at room temperature, in the presence of polymers used as additives. Parameters such as the dye concentration, the nature and concentration of the polymer, and the pH of the solution were varied. Six samples of microcrystals were therefore obtained and characterized by fluorescence microscopy and by electron microscopy (TEM and SEM). They differed by their content in microcrystals, the shape and size of which depends strongly on experimental conditions. Curiously, the UV/vis absorption spectra of the microcrystal suspensions were very different from one sample to another. As a result the emission spectra were also varied. The diversity of the optical response obtained was attributed to the presence of several dye populations in the microcrystal suspensions. A distinction was made between the intrinsic spectral properties of the microcrystals and artifacts due to the presence of the additives.
Introduction Until now, organic dyes have been essentially used as dispersed molecules, whether they were dissolved in solution, included in solids such as textile fibers or polymers, or grafted on biological substrates. Their use as nano- and microcrystals is very recent, and they already appear to be of real interest for various applications. Actually, nano- and microcrystals of organic dyes have been considered as functional materials for organic lightemitting diodes (OLEDs)1 and optical devices such as optical waveguides2 and memory systems based on photochromic molecules.3 They are also fluorescent sensors for the analysis of biological molecules such as DNA,4 proteins,5 or glucose6 and for the detection of pollutants such as chromium.7 In photo* Corresponding author. Telephone: +33 5 61 55 68 05. Fax: +33 5 61 55 81 55. E-mail:
[email protected]. † Laboratoire des Interactions Mole´culaires Re´activite´ Chimique et Photochimique. ‡ Laboratoire de Chimie de Coordination. (1) Mal’tsev, E. I.; Lypenko, D. A.; Shapiro, B. I.; Brusentseva, M. A.; Berendyaev, V. I.; Kotov, B. V.; Vannikov, A. V. Appl. Phys. Lett. 1998, 73, 3641–3643. (2) (a) Kaneko, Y.; Shimada, S.; Fukuda, T.; Kimura, T.; Yokoi, H.; Matsuda, H.; Onodera, T.; Kasai, H.; Okada, S.; Oikawa, H.; Nakanishi, H. AdV. Mater. 2005, 17, 160–163. (b) Kaneko, Y.; Onodera, T.; Kasai, H.; Okada, S.; Oikawa, H.; Nakanishi, H.; Fukuda, T.; Matsuda, H. J. Mater. Chem. 2005, 15, 253–255. (c) Yanagi, H.; Ohara, T.; Morikawa, T. AdV. Mater. 2001, 13, 1452–1455. (d) Yanagi, H.; Morikawa, T. Appl. Phys. Lett. 1999, 75, 187–189. (3) (a) Lim, S.-J.; An, B.-K.; Jung, S. D.; Chung, M.-A.; Park, S. Y. Angew. Chem., Int. Ed. 2004, 43, 6346–6350. (b) Spagnoli, S.; Block, D.; BotzungAppert, E.; Colombier, I.; Baldeck, P. L.; Ibanez, A.; Corval, A. J. Phys. Chem. B 2005, 109, 8587–8591. (4) (a) Jinshui, L.; Lun, W.; Feng, G.; Yongxing, L.; Yun, W. Anal. Bioanal. Chem. 2003, 377, 346–349. (b) Zhou, Y.; Bian, G.; Wang, L.; Dong, L.; Wang, L.; Kan, J. Spectrochim. Acta, Part A 2005, 61, 1841–1845. (c) Wang, L.; Xia, T.; Wang, L.; Chen, H.; Dong, L.; Bian, G. Microchim. Acta 2005, 149, 267–272. (5) Wang, L.; Wang, L.; Dong, L.; Bian, G.; Xia, T.; Chen, H. Spectrochim. Acta, Part A 2005, 61, 129–133. (6) Botzung-Appert, E.; Monnier, V.; Ha Duong, T.; Pansu, R.; Ibanez, A. Chem. Mater. 2004, 16, 1609–1611. (7) Wang, L.; Wang, L.; Xia, T.; Dong, L.; Bian, G.; Chen, H. Anal. Sci. 2004, 20, 1013–1017.
chemistry, they are efficient photosensitizers, less prone to photodegradation than isolated dye molecules.8 However, understanding and even more predicting the spectroscopic behavior of organic dye microcrystals is still very difficult. How a chemical structure should be modified to tune the spectroscopic properties of a dye in solution is quite well-known.9 However, in most cases, this experience is not directly transposable to the solid state. This can be explained by two main reasons. The first one is that, in the solid state, flexible molecules are frozen in a particular conformation that can affect directly the delocalization of the electron system. A consequence is the variation of the absorption spectrum with respect to that of the dissolved dye.10 This phenomenon can also lead to spectacular changes in crystal color, as nicely illustrated by color polymorphism.11 The second reason is that intermolecular interactions that take place between dye molecules, although weak for organic compounds, influence the energy levels.12 The presence of neighboring molecules stabilizes or destabilizes the excited states. The color and photophysical behavior of dyes in the solid state are thus closely related to molecular arrangement. The problem is even more complicated with microcrystals because of their very particular nature. It would be exaggerated to say that, like nanocrystals, microcrystals contain a large number of surface molecules with respect to inner molecules. However, surface molecules still can play a significant role, especially when microcrystals have a nanometric dimension (as it is the case in the following work where the microcrystals are about 80 nm thick). Surface molecules undergo weak packing forces and are often involved in surface defects where the crystal network can be (8) Kim, H. Y.; Bjorklund, T. G.; Lim, S.-H.; Bardeen, C. J. Langmuir 2003, 19, 3941–3946. (9) For example, see Krasovitskii, B. M.; Bolotin, B. M. Organic Luminescent Materials; VCH: Weinheim, 1988. (10) Fujita, S.; Kasai, H.; Okada, S.; Oikawa, H.; Fukuda, T.; Matsuda, H.; Tripathy, S. K.; Nakanishi, H. Jpn. J. Appl. Phys 1999, 38, L659–L661. (11) (a) Yu, L. J. Phys. Chem. A 2002, 106, 544–550. (b) Csiko´s, E.; Ferenczy, ´ ngya´n, J. G.; Bo¨cskei, Z.; Simon, K.; Go¨nczi, C.; Hermecz, I. Eur. J. G. G.; A Org. Chem. 1999, 2119–2125. (12) (a) Birks, J. B. Photophysics of Aromatic Molecules; Wiley: London, 1970. (b) Silinsh, E. A. Organic Molecular Crystals; Springer-Verlag: Berlin, 1980.
10.1021/la803549u CCC: $40.75 2009 American Chemical Society Published on Web 01/05/2009
1652 Langmuir, Vol. 25, No. 3, 2009 Chart 1. Chemical Structure of Compound 1
different. Besides, they are in contact with the outer medium. Their spectroscopic characteristics can then be different from those of inner molecules. Finally, it must be kept in mind that microcrystals are often prepared in the presence of additives used to control the reprecipitation process. It can be expected that the presence of these additives in the medium generates some artifacts. For example, some microcrystal molecules can remain in contact with the additive molecules that are included into the crystal structure or adsorbed at the surface. The number of parameters that must be taken into account explains why predicting the spectroscopic properties of dye microcrystals is so difficult and why problems of reproducibility are often encountered, while they do not arise when working with dye solutions. The aim of the present work is to give evidence for the diversity of the spectroscopic responses that can be obtained from suspensions of microcrystals made of the same dye, but prepared under different experimental conditions, and to understand the origin of these differences. To do so, we worked with an organic dye, specifically 4-n-octylamino-7-nitrobenzoxadiazole (1, Chart 1), which belongs to a well-known family of fluorescent probes.13 This compound was extensively used by our team during the past few years to study the formation of free-standing microcrystals by a solventexchange method, called “reprecipitation method.”14 The latter consists of dissolving an organic compound into a hydrophilic organic solvent and pouring a small volume of this concentrated solution into a large volume of water, which acts as a nonsolvent. As a result, the organic compound precipitates and possibly crystallizes, giving an aqueous suspension of microcrystals. The experimental parameters can be modified easily while keeping the same basic procedure. Interestingly, one of the most striking characteristics of compound 1 is that it crystallizes in a very versatile manner when changing the reprecipitation conditions. For instance, when a macromolecule (dendrimer,15,16 calf thymus DNA,17 or linear polymer18) was placed as an additive in the reprecipitation medium, we obtained microcrystals of 1 that strongly differed in size and habit according to the macromolecule used. These effects were explained by the fact that the macromolecules adsorb on the microcrystal surface and interfere with crystal growth along some directions. Another advantage of using a macromolecule as additive is that dye microcrystals (13) (a) Fery-Forgues, S.; Fayet, J.-P.; Lopez, A. J. Photochem. Photobiol. 1993, 70, 229–243. (b) Chattopadhyay, A. Chem. Phys. Lipids 1990, 53, 1–15. (c) Lin, S.; Struve, W. S. Photochem. Photobiol. 1991, 54, 361–365. (d) Mukherjee, P.; Cardinal, J. R. J. Phys. Chem. 1978, 82, 1620–1627. (e) Chattopadhyay, A.; London, E. Biochemistry 1987, 26, 39–45. (f) Chattopadhyay, A.; London, E. Biochem. Biophys. Acta 1988, 938, 24–34. (g) Rajarathnam, K.; Hochman, J.; Schindler, M.; Ferguson-Miller, S. Biochemistry 1989, 28, 3168–3176. (14) (a) Nakanishi, H.; Oikawa, H. In Single Organic Nanoparticles; Masuhara, H., Nakanishi, H., Sasaki, K., Eds.; Springer-Verlag: Berlin, 2003; Chapter 2, pp 17-31. (b) Van Keuren, E.; Georgieva, E.; Adrian, J. Nano Lett. 2001, 1, 141– 144. (15) Bertorelle, F.; Lavabre, D.; Fery-Forgues, S. J. Am. Chem. Soc. 2003, 125, 6244–6253. (16) Bertorelle, F.; Rodrigues, F.; Fery-Forgues, S. Langmuir 2006, 22, 8523– 8521. (17) Bıˆrla˘, L.; Bertorelle, F.; Rodrigues, F.; Pansu, R.; Badre´, S.; Fery-Forgues, S. Langmuir 2006, 22, 6256–6265. (18) Abyan, M.; Bertorelle, F.; Fery-Forgues, S. Langmuir 2005, 21, 6030– 6037.
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do not agglomerate. The suspension remains steady during many hours and can be studied easily by classical spectroscopic methods usually devoted to the study of genuine solutions. In the course of our work, we observed that the optical properties of the suspensions were quite different from one sample to another, depending on the experimental conditions used for reprecipitation. We now think that it is of interest to clarify and rationalize this observation. In the present work, we decided to study thoroughly six samples that differed by the physical characteristics of the microcrystals they contained and by their spectroscopic response. These samples were chosen after numerous combinations had been tried, and they seemed to us quite representative of the various possibilities encountered. They were prepared by the reprecipitation method using synthetic polymers as additives, under different conditions of concentration and pH. The microcrystals obtained as aqueous suspensions were first visualized by fluorescence microscopy as well as by transmission (TEM) and scanning electron microscopy (SEM). Spectroscopic studies such as UV/visible absorption and steady-state fluorescence were then directly carried out on these microcrystal suspensions.
Experimental Section Materials. The fluorescent dye 4-n-octylamino-7-nitrobenz-2oxa-1,3-diazole (1) was prepared as previously described19 according to a variant of the synthesis reported by Heberer et al.20 Absolute ethanol (Carlo Erba Reagenti) and high-pressure demineralized water (pH ) 4.9), prepared with a Milli-Q apparatus (Millipore), were used as solvents. Poly(acrylic acid) sodium salt (PAA, MW ) 5100 g mol-1) and poly(acrylamide) (MW ) 5 × 106-6 × 106 g mol-1) were purchased from Aldrich. Dextran (low fraction) was from Acros. Its molecular weight was checked by mass spectrometry and found to be 75 000 g mol-1. All polymers were used without further purification. Apparatus and Methods. The pH was measured directly in the aqueous polymer solution with pH-indicator strips (Merck). All spectroscopic and kinetics experiments were performed in a temperature-controlled cell. When not specified the temperature was 22 °C. The UV/vis absorption spectra were recorded on a HewlettPackard 8452A diode array spectrophotometer. Steady state fluorescence spectra were recorded with a Photon Technology International (PTI) Quanta Master 1 spectrofluorometer. All the fluorescence spectra were corrected. The size and shape of the microcrystals were observed from a droplet of the suspensions deposited between a slide and a cover glass after reprecipitation was complete. To determine the microcrystal content of the various samples, 25-40 observations were made randomly per sample and all the crystals present in the optical area were counted. According to the samples, the averages were calculated from 500 to 1500 microcrystals. A Zeiss Axioskop fluorescence microscope equipped with a standard camera was used. The objective was made up of lenses of either ×40 or ×100 magnification. The eyepiece had a lens of ×2.5 magnification. When not specified, the excitation wavelength was 450-490 nm, and the emission was gathered above 500 nm, by using suitable filters. Electron microscopy was performed at the “Service Commun de Microscopie de l’Universite´ Paul Sabatier.” A TEMSCAN 200CX scanning electron microscope was used for transmission electron microscopy (TEM). To prepare the sample, a droplet of the microcrystal suspension was deposited on a carbon grid and the excess liquid was drawn off with paper. The sample was then revealed with a drop of ammonium molybdate solution (2%) as a contrasting agent and allowed to dry for 24 h at room temperature. Scanning electron microscopy (SEM) was carried out on a JEOL JSM 840A apparatus equipped with a field-effect gun. (19) Galinier, F.; Bertorelle, F.; Fery-Forgues, S. C. R. Acad. Sci. 2001, 4, 941–950. (20) Heberer, H.; Kersting, H.; Matschiner, H. J. Prakt. Chem. 1985, 327, 487–504.
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Table 1. Composition of the Six Samples Studied (PAA ) Poly(acrylic acid) Sodium Salt; PA ) Poly(acrylamide); Dex ) Dextran; EtOH ) Ethanol/Water v/v) and Distribution of the Microcrystals Formed According to the Classification of Figure 2 microcrystal content (%) sample 1 2 3 4 5 6
composition -5
-5
[PAA] ) 5.9 × 10 M, [1] ) 2.0 × 10 M, EtOH ) 4% pH ) 6.3 [PAA] ) 5.9 × 10-5 M, [1] ) 1.9 × 10-4 M, EtOH ) 4% pH ) 6.3 [PAA] ) 5.9 × 10-4 M, [1] ) 2.0 × 10-5 M, EtOH ) 2% pH ) 8.3 [PAA] ) 5.9 × 10-4 M, [1] ) 2.0 × 10-5 M, EtOH ) 2% pH ) 2.0 [PA] ) 4.1 × 10-7 M, [1] ) 2.0 × 10-5 M, EtOH ) 2% pH ) 5.0 [Dex] ) 6.5 × 10-4 M, [1] ) 2.0 × 10-5 M, EtOH ) 2% pH ) 5.0
T1
T2
T3
T4
T5
T6
T7
T8
0
0
0
97.4
0
0
2.6
0
0
0
0.9
98.0
0
0
1.1
0
5.8
2.5
3.1
76.9
0
0
11.7
0
0.2
0
0.1
99.7
0
0
0
0
0.7
0.7
0.7
0
33.9
20.3
27.3
16.4
4.6
0
0.2
91.9
0
0
3.3
0
The microcrystal suspensions were prepared as usual and filtered using a Nylon membrane filter. The red deposit was rinsed with water, dried at 50 °C under vacuum for 2 h, and kept under vacuum at room temperature until use. For observation by SEM, the microcrystals were stuck on a piece of black self-adhesive tape. The X-ray powder diffraction patterns were collected in transmission mode, on capillary samples, on a θ-θ XPert Pro Panalytical diffractometer, with λ (Cu KR1, KR2) 1.54059 and 1.54439 Å. The extraction of peak positions for indexing was performed with the fitting program, available in the PC software package Highscore+ supplied by Panalytical. Data Processing. The program Origin 6.0 (Microcal Software) was used for the deconvolution of the absorption spectra.
Results Preparation of the Samples. All samples were made in the same way. A concentrated solution of dye 1 in absolute ethanol was first prepared. A small volume of this solution was then introduced into an aqueous solution of polymer under constant stirring. It must be underlined that the polymer was dissolved in water before mixing with the dye solution. From one sample to the other, the concentration of the dye solution, the concentration of the polymer solution, the nature of the polymer, and the pH of the reprecipitation medium were allowed to change. The precise experimental conditions used to prepare these samples are summarized in Table 1. The samples can roughly be divided in two groups. Those of the first group (samples 1-3) contain a charged polymer, specifically poly(acrylic acid) sodium salt used in conditions where the functional groups are anionic carboxylate groups. The samples that constitute the second group are based on uncharged polymers. Sample 4 contains poly(acrylic acid) sodium salt in pH conditions where the carboxylic groups should not be ionized. Samples 5 and 6 contain nonionic polymers such as poly(acrylamide) and dextran, a high-molecular-weight polymer of glucose. The dye concentration in the samples varies from 2.0 × 10-5 to 1.9 × 10-4 M. The solubility limit of the dye being 4 × 10-7 M in water containing 2% ethanol,15,19 the dye supersaturation, which is the driving force for crystallization, is thus high in every case. It can also be noted that the solution of PAA at 5.9 × 10-4 M has the same concentration of functional groups as the solution of poly(acrylamide). The solution at a pH of 2 was obtained by addition of concentrated hydrochloric acid. The dye can withstand a low pH medium because its amine group is weakly basic, due to the strong withdrawing effect of both the nitro and furazan groups.20 Reprecipitation was thus performed for the six samples at 22 °C. The duration of this process, monitored by UV/vis absorption spectroscopy (Figure 1), varied with the experimental conditions used. It was about 16 min in the presence of ionized poly(acrylic
Figure 1. Evolution of the UV/vis absorption spectrum of 1 (2 × 10-5 M) in water containing 2% ethanol, during the reprecipitation process, in the presence of poly(acrylamide) at a concentration of 4.1 × 10-7 M. One measurement every 6 min.
acid) sodium salt, 40 min with PAA at acidic pH, 45 min with dextran, and 1 h with poly(acrylamide), with the latter duration being similar to that observed in water.15 Observation by Fluorescence Microscopy. The six samples were observed by fluorescence microscopy when reprecipitation was complete. They contained various types of microcrystals previously encountered in other experimental conditions.15,16,18 These microcrystals differed by their shape, by their size, and sometimes by the way they fluoresce, as pictured in Figure 2. From a general viewpoint, all the samples displayed a majority of individual, flat, very thin microcrystals, the thickness of which was about 80-100 µm (microcrystals of type 1-6). The observation of these microcrystals with a polarization microscope revealed that they were monocrystals.21 Crystals made of several moieties arranged around a common axis (microcrystals of type 7 and 8) were also currently found. They were twinned crystals21 in the shape of a cross or a star. Fragmented plates were sometimes encountered when stirring was not strong enough. Most of these structures emit light much more strongly from their edges than from their surface. No sample was totally homogeneous, since the various types of microcrystals coexist. However, some structures were more present in some samples than in others. Their distribution is expressed as percentages in Table 1. The majority of the microcrystals obtained were roughly rectangular plates. Sample 2 displayed very regular crystals, most of them measuring 23 µm × 10 µm, with slightly concave edges, a distinct notch on the small side, and light emission arising from the whole periphery (type 4a, Figure 2). In sample 4, the crystals have the same shape, but their size is (21) Rodriguez-Otazo, M. Ph.D. Thesis, Universite´ de Paris 11, 2008.
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Figure 3. Fluorescence microscopy image of microcrystals of 1 obtained by the reprecipitation method. (a) Observation of a twinned crystal with λex ) 540-580 nm and λem > 580 nm. (b) Observation of sample 4 ([PAA] ) 5.9 × 10-4 M, [1] ) 2.0 × 10-5 M, EtOH/water ) 2% v/v, and pH ) 2.0) under standard conditions (λex ) 450-490 nm and λem > 500 nm).
Figure 4. Observation by TEM (a) and SEM (b-d) of the microcrystals obtained by reprecipitation of 1 in different experimental conditions. (a) [1] ) 1.9 × 10-4 M, [PAA] ) 5.9 × 10-5 M, ethanol/water 4% (sample 2); (b) [1] ) 2.0 × 10-5 M, [PAA] ) 5.9 × 10-4 M, ethanol/water 2% (sample 3); and (c,d) [1] ) 2.0 × 10-5 M, poly(acrylamide) ) 4.1 × 10-7 M, ethanol/water 2% (sample 5).
Figure 2. Observation by fluorescence microscopy of the different types of microcrystals obtained by reprecipitation of 1 in various experimental conditions.
much smaller (about 5 µm long) and they tend to agglomerate. The microcrystals obtained in the presence of dextran at 22 °C displayed one or two deep notches on their little side. Those obtained with poly(acrylamide) were either plain and elongated spatula-like structures (type 5) or strongly notched objects (type 6 and 8). Crystals presenting sharply delineated hourglass sectors (type 1) were currently found, especially in samples 3 and 6. Type 2 crystals also result from sector zoning. Let us note that this phenomenon has been observed for long in a variety of minerals and is clearly related to how fast or efficiently different parts of the crystal grow.22 We also observed in the three samples, and especially in sample 3, a minority of large microcrystals that emit red light from one of the small edges or by both (type 3a and 3b, respectively), a phenomenon that we attributed to waveguiding and light reabsorption.17 (22) (a) Leung, I. S. Am. Mineral. 1974, 59, 127–138, and refs cited. (b) Dowty, E. Am. Mineral. 1976, 61, 460–469.
It must be emphasized that all microcrystals were observable by exciting with green light (540-580 nm) and using for emission a filter that cuts light below 580 nm (Figure 3a). In contrast, no diffuse fluorescence of the aqueous phase was detected whatever the filters used. It is also important to note that, besides microcrystals, some particles with no precise shape were observed in the suspensions made with PAA at acidic pH. These particles were of various sizes, with some appearing as little dots and others being of the same size as the crystals or even bigger. Figure 3b shows one free particle and numerous particles stuck to an agglomerate of microcrystals. These particles emit strongly in the green or in the yellow-green. Observations by Transmission and Scanning Electron Microscopy. Three of the samples were also observed by electron microscopy, which allowed additional details to be obtained concerning the microcrystal structure. For instance, TEM performed on sample 2 revealed that the thickness of the microcrystals is uneven. The long sides of the crystals show a much sharper contrast than the small sides. It seems that microcrystals are constituted by layers that form sort of inclined planes on the sides (Figure 4a). SEM performed on samples 3 and 5 distinctly showed numerous small crystals whose shape is reminiscent of that of the crystals observed by fluorescence microscopy. For instance, microcrystals prepared with PAA either were perfectly rectangular or had a notch in the middle of the small side (Figure 4b). The microcrystals obtained with poly-
Optical Properties of Organic Microcrystals
(acrylamide) appeared as elongated structures, slightly curved widthwise (Figure 4c and d). X-ray Powder Diffraction Patterns. In view of the variety of the microcrystals observed, a question comes to mind: does this variety result from a change in crystal habit or from the formation of different polymorphs? The problem is that the possibility of polymorphism is hard to study in our case. All classical X-ray analyses were unsuccessful until now because of the small size of the microcrystals, and we did not succeed in growing a single crystal of sufficient size, so that the crystal structure of 1 is still unknown. However, the X-ray powder diffraction (XRPD) patterns of microcrystals of samples 2 and 6 were recorded and showed no difference between them. They were also very close to the XRPD pattern of 1 obtained as a dark red powder at the end of the synthesis, after evaporation from an organic phase. These patterns are given as Supporting Information. These experiments do not allow the possibility of polymorphism to be totally ruled out, but we can consider that in every sample a large majority of microcrystals have the same crystal structure. Actually, it is more likely that the difference observed between the microcrystals is due to different crystal habits resulting from the effect of the additives on crystal growth.23 For example, adsorbed polymers would impede the approach of dye molecules and inhibit the growth of some crystal faces. Let us recall that the presence of residual additives in rinsed microcrystals was shown previously by elemental microanalysis15,18 and by the measurement of surface potential.16-18,25 UV/VisAbsorptionPropertiesoftheMicrocrystalSuspensions. Before examining the optical properties of the microcrystals, we thought it was useful to record the absorption spectrum of dye 1 at 1.5 × 10-6 M in water containing 2% ethanol. This concentration is higher than the solubility limit, but the spectrum was steady during many hours. We consider it to be quite close to that of the dissolved dye. This spectrum displayed a band of moderate intensity at 346 nm, resulting from a π-π* transition, and a band of strong intensity at 482 nm, assigned to intramolecular charge transfer (Figure 5a). The spectra of the microcrystal suspensions were recorded once reprecipitation was complete. Strikingly, all absorption spectra showed a different profile. For example, the spectrum obtained with the microcrystals grown in the presence of PAA at pH ) 2 (sample 4) was quite close to that of the highly dilute dye, apart from a small additional band around 544 nm (Figure 5b). Sample 5, prepared with poly(acrylamide), gave a spectrum that resembles the former, but the small band at long wavelengths was now a well-defined shoulder (Figure 5b). From a general viewpoint, the spectrum of the four other samples (Figure 5c and d) was much wider than that of samples 4 and 5. Two peaks were clearly distinguished in the main band at long wavelengths. For sample 3, the peak situated at long wavelengths was quite narrow, whereas it was particularly wide for samples 1 and 2 that absorb strongly above 600 nm. The spectra of the suspensions formed in the presence of PAA with and without hydrochloric acid being quite different, it was interesting to know whether this effect was reversible. Reprecipitation was thus carried out in the conditions of sample 3, and when it was complete, dilute hydrochloric acid was added until pH ) 2 and the suspension was left to stir again during 20 min. The absorption spectrum remained very close to that recorded at the end of the (23) Chernov, A. A. Modern Crystallography III - Crystal growth; SpringerVerlag: Berlin, 1984. (24) Abyan, M.; Bıˆrla˘, L.; Bertorelle, F.; Fery-Forgues, S. C. R. Chimie 2005, 8, 1276–1281. (25) Abyan, M.; Bıˆrla˘, L.; Bertorelle, F.; Rodrigues, F.; Fery-Forgues, S. Int. J. Photoenergy 2006, 1–5, article ID 30937.
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Figure 5. (a) UV/vis absorption spectra of highly dilute dye 1 (1.5 × 10-6 M) in water containing 2% ethanol. Optical path length: 10 cm. UV/vis absorption spectra of the microcrystal suspensions obtained in different experimental conditions (see Table 1): (b) samples 4 (squares) and 5 (solid line); (c) samples 3 (circles) and 6 (solid line); and (d) samples 1 (solid line) and 2 (circles). The spectra of the microcrystal suspensions were normalized at 0.20. The maximum absorbance of the rough spectra ranged from 0.10 (sample 6) to 0.41 (sample 2). Optical path length: 1 cm.
reprecipitation process. This shows that the properties of the samples are quite steady, once the microcrystals are formed. The UV/vis absorption spectra of the suspensions prepared with charged additives showed little variation from one experiment to another. This was not the case with the experiments run with neutral additives that seemed to be quite sensitive to different parameters, and especially to temperature. For a better understanding of this effect, reprecipitation was performed at different temperatures in the conditions of sample 4 (with PAA in acidic medium) and sample 6 (with dextran). An effect was observed in both cases, but it was particularly spectacular for sample 6. In this case, the shape of the absorption spectrum of the microcrystal suspension at the end of the reprecipitation process displayed significant variations (Figure 6). Two bands were clearly visible when reprecipitation was performed around 20 °C, but the intensity of the band at 540 nm markedly decreased when increasing the temperature, until the band disappeared when working at 40 °C. Meanwhile, the band around 480-490 was slightly shifted to the red. The baseline was higher for suspensions generated at low temperature than for the others, a light-scattering phenomenon that indicates the
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Figure 6. UV/vis absorption spectra of microcrystal suspensions of 1 (2.0 × 10-5 M) obtained in the presence of dextran (6.5 × 10-4 M) in water with 2% ethanol (sample 6) at different temperatures. From top to bottom: 18, 22, 30, and 40 °C.
presence of a larger number of microcrystals. Actually, fluorescence microscopy confirmed this observation: the microcrystals obtained at 40 °C were significantly fewer, longer, and thinner than those obtained at 18 °C. Interestingly, small particles that emit bright green light and resembled those observed in sample 4 were clearly visible in the suspension obtained at 40 °C. Therefore, the strong absorption band near 480 nm is linked to the presence of these particles, while the absorption spectrum that widens toward long wavelengths characterizes a suspension that contains a large number of microcrystals. Deconvolution of the UV/Vis Absorption Spectra. At first sight, the absorption spectra were very different from one sample to another. Deconvolution allowed them to be analyzed more thoroughly. Gaussian functions were used, but very close results were also obtained using Lorentzian functions. For the spectrum of the dilute dye, a good correlation was obtained with three bands centered at 347, 468, and 491 nm (Figure 7a). It can be noted that these bands were also found in the spectrum of 1 dissolved in ethanol, with a slight shift in wavelengths. These three bands thus belong to the dissolved dye, and the two bands at long wavelengths are probably vibronic bands belonging to the same electronic transition. In contrast, five to six bands were necessary for the deconvolution of the spectra of the microcrystals: according to the sample, one or two bands (noted A and A′) were distinguished between 300 and 400 nm, in the area corresponding to the π-π* transition. The four other bands (B-E) were situated at long wavelengths, above 400 nm. Two examples of deconvolution are given in Figure 7b and c. The results of deconvolutions are gathered in Table 2. These results must of course be considered carefully, because deconvolution gives a minimum number of bands necessary to obtain a good fit, but other bands can be present in the spectrum. A shift of the bands can also be observed while the correlation coefficient is almost the same. Besides, there is no indication about the nature of the transitions responsible for the bands. However, a very interesting piece of information emerges from this data processing: all seven spectra of microcrystals were strictly composed of the same bands. The difference between the spectra came from the respective intensity of the bands. As could be expected, the more the spectrum resembled that of the dilute dye (samples 4 and 5), the stronger the contribution of bands B and C, around 470 and 490-500 nm. Conversely, when the spectrum was very different from that of the dilute dye (samples 1-3 and sample 6), the contribution of band D around 545 nm was increased. The same behavior was found for band A′ situated
Figure 7. Deconvolution with Gaussian functions of the absorption spectra. (a) Highly dilute dye 1 (1.5 × 10-6 M) in water containing 2% ethanol (optical path length: 10 cm), r2 ) 0.996. (b) Suspension of microcrystals of 1 (2.0 × 10-5 M) in water containing 2% ethanol with 4.1 × 10-7 M poly(acrylamide) (sample 5), r2 ) 0.998. (c) Suspension of microcrystals of 1 (2.0 × 10-5 M) in water containing 4% ethanol with 5.9 × 10-5 M poly(acrylic acid) (sample 1), r2 ) 0.999. Red curves are the sum of the bands issued from deconvolution.
around 380 nm, the presence of which seems to be correlated to that of D. Finally, all spectra showed a contribution in the red, particularly strong for samples 1, 2, and 6. This wide band, noted E, has a maximum moving between 573 and 605 nm. It cannot be attributed to Rayleigh light scattering, which is due to very small particles, because if it were the case, intensity would be increased at short wavelength. However, we can tentatively attribute this band to light scattering due to the large dye particles. From a general point of view, light scattering is also clearly visible in the deviation of the baseline. Fluorescence Properties. Before examining the fluorescence properties of the microcrystal suspensions, let us recall those of the solution of 1 at 1.5 × 10-6 M in water with 2% ethanol. In this case, the emission spectrum displays only one unresolved band, with a maximum at 560 nm (Figure 8, inset). The shape and position of this spectrum are independent of excitation wavelengths. Conversely, the excitation spectrum, which closely resembles the absorption spectrum, does not vary with emission
Optical Properties of Organic Microcrystals
Langmuir, Vol. 25, No. 3, 2009 1657
Table 2. Maximum Wavelength and Area of the Bands Issued from Deconvolution, and Correlation Coefficient, for the UV/Vis Absorption Spectrum of the Highly Dilute Dye and of the Different Microcrystal Samples 1-6 Recorded after Reprecipitation at 22 °C A
A′
B
C
D
E
λmax (nm) area (%) λmax (nm) area (%) λmax (nm) area (%) λmax (nm) area (%) λmax (nm) area (%) λmax (nm) area (%) dilute 1 2 3 4 5 6
347 357 357 353 350 354 350
23.4 14.7 16.2 10.4 20.8 19.2 7.9
385 387 374
5.5 8.5 6.9
378
8.9
468 463 471 470 468 477 465
55.3 9.3 22.3 19.3 52.2 56.3 12.2
Figure 8. Fluorescence spectra of a suspension of microcrystals of 1 (2 × 10-5 M) obtained in the presence of dextran (6.5 × 10-4 M) in water containing 2% ethanol at 22 °C (sample 6). (Left) normalized excitation spectra for different emission wavelengths: 570 nm (a), 660 nm (b), and 720 nm (c). (Right) normalized emission spectra for different excitation wavelengths: 480 nm (d), 510 nm (e) and 540 nm (f). (Inset) Excitation (λem ) 580 nm) and emission (λex ) 480 nm) spectra of a dilute solution of 1 (1.5 × 10-6 M) in water with 2% ethanol.
wavelengths. This indicates the presence of only one species that absorbs light and emits fluorescence in solution. It can be noted that a similar behavior was observed in ethanol, where the dye is perfectly soluble. Let us now look at the fluorescence properties of the microcrystal suspensions. When excited at 480 nm, the six samples gave an emission spectrum that exhibits only one unresolved band (Figure 8), with a maximum peaking between 567 (sample 4) and 577 nm (sample 1). The spectrum was therefore slightly red-shifted with respect to that of the dilute dye. When the excitation wavelength passed from 480 to 540 nm, the main emission band narrowed, and a shoulder appeared around 684 nm and progressively got more intense. Of course, the latter band was particularly intense when the compound absorbs strongly around 545 nm. It can be noted that, when exciting above 570 nm, no emission was detected, even when the suspension still absorbed strongly at these wavelengths, that is, for samples 1-3 and 6. This shows that absorption in the red part of the spectrum does not lead to emitting exciting states. It can be noted that we already found a similar fluorescence behavior for microcrystals of 1 formed in experimental conditions different from those used in the present work.17,24,25 For the six samples, the excitation spectrum obtained by gathering emission near 570 nm was close to that of the dilute dye. It showed a band of moderate intensity around 350 nm and an intense band peaking at 484 nm. Consequently, the excitation spectrum resembles the UV/vis absorption spectrum for samples 4 and 5, and it is very different for samples 1-3 and 6, since in this case it only reflects a part of the absorption spectrum. The excitation spectrum was very dependent on the emission wavelengths, and this was particularly visible for samples 1-3 and 6. When gathering emission at long wavelengths, an excitation
491 492 498 502 492 494 494
21.3 12.4 6.8 24.7 19.5 4.6 8.6
548 549 542 539 548 541
26.4 16.4 18.5 4.2 5.3 18.2
605 585 573 576 578 589
31.6 29.8 20.2 3.3 14.6 44.2
r2 0.996 0.999 0.999 0.994 0.998 0.998 0.997
band appeared near 545 nm. Emission at 570 nm can thus be correlated to excitation at 484 nm, while emission in the red, around 684 nm, originates from excitation around 545 nm. One question concerning fluorescence is to know whether it comes from the crystal molecules or from some dye molecules dissolved in the aqueous phase or adsorbed on the microcrystal surface. An additional experiment was undertaken as an attempt to clarify this point. A typical fluorescence quencher, sodium iodide, was added to a microcrystal suspension. A fluorescence decrease of 12% and 27% was observed in the presence of 1.96 × 10-2 M and 4.9 × 10-2 M NaI, respectively, when exciting at 480 nm. In contrast, no quenching effect was observed when exciting at 540 nm. This shows that a proportion of the molecules excited at 480 nm are accessible to the quencher, and thus in contact with the aqueous phase, while this is not the case for the molecules excited at 540 nm. Absorption and Fluorescence Spectra of Microcrystals Obtained in the Absence of Additives. As explained in the Introduction, we extensively used additives to prepare microcrystals with the reprecipitation method. The reason is that the microcrystals prepared in water without any additives agglomerate readily and form a red deposit that sticks at the bottom of the cell and on the magnetic stirrer. However, the intrinsic spectral characteristics of the microcrystals must be known in order to be distinguished from the possible artifacts due to the presence of additives. Therefore, we proceeded as follows: reprecipitation was carried out in the absence of additives, the suspension was then sonicated, and the absorption and fluorescence spectra were recorded immediately. These spectra were extremely close to those of the microcrystal suspensions obtained in the presence of charged polymers.
Discussion Knowing the spectral properties of the microcrystals prepared in the absence of additives allows us to understand the whole set of results obtained in this work. First of all, this observation confirms that the absorption spectrum of microcrystals is composed of several bands with a strong contribution at long wavelengths. This feature is reminiscent of the red shift observed during the formation of J-aggregates. In addition to the stabilization due to molecular arrangement in the solid state, the absorption spectrum is obviously influenced by many factors linked to the nature of the microcrystals. For instance, the refraction index and the extinction coefficient can depend on the orientation of the crystal, and the light scattering caused by defects and roughness complicates the analysis of the transmitted light. These effects should be considered very carefully if we were working with single crystals. However, the present work only focuses on suspensions that contain a very large number of microcrystals, which move freely in the aqueous medium and take every possible orientation with respect to the light beam. Consequently, our measurements must be considered as an average, a crude approximation of the spectroscopic properties
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of a population of microcrystals. We use these measurements to draw a comparison between our samples. It appears that microcrystals are composed of at least three well-distinct populations of molecules. The first population absorbs around 480 nm and emits strongly around 570 nm. The spectral characteristics of these molecules are close to those of dispersed dye molecules. The second population of molecules is characterized by its absorption around 545 nm and a rather weak fluorescence at 684 nm (the contribution of this population is visualized with the fluorescence microscope using a long-pass filter). Finally, a third population of molecules absorbs at high wavelengths and does not fluoresce. It seems reasonable to write that these three populations gather molecules located at different places in the crystal and thus differently stabilized. These properties are encountered for all microcrystals of 1, whatever the mode of preparation. Since dissolved dye molecules also absorb around 480 nm and emit around 560 nm, their contribution superimposes to the microcrystal spectra. However, knowing the solubility limit of the compound (4 × 10-7 M) and its molar extinction coefficient (26 500 M-1 cm-1),26 it can be calculated that the absorbance of dissolved molecules in the cell is around 0.010, meaning that their contribution should be less than 10% of the absorption signal. Due to specific quenching by water molecules, the quantum yield of dissolved molecules is low, in the same range of magnitude as that of microcrystals (1 × 10-3).17 The contribution of dissolved molecules to the suspension emission spectrum is therefore rather weak. This is in line with our quenching experiment that shows that only a small proportion of molecules are accessible to the quencher and thus at contact with the aqueous medium. Now, how can we explain that the six samples of microcrystals investigated exhibit very different UV/vis absorption spectra and consequently different emission spectra? In the bibliography, strong variations in the absorption spectra have been reported to take place between nanocrystals and amorphous nanoparticles made of the same compound.27 To our knowledge, such variations have never been reported when comparing only crystals, except in the case of polymorphism.11 However, polymorphism, if any, is quite unlikely to play a significant role in the present case. It would also be tempting to attribute given spectroscopic features to a particular crystal habit, supposing, for example, that some crystals could present more defects than others. The proportions of the different types of microcrystal actually vary from one sample to another, but to date we found no clear correlation between the crystal type and the absorption spectrum of the suspension. This does not mean that this hypothesis must be discarded definitively but the explanation is probably much simpler. Deconvolution revealed that all the UV/vis absorption spectra encompass the same bands, with different intensities. The spectral characteristics of the microcrystals can be found in every sample. The main difference between the spectra lies in the relative proportion of the absorption band centered near 480 nm with respect to the others. This band is particularly strong in suspensions prepared in the presence of PAA at acidic pH, poly(acrylamide), and dextran at 40 °C. These samples are precisely those in which the presence of bright green shapeless microstructures has been observed by fluorescence microscopy. Most likely, these particles strongly contribute to increasing the intensity of the absorption and fluorescence bands at short wavelengths. They probably
result from the interaction of 1 with neutral polymers, which are more compact and less soluble than charged polymers and therefore more able to trap some dye molecules. Curiously, for dextran, the formation of green-emitting particles was detected for reprecipitation experiments run at 40 °C and not at 18 °C. The formation of particles is therefore not directly related to the dextran solubility, which should increase when rising the temperature. It seems that we are in the presence of a more complex phenomenon, where the formation of microcrystals competes with the uptake of 1 by the polymer. It is now understandable why the experiments with neutral additives lack reproducibility when all parameters are not perfectly controlled. The incorporation of molecules of 1 in the polymers does not appear when working with charged polymers. It can be noted that we already gave evidence for an electrostatic interaction between anionic or cationic dendrimers and highly dilute 1.15,16 However, this interaction that generates an absorption band around 400 nm is not visible on the suspension spectra recorded here. Actually, the microcrystals obtained in the presence of charged polymers display the same spectral characteristics as the microcrystals obtained in water alone. It must also be noted that these spectral characteristics were also encountered in previous works where the microcrystals were prepared in the presence of anionic and cationic dendrimers,15,16 DNA17 (which is negatively charged), and with a neutral dendrimer terminated by glucose units.28 Consequently, the use of these additives induces no particular signature on the spectral characteristics of the microcrystal suspensions. Interestingly, this result is different from that published by Yao’s team, who has shown that the presence of poly(vinyl alcohol) has some influence on the optical properties of perylene nanocrystals generated by the reprecipitation method.29
(26) Bertorelle, F. Ph.D. Thesis, Universite´ Paul Sabatier, Toulouse, 2003. (27) Al-Kaysi, R. O.; Mu¨ller, A. M.; Ahn, T. S.; Lee, S.; Bardeen, C. J. Langmuir 2005, 21, 7990–7994. (28) Bertorelle, F.; Al-Ali, F.; Fery-Forgues, S. Int. J. Photoenergy 2004, 6, 221–225. (29) Xie, R.; Xiao, D.; Fu, H.; Ji, X.; Yang, W.; Yao, J. New J. Chem. 2001, 25, 1362–1364.
Supporting Information Available: XRPD patterns of microcrystals of 1 obtained in the presence of dextran (sample 6) and poly(acrylic acid) (sample 2), and powdered compound 1 directly issued from synthesis. This material is available free of charge via the Internet at http://pubs.acs.org.
Conclusion The usefulness of the reprecipitation method to produce suspensions of micro- and nanocrystals is well-recognized, as well as the possibility to influence the crystallization process by modifying some experimental parameters. However, this method still relies on a very empirical basis. There is a huge lack of data to know how each parameter must be controlled to get the desired result, and the precise role of the additives has been scarcely investigated until now. This is particularly true when considering a field of very recent interest, such as the preparation of photoactive microcrystals, and the need for systematic studies on this topic is real. Dye 1 lends itself perfectly to this exercise. Its crystallization is extremely sensitive to experimental conditions, and the variations of its spectroscopic properties are easy to monitor. This dye allowed us to show how polymers used as additives may influence the optical properties of the microcrystal suspensions formed, by leading to spectacular artifacts. Being aware of these drawbacks is particularly important when the microcrystal suspensions must be used for subsequent applications linked to interaction with light. There is no doubt that the interest of these applications should motivate further developments of the reprecipitation method in the near future. Acknowledgment. We want to express our thanks to Dr. Clara Fournier-Noe¨l for the measurement of the molecular weight of dextran and interesting discussions about polymers. We are also indebted to Dr. Charles-Louis Serpentini for judicious technical advice.
LA803549U
