Use of Linear Polymers To Control the Preparation of Luminescent

Microcrystals of an organic fluorescent dye, 4-octylamino-7-nitrobenz-2-oxa-1,3-diazole, were generated using the reprecipitation method, which is a s...
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Use of Linear Polymers To Control the Preparation of Luminescent Organic Microcrystals Mouhammad Abyan, Franck Bertorelle, and Suzanne Fery-Forgues* Laboratoire des Interactions Mole´ culaires Re´ activite´ Chimique et Photochimique, UMR CNRS 5623, Universite´ Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France Received December 17, 2004. In Final Form: March 23, 2005 Microcrystals of an organic fluorescent dye, 4-octylamino-7-nitrobenz-2-oxa-1,3-diazole, were generated using the reprecipitation method, which is a solvent exchange process. In the presence of polymers, namely, poly(acrylic acid), molecular weight 5100 g mol-1 and 15 000 g mol-1, and poly(acrylic acid-co-maleic acid), average molecular weight about 50 000 g mol-1, used as their sodium salts, the reprecipitation process was strongly accelerated. The reprecipitation kinetics was monitored by UV/vis absorption spectroscopy and revealed a three-step mechanism, each step being influenced by the polymer. The size and shape of the microcrystals were analyzed by fluorescence microscopy. The microcrystals obtained in the presence of polymers were smaller and more regular than those prepared in water alone and were not agglomerated. When the polymer was placed in the reprecipitation medium before introducing the dye solution, the microcrystals displayed a rectangular shape. When the polymer was introduced 20 min after the beginning of the reprecipitation process, intricately structured flowerlike microcrystals were observed. Microanalysis revealed that the microcrystals contained noticeable amounts of polymer. The measurement of the surface electric ζ potential suggested that a proportion of the polymer was present at the microcrystal surface. This work gives a thorough insight into a field where trials have until now been performed in an empirical way. It opens new perspectives to produce low-cost organic microcrystals, potentially useful in optics or pharmaceutical sciences.

Introduction Organic-molecule-based microcrystals have been commonly exploited and studied for decades, especially in the pharmaceutical industry where they are used for improving drug bio-availability.1,2 But, very recently, they have motivated a new burst of interest, because they are now seen as photocatalysts,3 luminescent probes for bioanalysis,4 and novel advanced materials with potential applications in photonics and microelectronics.5 However, the ability to produce them with controllable dimensions and spatial properties is still very limited, while it is a major requirement for any technological use. The elaboration of homogeneous organic microcrystals, identically replicated and available in large quantities, so that they can be manipulated and considered as pure macromolecular substances, is, therefore, a real challenge for the forthcoming years. Organic compounds often show thermal instability, and the methods used to prepare ultrafine metal particles cannot generally be applied. In this context, the reprecipitation method, widely developed during the past decade by Nakanishi’s team, offers an interesting alternative.5,6 This method consists of dissolving the organic compound in a good solvent. Then this concentrated solution is poured into a large volume of a second liquid, miscible with the * Corresponding author. E-mail: [email protected]. (1) Shekunov, B. Yu.; York, P. J. Cryst. Growth 2000, 211, 122-136. (2) Rasenack, N.; Hartenhauer, H.; Mu¨ller, B. W. Int. J. Pharm. 2003, 254, 137-145. (3) Kim, H. Y.; Bjorklund, T. G.; Lim, S.-H.; Bardeen, C. J. Langmuir 2003, 19, 3941-3946. (4) Jinshui, L.; Lun, W.; Feng, G.; Yongxing, L.; Yun, W. Anal. Bioanal. Chem. 2003, 377, 346-349. (5) Oikawa, H.; Kasai, H.; Nakanishi, H. In Anisotropic Organic Materials; Glaser, R., Kasizynski, P., Ed.; ACS Symposium Series 798; American Chemical Society: Washington, DC, 2002; Chapters 11 and 12, pp 158-178. (6) Van Keuren, E.; Georgieva, E.; Adrian, J. Nano Lett. 2001, 1, 141-144.

first one, which acts as a nonsolvent for the organic compound. Most currently, ethanol, acetone, or tetrahydrofuran are used to dissolve the organic compound, and this solution is mixed with water. Precipitation occurs, which can subsequently lead to the formation of microcrystals. It has been shown that the size and shape of the microcrystals produced by this method can be influenced by different factors. Among them are principally the concentration of the organic solution7-9 and the temperature.8-10 The two factors can of course be combined.11 Subsequent freeze and thaw cycles,12 microwave irradiation,13 sonication,14 and the time left for the suspension to age15,16 can also have an influence. Small molecules, (7) (a) Katagi, H.; Kasai, H.; Okada, S.; Oikawa, H.; Matsuda, H.; Nakanishi, H. J. Macromol. Sci., Pure Appl. Chem. 1997, 34, 20132024. (b) Katagi, H.; Oikawa, H.; Okada, S.; Kasai, H.; Watanabe, A.; Ito, O.; Nozue, Y.; Nakanishi, H. Mol. Cryst. Liq. Cryst. Sci. Technol., Sect. A 1998, 314, 285-290. (c) Katagi, H.; Kasai, H.; Okada, S.; Oikawa, H.; Komatsu, K.; Matsuda, H.; Liu, Z.; Nakanishi, H. Jpn. J. Appl. Phys. 1996, 35, L1364-L1366. (8) Nakanishi, H.; Katagi, H. Supramol. Sci. 1998, 5, 289-295. (9) Nalwa, H. S.; Kasai, H.; Okada, S.; Oikawa, H.; Matsuda, H.; Kakuta, A.; Mukoh, A.; Nakanishi, H. Adv. Mater. 1993, 5, 758-760. (10) (a) Kasai, H.; Kamatani, H.; Okada, S.; Oikawa, H.; Matsuda, H.; Nakanishi, H. Jpn. J. Appl. Phys. 1996, 35, L221-L223. (b) Kasai, H.; Oikawa, H.; Okada, S.; Nakanishi, H. Bull. Chem. Soc. Jpn. 1998, 71, 2597-2601 and references therein. (c) Kasai, H.; Yoshikawa, Y.; Seko, T.; Okada, S.; Oikawa, H.; Mastuda, H.; Watanabe, A.; Ito, O.; Toyotama, H.; Nakanishi, H. Mol. Cryst. Liq. Cryst. 1997, 294, 173176. (11) (a) Fu, H.-B.; Yao, J.-N. J. Am. Chem. Soc. 2001, 123, 14341439. (b) Fu, H.; Xiao, D.; Xie, R.; Ji, X.; Yao, J.-N. Can. J. Chem. 2003, 81, 7-13. (12) Iida, R.; Kamatani, H.; Kasai, H.; Okada, S.; Oikawa, H.; Matsuda, H.; Kakuta, A.; Nakanishi, H. Mol. Cryst. Liq. Cryst. 1995, 267, 95-100. (13) (a) Baba, K.; Kasai, H.; Okada, S.; Oikawa, H.; Nakanishi, H. Opt. Mater. 2002, 21, 591-594. (b) Baba, K.; Kasai, H.; Okada, S.; Oikawa, H.; Nakanishi, H. Jpn. J. Appl. Phys. 2000, 39, L1256-L1258. (c) Onodera, T.; Kasai, H.; Okada, S.; Oikawa, H.; Mizuno, K.; Fujitsuka, M.; Ito, O.; Nakanishi, H. Opt. Mater. 2002, 21, 595-598. (14) Kang, P.; Chen, C.; Hao, L.; Zhu, C.; Hu, Y.; Chen, Z. Mater. Res. Bull. 2004, 39, 545-551.

10.1021/la046877j CCC: $30.25 © 2005 American Chemical Society Published on Web 04/30/2005

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Chart 1. Chemical Structure of Compound 1

very relevant, because in the references only a few articles deal with the effect of polymers upon the production of organic microcrystals by the reprecipitation method.20,21 It appears that polymers are empirically used as stabilizers in the crystallization of compounds of pharmaceutical interest, but there is obviously a lack of knowledge concerning the mechanism involved. The reprecipitation of dye 1 was, therefore, investigated in the presence of three polymers, used as additives. Two homopolymers were of the poly(acrylic acid) (PAA) type, used in the form of their sodium salt. Their average molecular weights were 5100 g mol-1 and 15 000 g mol-1, so that they were respectively made of 54 and 159 constitutive units, each unit bearing a carboxylate group. A copolymer, poly(acrylic acid-co-maleic acid) (PAAM), with an average molecular weight of 50 000 g mol-1, was also used. In this copolymer, the constitutive units [-CH2CH(COOR)-] and [-CH(COOR)-CH(COOR)-] are linked randomly. We supposed that R only designated Na ions, although the manufacturer indicates that some carboxylic groups can be protonated (R ) H). By assuming the same proportion of acrylic acid and maleic acid residues, the number of carboxylate groups per molecule was calculated to be around 588. The three polymers were used at different concentrations. The effect on the kinetics was regarded first and then the morphology of the microcrystals, as well as their composition and surface properties, was studied.

such as surfactants, can be used as additives and mainly act as crystal growth inhibitors.8,9,17 In a recent work, we showed for the first time that starburst dendrimers, used as additives, were able to control the size and shape of organic microcrystals obtained by the reprecipitation method.18 These dendrimers belong to the polyamidoamine family and were terminated by carboxylate groups with sodium contraions. There were placed into the reprecipitation medium prior to injection of the organic compound. The latter was a dye, 4-n-octylamino-7-nitrobenzoxadiazole (1; Chart 1). Because this compound forms microcrystals quite slowly, it allowed the different steps of the crystallization process to be distinguished. It can also be recalled that dyes of the aminonitrobenzoxadiazole (amino-NBD) family display remarkable UV/vis absorption and fluorescence properties, particularly sensitive to the polarity and acidity of their environment.19 This first approach showed that it was possible to influence the recrystallization process of our dye. But, it also raised many questions, in particular concerning the role played by the additive. For instance, it was difficult to understand why the reprecipitation process was so drastically accelerated in the presence of dendrimers. It was very tempting to attribute this effect to the very nature of the dendrimers. In fact, due to their highly ordered, fractal structure that generates nanoscopic host compartments, dendrimers are able to accommodate several dye molecules and, thus, could constitute special nucleation sites. However, this hypothesis was not in line with the spectroscopic evidence that the NBD dye did not enter the dendrimer core. Consequently, the fundamental question of the mechanism was not resolved. Besides, there was a concern of a more practical nature. The dendrimers used were commercially available, but their high price did not allow them to be employed for preparing large amounts of microcrystals. This was a limitation in our study and could be a serious disadvantage for future applications. All this motivated us to carry out a new investigation, to see whether the dendrimers could be replaced by linear polymers. Moreover, this work seemed (15) (a) Fu, H.-B.; Wang, Y.-Q.; Yao, J.-N. Chem. Phys. Lett. 2000, 322, 327-332. (16) Xiao, D.; Xi, L.; Yang, W.; Fu, H.; Shuai, Z.; Fang, Y.; Yao, J. J. Am. Chem. Soc. 2003, 125, 6740-6745 and references therein. (17) (a) Onodera, T.; Oshikiri, T.; Katagi, H.; Kasai, H.; Okada, S.; Oikawa, H.; Terauchi, M.; Tanaka, M.; Nakanishi, H. J. Cryst. Growth 2001, 229, 586-590. (b) Ji, X.; Ma, Y.; Cao, Y.; Zhang, X.; Xie, R.; Fu, H.; Xiao, D.; Yao, J. Dyes Pigm. 2001, 51, 87-91. (c) Oshikiri, T.; Kasai, H.; Katagi, H.; Okada, S.; Oikawa, H.; Nakanishi, H. Mol. Cryst. Liq. Cryst. Sci. Technol., Sect. A 1999, 337, 25-30. (18) Bertorelle, F.; Lavabre, D.; Fery-Forgues, S. J. Am. Chem. Soc. 2003, 125, 6244-6253. (19) (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. Biochim. Biophys. Acta 1988, 938, 24-34. (g) Rajarathnam, K.; Hochman, J.; Schindler, M.; Ferguson-Miller, S. Biochemistry 1989, 28, 3168-3176. (h) Mukherjee, S.; Chattopadhyay, A.; Samanta, A.; Soujanya, T. J. Phys. Chem. 1994, 98, 2809-2812.

Experimental Section Materials. The fluorescent dye 4-n-octylamino-7-nitrobenz2-oxa-1,3-diazole (1) was prepared as described elsewhere,22 according to a variant of the synthesis reported by Heberer et al.23 Polymers PAA (sodium salt; molecular weight, MW ) 5100 g mol-1 and 15 000 g mol-1) and PAAM (sodium salt; MW ) 50 000 g mol-1) were purchased from Aldrich and used without further purification. PAA, MW 15 000 g mol-1, was purchased as a 35 wt % solution in water; the two other acids were solids. Acrylic and malonic acids were from Fluka and Merck, respectively. Absolute ethanol was purchased from Carlo Erba Reagenti. Ultrapure water with a resistivity of 16 MΩ‚cm was produced using a Milli-Q apparatus (Millipore). Apparatus. UV/vis absorption spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer. The measurements were conducted at 25 °C in a thermostated cell. The size and shape of the microcrystals were observed with a Zeiss MC80DX fluorescence microscope. The objective was made up of lenses of either ×40 or ×100 magnification; the eyepiece had a fixed lens of ×2.5 magnification. The microscope was equipped with a standard camera. The excitation wavelength was 430-450 nm, and the emission wavelength was set at around 500-530 nm, by using adequate filters. Confocal fluorescence microscopy was carried out in the Service de Microscopie Confocale of the Centre de Biologie du De´veloppement of Toulouse, using a confocal Leica TCS2 microscope. The sample preparation was the same for classical and confocal fluorescence microscopy. When reprecipitation was completed, a droplet of the suspensions used for spectroscopic measurements was deposited between a slide and a cover glass, and the latter was subsequently sealed with nail varnish. The microanalyses were obtained with a EA1110 elemental analyzer from CE Instruments. The surface electric ζ-potential measurements were conducted at room temperature with a Zetasizer 3000HS apparatus from Malvern Instruments. (20) Johnson, B. K.; Prud’homme, R. K. Aust. J. Chem. 2003, 56, 1021-1024. (21) Xie, R.; Xiao, D.; Fu, H.; Ji, X.; Yang, W.; Yao, J. New J. Chem. 2001, 25, 1362-1364. (22) Galinier, F.; Bertorelle, F.; Fery-Forgues, S. C. R. Acad. Sci., Paris 2001, 4, 941-950. (23) Heberer, H.; Kersting, H.; Matschiner, H. J. Prakt. Chem. 1985, 327, 487-504.

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Preparation of the Solutions. A solution of dye 1 in absolute ethanol was prepared, at a concentration of 10-3 M. Then, a small volume of the dye solution was introduced into water or into the aqueous solution of polymer. The proportion of ethanol in water was then 2% (v/v). For instance, for absorption measurements, 40 µL of dye solution was transferred to a cell containing 1.96 mL of water, containing an additive or not. For preparing a larger amount of microcrystals necessary to perform elemental analysis, 20 mL of the dye solution was poured under stirring into 980 mL of water containing 3.2 × 10-2 M PAA (average MW ca. 5100 g mol-1). Absorption Data Analysis. An apparent molar absorption coefficient i(app) was assigned to each species i. Absorbance A was related to concentrations Ci using the relationship: A ) l∑(i(app)Ci) where l is the optical path length. The system of differential equations with independent variables was numerically integrated by a semi-implicit Runge-Kutta method.24 The sum of the squares of the differences between the experimental values and those of the numerical calculation was minimized by a Powell nonlinear minimization algorithm.

Results Dye Reprecipitation Monitored by UV/Vis Absorption Spectroscopy. Compound 1 is poorly soluble in aqueous solution at neutral pH, where it is not ionized.23 In a mixture of water with 2% ethanol (v/v), as will be used throughout this work, its solubility limit is 4 × 10-7 M.22 Below this concentration, solutions of 1 give an UV/ vis absorption spectrum whose intensity is steady and where two bands can be distinguished. The first one, situated around 346 nm, is of moderate intensity and corresponds to a π-π* transition. The second band, with a maximum at 482 nm, is more intense. It has been assigned to a charge transfer (CT) transition occurring from the electron-donating amino group to the electronwithdrawing moiety, that is, the nitro and the furazan groups.25 For reprecipitation experiments, a small amount of a concentrated solution of dye in ethanol was rapidly introduced into water under continuous stirring (see Experimental Section). The final solution contained 2% (v/v) ethanol in water and 2 × 10-5 M dye, which makes the dye concentration 50 times higher than the solubility limit. The reprecipitation process of 1 in water alone was previously described.18 This experiment was repeated here. After introduction of the dye in ethanol, the initial solution was yellow. Then it became discolored as the organic compound precipitated, and red agglomerated microcrystals were obtained on the stirrer. The whole process took about 1 h. Meanwhile, the UV/vis absorption spectrum underwent major variations. Just after mixing, the bands were situated at 344 and 476 nm, which indicates that the dye was surrounded by an ethanol-rich water shell. Then, the bands rapidly shifted to 346 and 482 nm, revealing that the dye was now surrounded by a homogeneous mixture of water with 2% ethanol. Then, the position of the bands was unchanged, but their intensity decreased until completion of the reprecipitation process. In the present paper, reprecipitation was successively carried out in the presence of the three anionic polymers, PAA 5100 g mol-1, PAA 15 000 g mol-1, and PAAM. They were placed in water before the ethanol solution of 1 was introduced, at the same dye concentration as before. For the sake of comparison, the polymer concentration is represented by the concentration of carboxylate groups. At low polymer concentrations (around 4.3 × 10-4 M in carboxylate groups), the reprecipitation process was (24) Kaps, K.; Rentrop, P. Comput. Chem. Eng. 1984, 8, 393-396. (25) Heberer, H.; Matschiner, H. J. Prakt. Chem. 1986, 328, 261274.

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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 PAA 5100 g mol-1 at a concentration of 5.9 × 10-4 M (3.2 × 10-2 M in carboxylate groups). One measurement every 2 min.

comparable to that encountered in water alone, except that it was slightly faster. When the polymer concentration was increased, a different type of behavior was observed. The initial yellow solution was rapidly discolored, becoming light pink. In our previous work, this effect was attributed to the presence of very small crystals remaining in suspension in the cell. The UV/vis absorption spectrum showed strong variations (Figure 1). The π-π* band and the CT band were respectively shifted to 364 and 496 nm, and their intensity was greatly decreased. Meanwhile, a new peak appeared at 546 nm, and the baseline moved markedly upward, due to the presence of particles in suspensions that lead to a scattering phenomenon. The evolution of the UV/vis absorption spectrum confirms that the reprecipitation process was drastically accelerated. For example, the process was completed within 16 min in the presence of PAA 5100 g mol-1 at a concentration of 5.9 × 10-4 M (3.2 × 10-2 M in carboxylate groups). The subsequent small decrease in absorbance was only due to sedimentation. Processing of the Spectroscopic Data. The strong variations exhibited by the UV/vis absorption spectra allow reliable monitoring of the reprecipitation kinetics. The kinetics of the reprecipitation carried out in the absence of additive has been studied in a previous work.18 The absorbance at three different wavelengths was recorded against time. The curves obtained were analyzed using a model involving three steps, two of which are autocatalytic:

A f B r1 ) k1[A]

(I)

B 98 C r2 ) k2[B](1 + kC[C])

(II)

C

D

C 98 D r3 ) k3[C](1 + kD[D])

(III)

In these equations, letters A-D refer to different organizational states of dye 1. On the basis of transmission electron microscopy (TEM) and fluorescence microscopy, state A has been assigned to ethanol-surrounded molecules, which subsequently form small aggregates (B). The latter assemble as large aggregates (C) and then lead to the formation of microcrystals (D). In our model, each organizational state possesses distinct spectroscopic properties and is characterized by an apparent molar absorption coefficient (app) at the wavelength considered. In the present work, the reprecipitation process was analyzed as observed in the presence of polymers. The absorbance was monitored at 476, 502, and 546 nm, three wavelengths where the absorbance variation is strong,

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Then, it was proposed that steps II and III coexist with steps IV and V, accelerated by the polymer. A reversible sedimentation step (VI) was also added, to take into account the fact that D′ remains in suspension and contributes to absorbance. The model can then be written as

A f B r1′ ) k1′[A]

(I′)

B 98C r2 ) k2[B](1 + kC[C])

(II)

C

D

C 98 D r3 ) k3[C](1 + kD[D]) C′

B 98 C′ r4 ) k4[B](1 + kC′[C′]) D′

Figure 2. Kinetics of the reprecipitation process monitored at 476, 502, and 546 nm in the presence of PAA 15 000 g mol-1, at a concentration of 2.7 × 10-6 M (a), 2.0 × 10-5 (b), and 2.0 × 10-3 M (c) (that is, 4.3 × 10-4 M (a), 3.2 × 10-3 M (b), and 3.2 × 10-1 M (c) in carboxylate groups). The points are experimental, and the curves were calculated by the model. The rate constants k1′, k4, and k5 are gathered in Table 1 (other constants are given in ref 18).

and which were selected to provide maximum information. The kinetics profiles obtained were particular. For instance, at 476 nm, the curves twice showed an abrupt decrease followed by a slowing down (Figure 2). This profile was close to that already encountered for 1 in the presence of anionic dendrimer.18 Therefore, the same model was used to analyze our curves. In the presence of polymers, the apparent molar coefficient of the molecular arrangements noted A and B was close to that found in water alone. Consequently, it seems that, during the first step, the presence of polymer does not affect the organizational state of the dye but only accelerates the formation of B. For the subsequent steps, it was observed that the spectroscopic properties of the solution varied when the latter contained some polymer. In this case, some new particles, noted C′ and D′, respectively, were detected by the side of C and D. They differed by the value of app.

(III) (IV)

C′ 98 D′ r5 ) k5[C′](1 + kD′[D′])

(V)

D′ / E r6 ) k6[D′] - k7[E]

(VI)

Constants k2, k3, kC, and kD were taken identical to those previously determined for the kinetics in water alone. Moreover, it was considered that the polymers did not influence the autocatalysis factors, and it was written that kC ) kC′ and kD ) kD′. The corresponding differential equations were similar to those published in a previous work from our team concerning the use of dendrimers as additives.18 Each kinetics experiment was repeated three times, and fitting was performed simultaneously on the curves obtained at the three wavelengths. Very good fits were obtained, as shown by Figure 2. This indicates that the three-step model used previously can be perfectly applied to describe the reprecipitation process in the presence of polymers. Just like the dendrimers, the polymers accelerate each of these steps and generate the formation of two new molecular arrangements (C′ and D′), characterized by their own app value. If looking more closely at the values of constants k1′, k4, and k5, small differences can be noted in the behavior of the three polymers (see Table 1). With PAAM, a threshold value is rapidly attained. With PAA 5100 g mol-1, it seems that the rate constants reach a peak for a concentration of 5.9 × 10-4 M (3.2 × 10-2 M in carboxylate groups), while they go on increasing at high concentrations of PAA 15 000 g mol-1. Reprecipitation in the Presence of Small Molecules. The influence of the polymers upon the reprecipitation process could be explained by a disturbance of the ionic strength of the medium by the carboxylate groups. To test this hypothesis, reprecipitation was carried out in the presence of acrylic acid (3.2 × 10-2 M) and malonic acid (1.6 × 10-2 M, that is, 3.2 × 10-2 M in carboxylate groups). These concentrations are directly comparable to the concentrations of carboxylate groups in the polymers solutions. In the presence of these small molecules, only a very weak acceleration of the reprecipitation process was observed. This indicates that the presence of carboxylate groups alone does not account for the effect observed with the polymers. Influence of pH upon Reprecipitation. To know whether the nature of the functional groups of the polymers is responsible for the effect observed, reprecipitation was carried out at acidic pH. PAA (MW 5100 g mol-1) was dissolved in water, the polymer concentration being 5.9 × 10-4 M (3.2 × 10-2 M in carboxylate groups). Then, hydrochloric acid was added to the solution, and the pH was measured precisely with a pH-meter. The reprecipitation experiment was then repeated as above, by introducing the ethanol solution of dye into the acidic

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Table 1. Values of the Rate Constants k1′, k4, and k5 Obtained by Processing the UV/Vis Absorption Data for the Reprecipitation of 1 (2 × 10-5 M) in the Presence of the Three Linear Polymers at Various Concentrations polymer PAA 5100 g mol-1

PAA 15 000 g mol-1

PAAM

concentration, mol/L

concentration, mol/L COO-

k1′, s-1

k4, s-1

k5, s-1

7.9 × 10-6 5.9 × 10-5 5.9 × 10-4 5.9 × 10-3 2.7 × 10-6 2.0 × 10-5 2.0 × 10-4 2.0 × 10-3 7.3 × 10-7 5.4 × 10-6 5.4 × 10-5 5.4 × 10-4

4.3 × 10-4 3.2 × 10-3 3.2 × 10-2 3.2 × 10-1 4.3 × 10-4 3.2 × 10-3 3.2 × 10-2 3.2 × 10-1 4.3 × 10-4 3.2 × 10-3 3.2 × 10-2 3.2 × 10-1

3.7 × 10-3 6.6 × 10-3 1.4 × 10-2 1.2 × 10-2 1.8 × 10-3 7.7 × 10-3 6.8 × 10-3 1.6 × 10-2 7.8 × 10-3 1.4 × 10-2 1.6 × 10-2 1.4 × 10-2

1.6 × 10-3 2.1 × 10-3 6.1 × 10-3 3.7 × 10-3 1.5 × 10-3 1.7 × 10-3 3.1 × 10-3 1.1 × 10-2 2.4 × 10-3 4.3 × 10-3 5.5 × 10-3 4.6 × 10-3

7.5 × 10-4 6.5 × 10-4 1.4 × 10-3 1.0 × 10-4 3.2 × 10-4 8.5 × 10-4 9.7 × 10-4 1.6 × 10-3 8.3 × 10-4 1.2 × 10-3 1.9 × 10-3 1.1 × 10-3

polymer solution. When the pH value was between 3 and 4, the variations of the UV/vis absorption spectrum were qualitatively close to those observed in the absence of acid, that is, for a polymer solution whose pH is 8.7. Only a moderate slowing down was observed in the duration of the reprecipitation process. In contrast, when the pH was lowered between 2 and 3, the UV/vis absorption spectrum got closer and closer to that obtained in water alone, in the absence of polymer. The π-π* and the CT bands were shifted to 352 and 488 nm, respectively, while the band at 546 nm was hardly distinguishable. The duration of the process was markedly increased. At pH 2.0 (Figure 3), the kinetics was not totally completed within 40 min (instead of 16 min in the absence of acid), the absorbance at 488 nm still decreasing smoothly after this period of time. It must be noted that, whatever the pH, all the curves representing the variation of absorbance as a function of time showed the same profile as before, indicating that the reprecipitation process still involves a three-step mechanism in the presence of acid. Morphology of the Microcrystals Obtained in the Presence of Polymers. As seen above, the presence of polymers strongly influences the kinetics of the reprecipitation process. It is now interesting to see if this phenomenon is accompanied by the formation of microcrystals and, if so, whether they are different in size and shape, with respect to the microcrystals obtained in water alone or in the presence of dendrimer. The suspensions obtained at the end of the reprecipitation process, in the presence of each of the three polymers at the four concentrations used above, were analyzed by fluorescence microscopy. In every case, small rectangular objects were detected. All of them were extremely thin. In a previous

Figure 3. Evolution of the UV/vis absorption spectrum of 1 (2 × 10-5 M) in water containing 2% ethanol, at pH 2.0, during the reprecipitation process, in the presence of PAA 5100 g mol-1 at a concentration of 5.9 × 10-4 M (3.2 × 10-2 M in carboxylate groups). One measurement every 4 min.

work, such objects were identified as microcrystals by electron diffraction using a transmission electron microscope.18 Differences in the shape and size were observed here when varying the polymer concentration. For example, when using a solution of PAA 5100 g mol-1 at a concentration of 5.9 × 10-5 M (3.2 × 10-3 M in carboxylate groups), most of the microcrystals showed an X-shaped pattern at the center (Figure 4a) and could be roughly distributed in three main populations, with sizes of approximately 40 × 15, 10 × 3, and 7 × 3 µm. The largest microcrystals only represented less than 1% of the total number of crystals, while the two populations of small microcrystals together accounted for 90%. Among the remaining microcrystals, 7.7% had slightly different dimensions, and 1.3% did not exhibit the characteristic X-shaped pattern. These microcrystals are very close to those formed in the presence of dendrimer, at a comparable carboxylate group concentration. It must be noted that the proportion of well-separated, individual microcrystals increases with the polymer concentration. Let us recall that only shapeless agglomerates of microcrystals were observed in water alone. It is also interesting to note that the microcrystals grown in the presence of polymer retain the same appearance after 1 month aging in suspension.

Figure 4. Fluorescence microscopy image of microcrystals of 1 grown in the presence of polymer PAA 5100 g mol-1 (5.9 × 10-5 M, that is, 3.2 × 10-3 M in carboxylate groups) introduced from the beginning of the reprecipitation process (a) or 20 min after (b-d). Observation with an ordinary fluorescence microscope (a-c) and confocal microscope (d).

Preparation of Organic Microcrystals with Polymers Table 2. Results of the Elemental Composition Analysis for C14H20N4O3 (MW ) 292.34 g mol-1)a found, % C H N

calculated, %

water

+PAA, t ) 0

+PAA, t ) 20 min

57.53 6.85 19.17

56.82 6.98 18.91

55.48 6.78 17.07

55.56 6.95 15.81

a The microcrystals were grown in water alone or in the presence of PAA 5100 g mol-1 (5.9 × 10-4 M, that is, 3.2 × 10-2 M in carboxylate groups), introduced at different times.

Another interesting observation is the following. In the above experiments, the polymers were placed in the reprecipitation process before the ethanol solution of dye was introduced. Now this protocol was changed. The reprecipitation process was conducted in water alone for 20 min, then polymer PAA 5100 g mol-1 was introduced into the medium, so that its concentration was 5.9 × 10-5 M, that is 3.2 × 10-3 M in carboxylate groups. The microcrystals obtained show a notched shape (Figure 4b), but above all, a large amount of intricately structured microcrystals, with a flowerlike appearance, were produced (Figure 4c). The diameter of these flowerlike arrangements was about 10-20 µm. Confocal fluorescence microscopy, which allows the microcrystals to be observed in all three dimensions, revealed that they were actually made of very thin petals (Figure 4d). Composition of the Microcrystals Grown in the Presence of Polymers. It is important to know whether these microcrystals are composed of pure NBD dye or have incorporated some polymer. To do so, a sufficient amount of microcrystals was prepared so that elemental composition analysis could be performed. Microcrystals were grown in the presence of PAA 5100 g mol-1 (5.9 × 10-4 M, that is, 3.2 × 10-2 M in carboxylate groups), placed in water before the reprecipitation process began. A second set of microcrystals was prepared by adding the same amount of polymer only 20 min after the beginning of the reprecipitation process. The comparison was made with microcrystals obtained in water alone. In every case, the microcrystals were filtered, carefully rinsed with water, and dried at 50 °C under vacuum. The results are gathered in Table 2. The composition of the microcrystals obtained in water alone indicates the presence of 1.3% of residual water. It was assumed that this amount of water was the same for the microcrystals grown in the presence of polymer. The composition of a constitutive unit of PAA is C, 38.31; H, 3.21; O, 34.02; and Na, 24.44 for C3H3O2Na. The amount of PAA contained in the microcrystals can, therefore, be determined. For example, for the microcrystals obtained with the polymer placed at the beginning of the reprecipitation process, it can be written that, for C, 56.82x + 38.31(1 - x) ) 55.48, hence, x ) 0.927. This means that these microcrystals contain, in weight, about 93% dye 1 and 7% polymer. The same ratio was obtained by taking into account the analysis of all three elements (C, H, N). Similar results were obtained for the microcrystals grown in the presence of PAA added during the reprecipitation process, although the results given by the analysis of the three elements were less homogeneous. It is, thus, likely that a small amount of polymer remains with the microcrystals, and this amount does not depend on the moment the additive is placed in the reprecipitation medium. Measurement of the ζ Potentials. To get further information about the nature of the microcrystals, the surface electric ζ potential was measured. It represents

Langmuir, Vol. 21, No. 13, 2005 6035

the charge that a particle acquires when it is placed in a solution. Measurements were made using a capillary electrophoresis technique based on the Doppler effect, which takes into account the horizontal shift of the microparticles in an electric field. Suspensions of microcrystals were prepared, with the total dye concentration being 2 × 10-5 M. For the microcrystals grown in water alone, the ζ potentials obtained varied strongly according to the preparation conditions (volume, stirring, etc.). This effect was attributed to decantation of heavy agglomerates which greatly disturbs the measurements. In contrast, decantation problems were not noted for the microcrystals grown in the presence of PAA (MW 5100 g mol-1, 5.9 × 10-4 M, that is, 3.2 × 10-2 M in carboxylate groups), which are small and remain in suspension. Suspensions were prepared exactly like for absorption measurements. They gave very stable and reproducible values, and the ζ potential was found to be -24.7 mV. Therefore, the microcrystals grown in the presence of polymers bear a negative charge in water. Discussion In our previous work, transmission electron microscopy allowed the particles formed during the reprecipitation process to be characterized.18 Therefore, the different steps of the kinetics could be interpreted. We proposed that, at the beginning of the process, just after the organic solution was poured into water, molecules of dye 1 are dispersed in an ethanol-rich environment. These molecules were noted A in our model. Then, as ethanol diffuses, small aggregates are formed, noted B. They assemble to give bigger aggregates (C), which finally generate microcrystals (D). The presence of these different molecular arrangements explains that three steps are necessary to fit the kinetics data, and it is then easily understood why steps II and III are autocatalyzed. This basic mechanism was encountered for both the experiments run in water alone and those run with the assistance of dendrimers. In the present work, the kinetics profiles resemble those obtained previously, and the curves are nicely fitted using the same model as before. This shows that a three-step mechanism also takes place here. Moreover, the rate constants obtained are very close to those determined for the kinetics run in the presence of dendrimers, at a comparable carboxylate ion concentration. It must also be noted that the microcrystals obtained are identical in the two cases. Overall, this indicates that dendrimers and polymers play the same role as additives in the reprecipitation process. Two important consequences can be drawn from this observation. First, the hypothesis that the inner cavities of the dendrimer act as special nucleation sites can be ruled out. This supports our previous fluorescence spectroscopy experiments, which showed that the NBD dyes remained outside the dendrimer.18 Second, it can be logically thought that the activity of the macromolecule (polymer or dendrimer) is determined by its surface. There is no doubt that the nature of the terminal groups is a key factor. The acidification of the medium clearly shows that swapping carboxylate groups for carboxylic acid groups affects the process deeply. The functional groups must also be gathered on a macromolecule, because when they are borne by small molecules, their presence has very little effect. Their density on the polymeric backbone also has an influence, as shown by the comparison of the kinetics constants between the homopolymers and the copolymer. It can also be recalled that spectroscopic evidence was previously given for an interaction between the highly polar NBD dye and the carboxylate group.18 This leaves wide open the question

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of how the charged polymer surface can affect the reprecipitation process of the NBD dye. According to the analysis of the kinetics data, the polymers influence the three steps of reprecipitation. Concerning the early stage of this process, at least two hypotheses can account for the effect observed. (i) The polymer has a general effect upon the aqueous medium. Water is strongly structured around it so that the number of water molecules available to solvate the dye is reduced. (ii) The polymer interacts specifically with the dye molecules. Consequently, the NBD molecules are attracted near the polymer surface, which creates a local increase in dye concentration, hence, locally raising the degree of supersaturation. It is difficult to choose between these hypotheses for the time being. But, it can be noticed that both would lead to the same result, that is, an enhancement of the primary aggregates formation, compared to what happens in water alone. This is in line with the acceleration of the first step of the reprecipitation process. Let us now consider the last stage of reprecipitation, that is, the formation of microcrystals, and let us examine which role the polymer may play there. According to the microanalysis results, the microcrystals contain a significant amount of polymer. This was also the case with dendrimers (17% in weight) in our previous work. The 7% polymer fraction measured here by the microanalysis method seems quite large, especially if considering the formation of a thin polymer layer around the crystal, and it could be overestimated. The measurement of ζ potentials reveals that the surface of the microcrystals grown in the presence of PAA is negatively charged. No direct comparison can be made with the microcrystals grown in water alone, but it is likely that the ζ potential of these particles would be close to neutral, because the NBD dye is a neutral molecule. The negative ζ potential observed for the microcrystals grown in the presence of PAA could then be attributed to the presence of polymer at the surface. However, it can be argued that some neutral molecules may exhibit a negative charge in water, due to the selective adsorption of hydroxyl anions that originate from ionization of the surrounding water.16 If this were the case here, the negative potential measured for the microcrystals grown in the presence of PAA would be that of genuine NBD microparticles. One additional observation allows us to refute this last hypothesis. When microcrystals were grown in the presence of another type of polymer, that is, poly(acrylamide), very small microcrystals were obtained too, and their ζ potential was found to be almost neutral. There is a direct link between the nature of the polymer functional groups and the ζ potential of the microcrystals grown in the presence of the polymer. This reveals that a certain amount of polymers are present at the microcrystal surface. However, there is no indication that the polymer is exclusively found at this place. In particular, this does not rule out the hypothesis that polymer can play the role of a template at the beginning of crystallization and that a proportion of polymer is included in the crystal interior. The fact that the polymer interacts with the microcrystals, and especially with its surface, allows the shape of the microcrystals to be better understood. If the polymer is preferentially adsorbed to one face, it retards crystal growth perpendicular to that face, which can explain the fact that the microcrystals produced are very flat and thin. The fact that the polymer interferes with the crystal growth is nicely pictured by comparing part a and part b of Figure 4. It can be seen there that placing the additive in the reprecipitation medium while aggregates and small crystals are already formed is enough to modulate the shape of the microc-

Abyan et al.

rystals. It can be thought that the large number of functional groups borne by the polymer makes the affinity of this macromolecule for the microcrystal surface very strong. This explains why little effect was encountered using small isolated molecules and why the polymer remains with the microcrystals after rinsing. Moreover, the fact that all the microcrystals grown in the presence of polymer do not agglomerate agrees well with the steric shielding due to the macromolecule. As for the intermediate stage of reprecipitation, there is no proof that the hydrophilic polymer can adsorb to the aggregate surface, before crystallization has begun. If this were the case, it would probably result in a decrease of the interfacial tension and then in a lowering of the aggregate critical radius, that is, the radius below which the aggregate is unstable and dissolves again.26 This would favor the appearance of numerous small particles, which is in agreement with our observations. It can be noted that, in the bibliography, the stabilization of organic colloidal particles by polymers has been the topic of numerous theoretical investigations, particularly about the adsorption of polyelectrolytes on oppositely charged surfaces.27 Finally, an interesting point is that microcrystals displaying a flower shape were reproducibly produced by adding the polymer 20 min after reprecipitation had begun. These structures were never observed by us previously. It is very tempting to make a connection between this observation and a theoretical work from Schiessel et al.28 These authors have recently calculated that when a wormlike chain interacts with a spherical organizing center, it can form a “rosette” structure. This could occur when the PAA polymer is added in the course of the reprecipitation process and wraps around a small dye colloid. This “rosette” structure could subsequently gather many very small aggregates, which further grow as microcrystals, thus, leading to complex structures such as the flowers observed. It can also be recalled that, in the case of inorganics, polymers can act as a binder for primary crystallites, resulting in superstructures.29 The results of this work can now be compared to those reported in the literature. The idea that polymers bind to the surface of organic microcrystals has been proposed many times in the bibliography. For example, it has been invoked to explain that polystyrene-block-poly(ethylene oxide) and poly(vinyl alcohol), used respectively in the reprecipitation of β-carotene20 and perylene,21 protect the microcrystals from aggregation. However, to our knowledge, the composition of the microcrystals obtained and the kinetics behavior have not been reported yet. In the field of inorganic compounds, the use of polymers as additives is common in the specific control of growth and in the prevention of agglomeration of metal particles, once they have been synthesized. But, with inorganics, the question no longer concerns the reprecipitation method. It can be noted, however, that it is generally considered that polymer adsorption at the colloid surface is a key event in this process29-31 and that the resulting inorganic particles are polymer-coated.26 In this respect, some careful comparisons with our findings can be attempted. However, a major difference must be kept in mind. In the case of (26) Fo¨rster, S.; Antonietti, M. Adv. Mater. 1998, 10, 195-217 and references therein. (27) Horn, D.; Rieger, J. Angew. Chem., Int. Ed. 2001, 40, 43304361 and references therein. (28) Schiessel, H.; Rudnick, J.; Bruinsma, R.; Gelbart, W. M. Europhys. Lett. 2000, 51, 237-243. (29) Qi, L.; Co¨lfen, H.; Antonietti, M. Angew. Chem., Int. Ed. 2000, 39, 604-607.

Preparation of Organic Microcrystals with Polymers

inorganic compounds, the presence of polymers generally slows down or inhibits the formation of microcrystals,32 while in our case, a drastic acceleration was observed. The explanation is probably that, in the case of our organic compounds and within the context of the reprecipitation method, the polymers do not simply play the role of crystal growth inhibitors but act on the whole reprecipitation process. Conclusion In the present work, the NBD dye appeared again as a good tool for the detailed study of the reprecipitation process. Its crystallization rate is slow enough to allow the analysis of each kinetics step. Experiments are underway to check whether this three-step kinetics is a general phenomenon for organic compounds. This study of the reprecipitation process in the presence of polymers confirms our previous findings and provides a deeper insight into the mechanism involved. Polymers have empirically been used as additives in the reprecipitation of organic compounds. This is definitely a convenient and cheap way to obtain populations of non-agglomerated microcrystals, with homogeneous size and morphology. For the first time, a detailed kinetics approach was proposed here, as well as a mechanism to account for the complex role played by the polymers. In particular, it was (30) (a) Amjad, Z. Langmuir 1993, 9, 597-600. (b) O ¨ ner, M.; Norwig, J.; Meyer, W. H.; Wegner, G. Chem. Mater. 1998, 10, 460-463. (c) Co¨lfen, H.; Qi, L.; Mastai, Y.; Bo¨rger, L. Cryst. Growth Des. 2002, 2, 191-196. (d) Co¨lfen, H.; Qi, L. Chem.sEur. J. 2001, 7, 106-116. (e) Co¨lfen, H. Macromol. Rapid Commun. 2001, 22, 219-252. (f) Yu, S.-H.; Co¨lfen, H.; Antonietti, M. Chem.sEur. J. 2002, 8, 2937-2945. (g) Co¨lfen, H.; Antonietti, M. Langmuir 1998, 14, 582-589. (h) Antonietti, M.; Breulmann, M.; Go¨ltner, C. G.; Co¨lfen, H.; Wong, K. K. W.; Walsh, D.; Mann, S. Chem.sEur. J. 1998, 4, 2493-2500. (i) Qi, L.; Co¨lfen, H.; Antonietti, M. Chem. Mater. 2000, 12, 2392-2403. (31) Sedla´k, M.; Antonietti, M.; Co¨lfen, H. Macromol. Chem. Phys. 1998, 199, 247-254. (32) (a) Naka, K.; Tanaka, Y.; Chujo, Y.; Ito, Y. Chem. Commun. 1999, 1931-1932. (b) Naka, K.; Chujo, Y. C. R. Chim. 2003, 6, 11931200. (c) Kato, T.; Suzuki, T.; Amamiya, T.; Irie, T.; Komiyama, M.; Yui, H. Supramol. Sci. 1998, 5, 411-415.

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shown that the polymers influence each step of the reprecipitation process. At the end of the process, a small amount of them remains with the microcrystal, especially at the surface. A study is presently underway to control the parameters that allow homogeneous microcrystals to be produced and to investigate their spectroscopic properties. It will further be interesting to know whether these properties can be modulated by the nature of the polymer. The functionalization of the latter could lead to different applications in the fields of optics and molecular recognition. This work also opens new perspectives in other fields, for example, that of pharmaceutics. Actually, it is widely acknowledged that the rate of dissolution of a hydrophobic drug is directly linked to the particle surface area.2,20 Controlling the size and morphology of the drug particles could be an efficient way to improve their bio-availability. Polymers can be used for this purpose. When choosing a polymer as an additive to prepare drug microcrystals by the reprecipitation method, it is necessary to take many parameters into account. The first parameter is the affinity of the polymer for the drug microcrystals so that interaction takes place. The second is the effect that can be obtained on the microcrystal size and morphology. The third is the intrinsic physiological effect of the polymer. For example, PAA is known to be a mucoadherent for enhanced drug retention, and poly(ethylene oxide) extends the lifetime of particles in the blood stream.20 A delicate balance between these three parameters could lead to a significant improvement in drug distribution and bioavailability. This will certainly motivate further investigations in this direction. Acknowledgment. Dr. Philippe Cochard (“Service de Microscopie Confocale” of the “Centre de Biologie du De´veloppement” of Toulouse) is gratefully acknowledged for his assistance with the confocal microscope. We are also indebted to Dr. Emile Perez for kindly helping us in ζ potential measurements. LA046877J