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Structure of supersaturated solution and crystal nucleation induced by diffusion
Journal of Crystal Growth ] (]]]]) ]]]–]]]
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Structure of supersaturated solution and crystal nucleation induced by diffusion Hiroshi Ooshima n, Koichi Igarashi, Hideo Iwasa, Ren Yamamoto Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138, Sugimoto-cho, Sumiyoshi-ku, Osaka 558-8585, Japan
a r t i c l e i n f o
Keywords: A1. Nucleation A1. Diffusion A2. Seed crystals B1. Organic compound
abstract The effect of a seed crystal on nucleation of L-alanine from a quiescent supersaturated solution was investigated. When a seed crystal was not used, nucleation did not occur at least for 5 h. When a seed crystal was introduced into the supersaturated solution with careful attention to avoid convection of the solution, fine crystals appeared at the place far from the seed crystal. At that time, there was no convection at the place that fine crystals appeared. Namely, there was no possibility that those fine crystals came from the surface of seed crystal. We supposed that nucleation was induced by directional diffusion of solute molecules caused by growth of the seed crystal. In order to prove this hypothesis, we designed an experiment using an apparatus composed of two compartments divided by a dialysis membrane that L-alanine molecules could freely permeate. Two supersaturated solutions having a supersaturation ratio of 1.2 and a smaller ratio were placed in the two compartments in the absence of seed crystals. This apparatus allowed the directional diffusion of solute molecules between two solutions. Nucleation occurred within 30 min. The frequency of nucleation among 7-times repeated experiments was in proportion to the difference of supersaturation ratio between the two solutions. This result poses a new mechanism of the secondary nucleation that the directional diffusion caused by growth of existing crystals induces nucleation. & 2012 Elsevier B.V. All rights reserved.
1. Introduction The important characteristics of crystals, for example polymorphism, crystal size and its distribution, solvation, etc., are dominated or deeply influenced by nucleation rather than crystal growth. Nucleation has been studied for a long time, but it is still unclear. Nucleation has been classified into primary nucleation and secondary nucleation. In the cooling crystallization without seed crystals, we often experience a long induction period before nucleation. The induction period in crystallization of p-acetanisidide under agitation using chloroform as a solvent was over 17 h [1]. Then, once crystallization began, it rapidly proceeded and was completed after a short time. This induction period is not the same as the waiting time required for the birth of many nuclei and growth of nuclei to detectable or visible large crystals. In order to understand the induction period and the subsequent rapid crystallization, we need to understand the mechanism of primary and secondary nucleation. The mechanism of nucleation may become clear from the analyses of structure of the supersaturated solution. Saito et al. [1] have investigated the structure of supersaturated solution of p-acetanisidide. By analyzing the interactions between p-acetanisidide molecules from the change of chemical shift (d), the nuclear Overhauser effect (NOE), and the spin-lattice relaxation
n
Corresponding author. Tel.: þ81 6 6605 2700; fax: þ 81 6 6605 2701. E-mail address: [email protected] (H. Ooshima).
time (T1) in Nuclear Magnetic Resonance (NMR) measurements, it was found that p-acetanisidide molecules are associated in the supersaturated solution and even in the under-saturated solution. The structure of associates, namely the intermolecular interaction, was not the same as that of crystal, but similar. It means that molecular associates must change the structure of associates to that of crystals for nucleation. It was also found that solute molecules had taken a conformation similar to that taken in appearing crystals (polymorphs) before nucleation in solution [2,3]. Namely, there is not so big freedom in the conformation of molecules in solution as supposed from a viewpoint of a characteristic of s-bond between carbons, i.e. free rotation ability. A possible mechanism of primary nucleation suggested from these previous experimental results was presented in Fig. 1. Since the molecular associates themselves are not necessarily nuclei, the structure of associates must be rearranged to that of crystals for nucleation (we name it the structure transformation of associates). The induction period must be the time required for beginning of the structure transformation of molecular associates. The structure transformation itself must be nucleation. However, it is still unknown what the trigger of the structure transformation is. On the other hand, it is well known that existing crystals induce nucleation, that is, secondary nucleation. Secondary nucleation has been concerned in crystallization from agitated solution [4–6]. However, there are several reports that secondary nucleation occurs even in quiescent solutions. Denk and Botsaris [7] investigated the effect of seed crystals in the crystallization of sodium chlorate from a
0022-0248/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcrysgro.2012.12.008
Please cite this article as: H. Ooshima, et al., Journal of Crystal Growth (2013), http://dx.doi.org/10.1016/j.jcrysgro.2012.12.008i
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H. Ooshima et al. / Journal of Crystal Growth ] (]]]]) ]]]–]]]
Molecular associates having a Supersaturated structuresimilartothestructure Solution (molecular arrangement) of crystal Induction period Nuclei
Structure transformation of the molecular associates (The primary nucleation)
Fig. 1. A possible mechanism of primary nucleation.
quiescent solution. They elucidated by a clever experiment using enantiomorphic seed crystal that at moderately high supersaturation secondary nuclei originated from seed crystal and at higher supersaturation spontaneous nucleation begin to take place. They explained the birth of secondary nuclei originated from seed crystal in a quiescent solution with the growth and detachment of irregularities such as dendrites on the surface of the seed crystal, and emphasized the importance of the boundary layer near the growing crystal for secondary nucleation in the presence of impurity. In that study, the effect of convection caused by the growth of seed crystals on the appearance of new crystals has not been discussed. In the present work, we propose a novel mechanism of secondary nucleation acting in a quiescent solution. When Lalanine was crystallized from a quiescent supersaturated solution in the presence of a seed crystal, the seed crystal induced nucleation at a place far from the seed crystal, although there was no convection. We will discuss the nucleation induced by diffusion of solute molecules.
2. Experimental procedures 2.1. Materials L-Alanine used was of reagent grade and purchased from Wako Pure Chemical Industries Ltd. Super-purified water was prepared by Nanopure diamond (Barnstead) and used as a solvent.
2.2. Crystallizer Two kinds of crystallizer were used as shown in Fig. 2. Crystallizer-1 was composed of two parts, namely a crystallization cell and a side tube for introduction of a seed crystal. The crystallization cell was a screw-capped glass-cell (10 10 40 mm in inner size). The side glass tube (20 mm in length, 5 mm in inner diameter) was attached to the side of the crystallization cell horizontally at the point of 10 mm height from bottom of the cell and sealed with a rubber plug. A thin wire used for the introduction of a seed crystal was set through the rubber plug. Crystallizer-2 was composed of two screw-capped and jacketed glass-tubes of 85 mm in length and 10 mm in inner diameter, a dialysis cellulose membrane of 12,000–16,000 in molecular weight cutoff, silicon rubber sheets and a horseshoe-shaped clamp. 2.3. Crystallization of L-alanine 2.3.1. Crystallization of L-alanine using Crystallizer-1 A given amount of L-alanine was dissolved in water to prepare the solution with a supersaturation ratio of 1.25 at 20 1C. The solubility of L-alanine is 157.9 mg/mL at 20 1C. The solution was filtrated with a 0.45 mm membrane filter and incubated at 40 1C
Fig. 2. Schematic diagrams of the crystallizers: (a) Crystallizer-1: 1, laser light source; 2, laser beam (standing layer); 3, water bath; 4, glass cell (crystallization cell and side tube); 5, seed crystal; 6, seed introduction motor and 7, board. (b) Crystallizer-2: 1, crystallization glass tube; 2, water jacket; 3, silicon-rubber gasket and 4, dialysis membrane.
for 24 h to completely dissolve L-alanine and to make the initial condition of the solution same in all experiments. A washed single crystal of L-alanine with a size of about 0.5 mm was used for a seed crystal. The seed crystal was fixed to the tip of thin wire with glue and set near the rubber plug in the side tube of Crystallizer1. Three milliliters of the L-alanine solution was placed in Crystallizer-1. At this time, care was taken not to immerse the seed crystal in solution. Namely, the seed crystal was maintained in a small air space formed between the solution and the rubber plug. Crystallizer-1 was placed in a water bath made of acrylic plastic and the temperature was controlled at 20 70.03 1C. The whole apparatus was incubated in a dark dry-incubator. After 1 h, the seed crystal was introduced to the solution at a constant slow speed (60 mm/s) by remote control. To detect appearance of crystals, two lying sheet-like laser beams (5 1 mm) were irradiated to the solution from the opposite side of the side tube of Crystallizer-1, although one standing laser beam used for the another experiment (Section 2.3.3) is illustrated in Fig. 2. The diffuse reflection of laser from fine crystals appeared was monitored by CCD camera (1.5 million in pixel counts; Keyence Co. Ltd.). We checked the clear diffuse reflection of laser in advance by using polystyrene latex beads with a size of 460 nm. The image was recorded once in every second. 2.3.2. Crystallization of L-alanine using Crystallizer-2 The L-alanine solution of 189.5 mg/mL (saturation temperature: 38.9 1C; supersaturation ratio C/Cs¼1.2 at 20 1C) was preincubated at 70 1C for 12 h and then at 40 1C for 1 h. Then, the solution was placed into the compartment-I of Crystallizer-2. On the other hand, the solution with different supersaturation ratios (C/Cs¼1.0, 1.05, 1.1, 1.15, 1.2 at 20 1C) was placed to the compartment-II. Temperature of the solutions was controlled at 20 1C by circulating water to the jacket. Seed crystals were not used in this experiment. The appearance of visible crystals was recorded by CCD camera at intervals of 5 min. 2.3.3. Visualization of convection flow in Crystallizer-1 To visualize the convection flow that might be spawned in the crystallization cell of Crystallizer-1 during crystallization, a small amount of polystyrene latex beads (460 nm) was added to the
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supersaturated solution and one standing laser beam was irradiated as shown in Fig. 2. The diffuse reflection from the latex beads was monitored by CCD camera and was recorded at intervals of 1 s. The speed of latex beads moving in the supersaturated solution was determined from changes in the position of latex beads arbitrary picked up.
3. Results and discussion 3.1. Crystallization of L-alanine using Crystallizer-1 The crystallization of L-alanine was carried out by using Crystallizer-1 at a supersaturation ratio of 1.25 at 20 1C for 2 h under completely quiescent condition. In the absence of a seed crystal, nucleation did not occur even when 5 h elapsed and this result was reproducible. On the other hand, when a single seed crystal was introduced into the quiescent supersaturated solution at the side tube of Crystallizer-1, nucleation was induced and fine crystals appeared in the crystallization cell far from the seed crystal. As an example, the appearance of fine crystals in the crystallization cell within 2 h was illustrated on a digital image taken by CCD camera in Fig. 3. The seed crystal does not come into view in Fig. 3 and should be in the right side of a thick arrow. In Fig. 3, the reflected lights from fine crystals are also not shown, but the sequence of appearance and positions of 12 fine crystals during the 2 h of crystallization are illustrated with numbers and arrows, respectively. The downward-pointing arrow means that the fine crystal moved downwards. Fine crystals appeared over the wide range of cross section of the crystallization cell as shown in cases of no.5 and no.11 and always dropped downwards. Fig. 4 shows a seed crystal used for the experiment shown in Fig. 3. Panel A is the seed crystal before introducing to the solution and Panel B is after growth. During crystallization for 2 h, the seed crystal grew about 4 times in length. It means that the growth of seed crystal in the side tube and nucleation of new crystals in the crystallization cell simultaneously occurred. The same experiment was repeated 5 times and similar results were obtained as summarized in Table 1. A crystal, no.9 in Run 2 moved upwards at a 1.7 mm distance from the right wall of the cell. The number of crystals appearing within 2 h was in the range of 6–12. The data shown in Table 1 were rearranged in Table 2 to elucidate when new crystals
11 2 6
Laser beam
8 9
7
Glass cell (Crystallizer-1)
Side tube
1 12 4
Seed 10 5 3
Fig. 3. Positions at which crystals appeared in the crystallization cell of Crystallizer-1 and those sequence. The downward-pointing arrow means that the fine crystal moved downwards. A seed crystal is in the right side of a thick arrow.
Fig. 4. Seed crystal used for the experiment: (a) before introducing to the solution and (b) after growth.
Table 1 Numbering of fine crystals and appearance time. Number of fine crystals
1 2 3 4 5 6 7 8 9 10 11 12 a b
Appearance time (s) Run 1a
Run 2
Run 3
Run 4
Run 5
390 390 690 1890 3390 3720 4110 4620 5130 5310 5880 6390
130 367 2392 2721 3601 4300 4407 4523 6689b
3072 4692 5704 5817 6050 6086 6100
259 1527 5065 5111 5112
200 347 780 976 1720 2259 2475 4398 5631 6053
The experiment shown in Fig. 3. This crystal moved upwards.
Table 2 Summary of 5-times repeated crystallization of L-alanine. Range of time at which fine crystals appeared (s)
Number of fine crystals
1–1000 1000–2000 2000–3000 3000–4000 4000–5000 5000–6000 6000–7200
10 3 4 4 7 9 6a
a
One crystal moved upwards.
appeared. The frequency of nucleation seems to be even during 2 h of crystallization.
3.2. Convection of the supersaturated solution in Crystallizer-1 during crystallization As presented in Table 1, a crystal moved upwards near the right wall of the cell. This observation suggested that the convection was spawned in the crystallization cell by 6689 s. The convection should be due to the local density change of the solution caused by growth of the seed crystal placed in the side tube of the cell. The convection called into question about the experimental data shown in Table 1. Namely, the question was if fine crystals listed up in Table 1 were generated in a quiescent solution or carried from the seed crystal by convection flow.
Please cite this article as: H. Ooshima, et al., Journal of Crystal Growth (2013), http://dx.doi.org/10.1016/j.jcrysgro.2012.12.008i
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Consequently, the convection in the crystallization cell was examined. Polystyrene latex beads were added to the supersaturated solution to visualize the flow of the solution. In the absence of seed crystal, the latex beads did not move except for the randomdirection micromotion in the rate range of 0.3–0.6 mm/s for at least 2 h. This meant that the convection did not arise. When a seed crystal was introduced into the supersaturated solution, a small convection flow of a speed of about 2 mm/s arose near the seed crystal, but it was limited in the side tube until 1110 s elapsed after the introduction of the seed crystal. However, after 1110 s, the convection began to spread to the crystallization cell. Namely some latex beads began to move from the side tube to the crystallization cell and moved upwards along the cell wall and some latex beads began to move from the crystallization cell into the side tube at the speed of 2–4 mm/s. Then, the convection became gradually fast to about 250 mm/s. Similar results were obtained in the repeated same experiments. From these results, we concluded that the supersaturated solution in the crystallization cell had been kept in the quiescent state until the convection began to spread from the side tube to the crystallization cell and that the convection began to spread to the crystallization cell after the elapse of the time of about 1000 s from the introduction of a seed crystal. 3.3. Secondary nucleation induced by diffusion of solute molecules As presented in Table 2, 10 crystals appeared within 1000 s after introduction of a seed crystal into the supersaturated solution in the side tube. The place at which crystals appeared was in the crystallization cell far from the seed crystal (at a distance of 0.5–1.5 cm). As described in Section 3.2, there was no convection in the supersaturated solution of the crystallization cell until at least 1000 s elapsed. From these facts, we conclude that fine crystals appearing in the first term of 1000 s are not originated from the surface of seed crystal. Meanwhile, after 1000 s elapsed, the appearance of several crystals per a term of 1000 s was detected. It is not clear if these fine crystals are originated from the seed crystal or not, although the small frequency of appearance of crystals per a term of 1000 s may indicate that those are not originated from the seed crystal also. It should be noted that it is also true that all fine crystals listed in Tables 1 and 2 would not appear if a seed crystal had not been introduced at the side tube of Crystallizer-1. In order to understand this secondary nucleation in a quiescent solution, we presumed that the diffusion of solute molecules induced nucleation. The diffusion of solute molecules takes place by growth of the seed crystal. As described in Section 1, the solute molecules are forming associates in the supersaturated solution. In case of p-acetanisidide, the structure of associates, namely the interaction between molecules, was similar to that in crystal [1]. For nucleation, the structure transformation from associates to crystals is required. The factor inducing the structure transformation may be the diffusion of solute molecules. In other word, the diffusion of molecular associates may be the trigger of nucleation. 3.4. Verification of a novel mechanism of secondary nucleation L-alanine was crystallized using Crystallizer-2. By fixing the supersaturation ratio of the solution placed in the compartment-I to 1.2 and changing the supersaturation ratio of the solution placed in the compartment-II, five kinds of crystallization were carried out. The crystallization under the same condition was repeated 7 times. L-alanine molecules in the compartment-I can diffuse to the compartment-II through a dialysis cellulose membrane of 12,000–16,000 in molecular weight cutoff. The
Fig. 5. Relationship between the probabilities of nucleation in the solution placed in the compartment-I and the supersaturation ratio of the solution placed in the compartment-II.
permeation of water from the compartment-II to the compartmentI by osmotic pressure must be negligibly small because of free permeability of L-alanine molecules through the membrane and a small headspace of the compartment sealed with a screw cap. Fig. 5 presents the relationship between the probability of nucleation in the solution placed in the compartment-I and the supersaturation ratio of the solution placed in the compartmentII, where the probability was defined as a percentage of experiments in which nucleation occurred in the compartment-I among 7-times repeated experiments. All nucleation occurred in the compartment-I, and never in the compartment-II. The larger the difference in supersaturation ratio between those two solutions, the larger the probability of nucleation became. When the supersaturation ratio was 1.20 in both the compartment-I and II, no nucleation occurred even when 5 h elapsed. These results can be explained by the difference in strength of the driving force of diffusion of L-alanine. Namely, the larger the driving force of diffusion, the higher the frequency of nucleation became. This result supports the novel mechanism of secondary nucleation that the diffusion of solute molecules accompanied by growth of existing crystal induces nucleation. The molecular associates may be deformed during diffusion, resulting in rearrangement of a part of associates into the structure of nucleus. Gong et al. [8] showed that nucleation was accelerated in a colloidal system with a large nucleation volume. For instance they observed many crystals in a droplet with 500 mm in diameter, although no crystal was observed in other droplets in neighborhood. They explained this as a result of the strong interactions among multiple nuclei and the effect of diffusion enhanced in neighborhood of crystallites. Our present result strongly supports the effect of diffusion in acceleration of nucleation observed in a colloidal system. In the present work, the effect of diffusion was also found to be widening not only in the vicinity of existing crystal but also over a distance of 0.5–1.5 cm from existing crystals. The induction of nucleation by diffusion must play an important role even in primary nucleation. As described in Section 1, the induction period often observed in crystallization of organic compounds must be the time required for beginning of the structure transformation of molecular associates and the structure transformation itself must be nucleated. If so, the trigger of the structure transformation should be the diffusion of molecular associates even in crystallization under agitation. Diffusion of molecules may be an essential matter for the rearrangement of structure of associates than convective movement by agitation,
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because in the former case molecules move by themselves and in the latter case molecules are usually riding on large vortexes.
4. Conclusions The mechanism of secondary nucleation was investigated. Crystallization of L-alanine from quiescent supersaturated solution was carried out in the absence and presence of a seed crystal. The same crystallization was repeated 5 times. In the absence of a seed crystal and at the supersaturation ratio of 1.25, nucleation did not occur even after 5 h. On the other hand, in the presence of a seed crystal, several fine crystals appeared within 1000 s at the place far from the seed crystal. In the 5-times repeated crystallization, totally 10 crystals appeared during the first 1000 s term. In that time, there was no convection that might carry nuclei from the surface of seed crystal. We concluded that those 10 crystals were generated at the place far from the seed crystal and they are not directly originated from the seed crystal. As a result, we hypothesized a novel nucleation mechanism that the diffusion of solute molecules accompanied by growth of seed crystal induced the secondary nucleation. This hypothesis was verified by an experiment arranged so as to make solute molecules diffuse in supersaturated solution without seed crystal. This secondary nucleation mechanism must be working in the primary nucleation, because nucleation induced by diffusion occurred even in the absence of seed crystal.
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Acknowledgment This study was supported by Grant-in-Aid for Scientific Research (C) (no. 21560781) from the Japan Society for the Promotion of Science (JSPS).
References [1] A. Saito, K. Igarashi, M. Azuma, H. Ooshima, Aggregation of p-acetanisidide molecules in the under-and super-saturated solution and its effect on crystallization, Journal of Chemical Engineering of Japan 35 (2002) 1133–1139. [2] S. Maruyama, H. Ooshima, Mechanism of the solvent-mediated transformation of Taltirelin polymorphs promoted by methanol, Chemical Engineering Journal 81 (2001) 1–7. [3] M.K. Hirano, K. Igarashi, K. Machiya, R. Tamura, H. Tue, H. Ooshima, Relationship between crystal polymorphism and solution structure of an imidazopyridine derivative as a drug substance for osteoporosis, Journal of Chemical Engineering of Japan 42 (2009) 204–211. [4] H.H. Ting, W.L. McCabe, Supersaturation and crystal formation in seeded solutions, Journal of Industrial and Engineering Chemistry 11 (1934) 1201–1207. [5] R.F. Strickland-Constable, R.E.A. Mason, Breeding of nuclei, 197, Nature, London, United Kingdom, 1963 897-898. [6] H.E.C. Powers, Nucleation and early crystal growth, Industrial Chemist and Chemical Manufacturer 39 (1963) 351–355. [7] E.G. Denk Jr, G.D. Botsaris, Fundamental studies in secondary nucleation from solution, Journal of Crystal Growth 13–14 (1972) 493–499. [8] T. Gong, J. Shen, Z. Hu, M. Marquez, Z. Cheng, Langmuir 23 (2007) 2919–2923.
Please cite this article as: H. Ooshima, et al., Journal of Crystal Growth (2013), http://dx.doi.org/10.1016/j.jcrysgro.2012.12.008i
Contents lists available at SciVerse ScienceDirect
Journal of Crystal Growth journal homepage: www.elsevier.com/locate/jcrysgro
Structure of supersaturated solution and crystal nucleation induced by diffusion Hiroshi Ooshima n, Koichi Igarashi, Hideo Iwasa, Ren Yamamoto Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138, Sugimoto-cho, Sumiyoshi-ku, Osaka 558-8585, Japan
a r t i c l e i n f o
Keywords: A1. Nucleation A1. Diffusion A2. Seed crystals B1. Organic compound
abstract The effect of a seed crystal on nucleation of L-alanine from a quiescent supersaturated solution was investigated. When a seed crystal was not used, nucleation did not occur at least for 5 h. When a seed crystal was introduced into the supersaturated solution with careful attention to avoid convection of the solution, fine crystals appeared at the place far from the seed crystal. At that time, there was no convection at the place that fine crystals appeared. Namely, there was no possibility that those fine crystals came from the surface of seed crystal. We supposed that nucleation was induced by directional diffusion of solute molecules caused by growth of the seed crystal. In order to prove this hypothesis, we designed an experiment using an apparatus composed of two compartments divided by a dialysis membrane that L-alanine molecules could freely permeate. Two supersaturated solutions having a supersaturation ratio of 1.2 and a smaller ratio were placed in the two compartments in the absence of seed crystals. This apparatus allowed the directional diffusion of solute molecules between two solutions. Nucleation occurred within 30 min. The frequency of nucleation among 7-times repeated experiments was in proportion to the difference of supersaturation ratio between the two solutions. This result poses a new mechanism of the secondary nucleation that the directional diffusion caused by growth of existing crystals induces nucleation. & 2012 Elsevier B.V. All rights reserved.
1. Introduction The important characteristics of crystals, for example polymorphism, crystal size and its distribution, solvation, etc., are dominated or deeply influenced by nucleation rather than crystal growth. Nucleation has been studied for a long time, but it is still unclear. Nucleation has been classified into primary nucleation and secondary nucleation. In the cooling crystallization without seed crystals, we often experience a long induction period before nucleation. The induction period in crystallization of p-acetanisidide under agitation using chloroform as a solvent was over 17 h [1]. Then, once crystallization began, it rapidly proceeded and was completed after a short time. This induction period is not the same as the waiting time required for the birth of many nuclei and growth of nuclei to detectable or visible large crystals. In order to understand the induction period and the subsequent rapid crystallization, we need to understand the mechanism of primary and secondary nucleation. The mechanism of nucleation may become clear from the analyses of structure of the supersaturated solution. Saito et al. [1] have investigated the structure of supersaturated solution of p-acetanisidide. By analyzing the interactions between p-acetanisidide molecules from the change of chemical shift (d), the nuclear Overhauser effect (NOE), and the spin-lattice relaxation
n
Corresponding author. Tel.: þ81 6 6605 2700; fax: þ 81 6 6605 2701. E-mail address: [email protected] (H. Ooshima).
time (T1) in Nuclear Magnetic Resonance (NMR) measurements, it was found that p-acetanisidide molecules are associated in the supersaturated solution and even in the under-saturated solution. The structure of associates, namely the intermolecular interaction, was not the same as that of crystal, but similar. It means that molecular associates must change the structure of associates to that of crystals for nucleation. It was also found that solute molecules had taken a conformation similar to that taken in appearing crystals (polymorphs) before nucleation in solution [2,3]. Namely, there is not so big freedom in the conformation of molecules in solution as supposed from a viewpoint of a characteristic of s-bond between carbons, i.e. free rotation ability. A possible mechanism of primary nucleation suggested from these previous experimental results was presented in Fig. 1. Since the molecular associates themselves are not necessarily nuclei, the structure of associates must be rearranged to that of crystals for nucleation (we name it the structure transformation of associates). The induction period must be the time required for beginning of the structure transformation of molecular associates. The structure transformation itself must be nucleation. However, it is still unknown what the trigger of the structure transformation is. On the other hand, it is well known that existing crystals induce nucleation, that is, secondary nucleation. Secondary nucleation has been concerned in crystallization from agitated solution [4–6]. However, there are several reports that secondary nucleation occurs even in quiescent solutions. Denk and Botsaris [7] investigated the effect of seed crystals in the crystallization of sodium chlorate from a
0022-0248/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcrysgro.2012.12.008
Please cite this article as: H. Ooshima, et al., Journal of Crystal Growth (2013), http://dx.doi.org/10.1016/j.jcrysgro.2012.12.008i
2
H. Ooshima et al. / Journal of Crystal Growth ] (]]]]) ]]]–]]]
Molecular associates having a Supersaturated structuresimilartothestructure Solution (molecular arrangement) of crystal Induction period Nuclei
Structure transformation of the molecular associates (The primary nucleation)
Fig. 1. A possible mechanism of primary nucleation.
quiescent solution. They elucidated by a clever experiment using enantiomorphic seed crystal that at moderately high supersaturation secondary nuclei originated from seed crystal and at higher supersaturation spontaneous nucleation begin to take place. They explained the birth of secondary nuclei originated from seed crystal in a quiescent solution with the growth and detachment of irregularities such as dendrites on the surface of the seed crystal, and emphasized the importance of the boundary layer near the growing crystal for secondary nucleation in the presence of impurity. In that study, the effect of convection caused by the growth of seed crystals on the appearance of new crystals has not been discussed. In the present work, we propose a novel mechanism of secondary nucleation acting in a quiescent solution. When Lalanine was crystallized from a quiescent supersaturated solution in the presence of a seed crystal, the seed crystal induced nucleation at a place far from the seed crystal, although there was no convection. We will discuss the nucleation induced by diffusion of solute molecules.
2. Experimental procedures 2.1. Materials L-Alanine used was of reagent grade and purchased from Wako Pure Chemical Industries Ltd. Super-purified water was prepared by Nanopure diamond (Barnstead) and used as a solvent.
2.2. Crystallizer Two kinds of crystallizer were used as shown in Fig. 2. Crystallizer-1 was composed of two parts, namely a crystallization cell and a side tube for introduction of a seed crystal. The crystallization cell was a screw-capped glass-cell (10 10 40 mm in inner size). The side glass tube (20 mm in length, 5 mm in inner diameter) was attached to the side of the crystallization cell horizontally at the point of 10 mm height from bottom of the cell and sealed with a rubber plug. A thin wire used for the introduction of a seed crystal was set through the rubber plug. Crystallizer-2 was composed of two screw-capped and jacketed glass-tubes of 85 mm in length and 10 mm in inner diameter, a dialysis cellulose membrane of 12,000–16,000 in molecular weight cutoff, silicon rubber sheets and a horseshoe-shaped clamp. 2.3. Crystallization of L-alanine 2.3.1. Crystallization of L-alanine using Crystallizer-1 A given amount of L-alanine was dissolved in water to prepare the solution with a supersaturation ratio of 1.25 at 20 1C. The solubility of L-alanine is 157.9 mg/mL at 20 1C. The solution was filtrated with a 0.45 mm membrane filter and incubated at 40 1C
Fig. 2. Schematic diagrams of the crystallizers: (a) Crystallizer-1: 1, laser light source; 2, laser beam (standing layer); 3, water bath; 4, glass cell (crystallization cell and side tube); 5, seed crystal; 6, seed introduction motor and 7, board. (b) Crystallizer-2: 1, crystallization glass tube; 2, water jacket; 3, silicon-rubber gasket and 4, dialysis membrane.
for 24 h to completely dissolve L-alanine and to make the initial condition of the solution same in all experiments. A washed single crystal of L-alanine with a size of about 0.5 mm was used for a seed crystal. The seed crystal was fixed to the tip of thin wire with glue and set near the rubber plug in the side tube of Crystallizer1. Three milliliters of the L-alanine solution was placed in Crystallizer-1. At this time, care was taken not to immerse the seed crystal in solution. Namely, the seed crystal was maintained in a small air space formed between the solution and the rubber plug. Crystallizer-1 was placed in a water bath made of acrylic plastic and the temperature was controlled at 20 70.03 1C. The whole apparatus was incubated in a dark dry-incubator. After 1 h, the seed crystal was introduced to the solution at a constant slow speed (60 mm/s) by remote control. To detect appearance of crystals, two lying sheet-like laser beams (5 1 mm) were irradiated to the solution from the opposite side of the side tube of Crystallizer-1, although one standing laser beam used for the another experiment (Section 2.3.3) is illustrated in Fig. 2. The diffuse reflection of laser from fine crystals appeared was monitored by CCD camera (1.5 million in pixel counts; Keyence Co. Ltd.). We checked the clear diffuse reflection of laser in advance by using polystyrene latex beads with a size of 460 nm. The image was recorded once in every second. 2.3.2. Crystallization of L-alanine using Crystallizer-2 The L-alanine solution of 189.5 mg/mL (saturation temperature: 38.9 1C; supersaturation ratio C/Cs¼1.2 at 20 1C) was preincubated at 70 1C for 12 h and then at 40 1C for 1 h. Then, the solution was placed into the compartment-I of Crystallizer-2. On the other hand, the solution with different supersaturation ratios (C/Cs¼1.0, 1.05, 1.1, 1.15, 1.2 at 20 1C) was placed to the compartment-II. Temperature of the solutions was controlled at 20 1C by circulating water to the jacket. Seed crystals were not used in this experiment. The appearance of visible crystals was recorded by CCD camera at intervals of 5 min. 2.3.3. Visualization of convection flow in Crystallizer-1 To visualize the convection flow that might be spawned in the crystallization cell of Crystallizer-1 during crystallization, a small amount of polystyrene latex beads (460 nm) was added to the
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supersaturated solution and one standing laser beam was irradiated as shown in Fig. 2. The diffuse reflection from the latex beads was monitored by CCD camera and was recorded at intervals of 1 s. The speed of latex beads moving in the supersaturated solution was determined from changes in the position of latex beads arbitrary picked up.
3. Results and discussion 3.1. Crystallization of L-alanine using Crystallizer-1 The crystallization of L-alanine was carried out by using Crystallizer-1 at a supersaturation ratio of 1.25 at 20 1C for 2 h under completely quiescent condition. In the absence of a seed crystal, nucleation did not occur even when 5 h elapsed and this result was reproducible. On the other hand, when a single seed crystal was introduced into the quiescent supersaturated solution at the side tube of Crystallizer-1, nucleation was induced and fine crystals appeared in the crystallization cell far from the seed crystal. As an example, the appearance of fine crystals in the crystallization cell within 2 h was illustrated on a digital image taken by CCD camera in Fig. 3. The seed crystal does not come into view in Fig. 3 and should be in the right side of a thick arrow. In Fig. 3, the reflected lights from fine crystals are also not shown, but the sequence of appearance and positions of 12 fine crystals during the 2 h of crystallization are illustrated with numbers and arrows, respectively. The downward-pointing arrow means that the fine crystal moved downwards. Fine crystals appeared over the wide range of cross section of the crystallization cell as shown in cases of no.5 and no.11 and always dropped downwards. Fig. 4 shows a seed crystal used for the experiment shown in Fig. 3. Panel A is the seed crystal before introducing to the solution and Panel B is after growth. During crystallization for 2 h, the seed crystal grew about 4 times in length. It means that the growth of seed crystal in the side tube and nucleation of new crystals in the crystallization cell simultaneously occurred. The same experiment was repeated 5 times and similar results were obtained as summarized in Table 1. A crystal, no.9 in Run 2 moved upwards at a 1.7 mm distance from the right wall of the cell. The number of crystals appearing within 2 h was in the range of 6–12. The data shown in Table 1 were rearranged in Table 2 to elucidate when new crystals
11 2 6
Laser beam
8 9
7
Glass cell (Crystallizer-1)
Side tube
1 12 4
Seed 10 5 3
Fig. 3. Positions at which crystals appeared in the crystallization cell of Crystallizer-1 and those sequence. The downward-pointing arrow means that the fine crystal moved downwards. A seed crystal is in the right side of a thick arrow.
Fig. 4. Seed crystal used for the experiment: (a) before introducing to the solution and (b) after growth.
Table 1 Numbering of fine crystals and appearance time. Number of fine crystals
1 2 3 4 5 6 7 8 9 10 11 12 a b
Appearance time (s) Run 1a
Run 2
Run 3
Run 4
Run 5
390 390 690 1890 3390 3720 4110 4620 5130 5310 5880 6390
130 367 2392 2721 3601 4300 4407 4523 6689b
3072 4692 5704 5817 6050 6086 6100
259 1527 5065 5111 5112
200 347 780 976 1720 2259 2475 4398 5631 6053
The experiment shown in Fig. 3. This crystal moved upwards.
Table 2 Summary of 5-times repeated crystallization of L-alanine. Range of time at which fine crystals appeared (s)
Number of fine crystals
1–1000 1000–2000 2000–3000 3000–4000 4000–5000 5000–6000 6000–7200
10 3 4 4 7 9 6a
a
One crystal moved upwards.
appeared. The frequency of nucleation seems to be even during 2 h of crystallization.
3.2. Convection of the supersaturated solution in Crystallizer-1 during crystallization As presented in Table 1, a crystal moved upwards near the right wall of the cell. This observation suggested that the convection was spawned in the crystallization cell by 6689 s. The convection should be due to the local density change of the solution caused by growth of the seed crystal placed in the side tube of the cell. The convection called into question about the experimental data shown in Table 1. Namely, the question was if fine crystals listed up in Table 1 were generated in a quiescent solution or carried from the seed crystal by convection flow.
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Consequently, the convection in the crystallization cell was examined. Polystyrene latex beads were added to the supersaturated solution to visualize the flow of the solution. In the absence of seed crystal, the latex beads did not move except for the randomdirection micromotion in the rate range of 0.3–0.6 mm/s for at least 2 h. This meant that the convection did not arise. When a seed crystal was introduced into the supersaturated solution, a small convection flow of a speed of about 2 mm/s arose near the seed crystal, but it was limited in the side tube until 1110 s elapsed after the introduction of the seed crystal. However, after 1110 s, the convection began to spread to the crystallization cell. Namely some latex beads began to move from the side tube to the crystallization cell and moved upwards along the cell wall and some latex beads began to move from the crystallization cell into the side tube at the speed of 2–4 mm/s. Then, the convection became gradually fast to about 250 mm/s. Similar results were obtained in the repeated same experiments. From these results, we concluded that the supersaturated solution in the crystallization cell had been kept in the quiescent state until the convection began to spread from the side tube to the crystallization cell and that the convection began to spread to the crystallization cell after the elapse of the time of about 1000 s from the introduction of a seed crystal. 3.3. Secondary nucleation induced by diffusion of solute molecules As presented in Table 2, 10 crystals appeared within 1000 s after introduction of a seed crystal into the supersaturated solution in the side tube. The place at which crystals appeared was in the crystallization cell far from the seed crystal (at a distance of 0.5–1.5 cm). As described in Section 3.2, there was no convection in the supersaturated solution of the crystallization cell until at least 1000 s elapsed. From these facts, we conclude that fine crystals appearing in the first term of 1000 s are not originated from the surface of seed crystal. Meanwhile, after 1000 s elapsed, the appearance of several crystals per a term of 1000 s was detected. It is not clear if these fine crystals are originated from the seed crystal or not, although the small frequency of appearance of crystals per a term of 1000 s may indicate that those are not originated from the seed crystal also. It should be noted that it is also true that all fine crystals listed in Tables 1 and 2 would not appear if a seed crystal had not been introduced at the side tube of Crystallizer-1. In order to understand this secondary nucleation in a quiescent solution, we presumed that the diffusion of solute molecules induced nucleation. The diffusion of solute molecules takes place by growth of the seed crystal. As described in Section 1, the solute molecules are forming associates in the supersaturated solution. In case of p-acetanisidide, the structure of associates, namely the interaction between molecules, was similar to that in crystal [1]. For nucleation, the structure transformation from associates to crystals is required. The factor inducing the structure transformation may be the diffusion of solute molecules. In other word, the diffusion of molecular associates may be the trigger of nucleation. 3.4. Verification of a novel mechanism of secondary nucleation L-alanine was crystallized using Crystallizer-2. By fixing the supersaturation ratio of the solution placed in the compartment-I to 1.2 and changing the supersaturation ratio of the solution placed in the compartment-II, five kinds of crystallization were carried out. The crystallization under the same condition was repeated 7 times. L-alanine molecules in the compartment-I can diffuse to the compartment-II through a dialysis cellulose membrane of 12,000–16,000 in molecular weight cutoff. The
Fig. 5. Relationship between the probabilities of nucleation in the solution placed in the compartment-I and the supersaturation ratio of the solution placed in the compartment-II.
permeation of water from the compartment-II to the compartmentI by osmotic pressure must be negligibly small because of free permeability of L-alanine molecules through the membrane and a small headspace of the compartment sealed with a screw cap. Fig. 5 presents the relationship between the probability of nucleation in the solution placed in the compartment-I and the supersaturation ratio of the solution placed in the compartmentII, where the probability was defined as a percentage of experiments in which nucleation occurred in the compartment-I among 7-times repeated experiments. All nucleation occurred in the compartment-I, and never in the compartment-II. The larger the difference in supersaturation ratio between those two solutions, the larger the probability of nucleation became. When the supersaturation ratio was 1.20 in both the compartment-I and II, no nucleation occurred even when 5 h elapsed. These results can be explained by the difference in strength of the driving force of diffusion of L-alanine. Namely, the larger the driving force of diffusion, the higher the frequency of nucleation became. This result supports the novel mechanism of secondary nucleation that the diffusion of solute molecules accompanied by growth of existing crystal induces nucleation. The molecular associates may be deformed during diffusion, resulting in rearrangement of a part of associates into the structure of nucleus. Gong et al. [8] showed that nucleation was accelerated in a colloidal system with a large nucleation volume. For instance they observed many crystals in a droplet with 500 mm in diameter, although no crystal was observed in other droplets in neighborhood. They explained this as a result of the strong interactions among multiple nuclei and the effect of diffusion enhanced in neighborhood of crystallites. Our present result strongly supports the effect of diffusion in acceleration of nucleation observed in a colloidal system. In the present work, the effect of diffusion was also found to be widening not only in the vicinity of existing crystal but also over a distance of 0.5–1.5 cm from existing crystals. The induction of nucleation by diffusion must play an important role even in primary nucleation. As described in Section 1, the induction period often observed in crystallization of organic compounds must be the time required for beginning of the structure transformation of molecular associates and the structure transformation itself must be nucleated. If so, the trigger of the structure transformation should be the diffusion of molecular associates even in crystallization under agitation. Diffusion of molecules may be an essential matter for the rearrangement of structure of associates than convective movement by agitation,
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because in the former case molecules move by themselves and in the latter case molecules are usually riding on large vortexes.
4. Conclusions The mechanism of secondary nucleation was investigated. Crystallization of L-alanine from quiescent supersaturated solution was carried out in the absence and presence of a seed crystal. The same crystallization was repeated 5 times. In the absence of a seed crystal and at the supersaturation ratio of 1.25, nucleation did not occur even after 5 h. On the other hand, in the presence of a seed crystal, several fine crystals appeared within 1000 s at the place far from the seed crystal. In the 5-times repeated crystallization, totally 10 crystals appeared during the first 1000 s term. In that time, there was no convection that might carry nuclei from the surface of seed crystal. We concluded that those 10 crystals were generated at the place far from the seed crystal and they are not directly originated from the seed crystal. As a result, we hypothesized a novel nucleation mechanism that the diffusion of solute molecules accompanied by growth of seed crystal induced the secondary nucleation. This hypothesis was verified by an experiment arranged so as to make solute molecules diffuse in supersaturated solution without seed crystal. This secondary nucleation mechanism must be working in the primary nucleation, because nucleation induced by diffusion occurred even in the absence of seed crystal.
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Acknowledgment This study was supported by Grant-in-Aid for Scientific Research (C) (no. 21560781) from the Japan Society for the Promotion of Science (JSPS).
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Please cite this article as: H. Ooshima, et al., Journal of Crystal Growth (2013), http://dx.doi.org/10.1016/j.jcrysgro.2012.12.008i