Oiling-Out Investigation and Morphology Control of Î²-Alanine Based

Article pubs.acs.org/crystal

Cite This: Cryst. Growth Des. XXXX, XXX, XXX−XXX

Oiling-Out Investigation and Morphology Control of β‑Alanine Based on Ternary Phase Diagrams Mengmeng Sun,†,‡ Shichao Du,†,‡ Mingyang Chen,†,‡ Sohrab Rohani,§ Huihui Zhang,†,‡ Ya Liu,†,‡ PanPan Sun,†,‡ Yaping Wang,†,‡ Peng Shi,†,‡ Shijie Xu,†,‡ and Junbo Gong*,†,‡ †

School of Chemical Engineering, Tianjin University, Tianjin 300072, China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300072, China § Department of Chemical and Biochemical Engineering, the University of Western Ontario, London, Ontario N6A 5B9, Canada ‡

S Supporting Information *

ABSTRACT: Crystallization is widely utilized as a purification technique in industries. Frequently in the course of crystallization, oiling-out, or termed liquid−liquid phase separation (LLPS), occurs and strongly influences the morphology of crystals. To research how to control and optimize the crystal habits when there is oiling-out, we investigated the β-alanine−water−isopropanol ternary system. Four ternary phase diagrams at 25, 30, 35, and 40 °C were constructed to provide a thermodynamic basis and guidance for the crystallization process. Moreover, to fully analyze the LLPS nucleation mechanism of β-alanine, the tie lines of the ternary diagram were constructed, and the site of nucleation was determined. Besides, on the basis of the phase diagram, evaporative crystallization was performed to investigate oiling-out crystallization. Furthermore, tabular crystals, octahedron shaped crystals, and spherical crystals were obtained respectively from normal crystallization, oiling-out crystallization, and quasi emulsion solvent diffusion crystallization.

1. INTRODUCTION The oiling-out phenomenon is well-known in protein processing,1 as well as in colloidal physics. However, in recent years, it has gained extensive attention in industrial crystallization because of its impacts on the crystal properties such as morphology of crystals, purity of product, crystal size distribution (CSD), and crystal forms. In most cases in industrial crystallization, crystals obtained from oiling-out crystallization would easily agglomerate and have higher impurity content than those obtained by normal crystallization (where there is no oiling-out).2,3 However, oiling-out phenomenon also has its own advantages. For example, crystal morphology can be controlled by oiling-out crystallization which has been reported in the literature.4−7 Janus droplets,8 which refer to multicomponent particles with different properties on opposite sides, could be fabricated by liquid− liquid phase separation resulting from evaporation and sandwich crystals of butyl paraben, which are characterized by an opaque and porous middle layer between two translucent nonporous layers, and could be prepared by controlling the volume fraction of the paraben-rich phase.9 Oiling-out is a common phenomenon in various amino acids, for example, βalanine. LLPS of β-alanine is found in numerous binary solvent mixtures. In the past few years, much attention was paid to βalanine (Figure 1) about its dissociation thermodynamics, dissolution thermodynamic property, phase transition on polymorphism, conformational behavior, nucleation, and © XXXX American Chemical Society

Figure 1. Chemical structure of β-alanine.

growth.10−22 However, there is no systematic research on the oiling-out phenomenon of β-alanine or crystal morphology studies. In the industry, crystal size and shape are important factors which can affect downstream processes and crystal properties. In the production of β-alanine, rhombohedral shapes of crystals are often obtained. However, when a second liquid appears during the crystallization process, that is, LLPS, the nucleation conditions of β-alanine change, which may influence the morphology of the products and then influence the properties of the product. Therefore, in this work, the oiling-out crystallization2,23−26 and morphology control of βalanine in water-isopropanol system were investigated. For the LLPS system of β-alanine−water−isopropanol, the ternary phase diagrams were determined at 25, 30, 35, and 40 °C. To further understand the oiling-out crystallization Received: September 13, 2017 Revised: November 25, 2017 Published: January 3, 2018 A

DOI: 10.1021/acs.cgd.7b01293 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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solution of β-alanine with different concentrations or a solvent mixture of water−isopropanol with different water mass fractions was added into a crystallizer, the temperature of which was controlled by the CF41. Then an isopropanol or aqueous solution of β-alanine with different concentrations was dropped into the crystallizer by a peristaltic pump (at the rate of 100 μL/min) with a mechanical stirring speed at 250 rpm. At first, liquid in the crystallizer was clear, and then it became cloudy with a certain mass of the second liquid being dropped into the crystallizer gradually. The changes of intensity of the transmitted laser piercing the solutions in the crystallizer were used to judge when the oil droplets occurred during the experiment, which was taken as the cloud points. Then, the dropwise addition would be stopped and the mass fractions of the three components: βalanine, water, and isopropanol were calculated based on the composition of solution in the crystallizer. All solubility data (for solid−liquid equilibrium lines) and liquid− liquid phase separation data (for liquid−liquid equilibrium lines) obtained from the above experiments were used to construct the ternary phase diagrams of β-alanine−water−isopropanol at different temperatures. 2.3.3. Determination of the Tie Lines in the Liquid−Liquid Phase Region. To determine the tie lines26 in the liquid−liquid phase region and then verify the accuracy of the ternary phase diagrams, three solutions with different overall compositions (Q1, Q2, and Q3 presented in Table 1) within the liquid−liquid phase region of the

mechanism of β-alanine, the tie lines of the phase diagram and nucleation site were determined and confirmed. It was found that nucleation in the oiling-out system took place in oil droplets and fit into the two-step nucleation theory. Besides, oiling-out crystallization induced by evaporation was performed to determine the critical binary solvent composition of the normal and oiling-out crystallization on the condition of evaporation. Furthermore, experiments on normal crystallization, oiling-out crystallization, and quasi emulsion solvent diffusion crystallization were carried out through antisolvent crystallization in order to study the morphology of β-alanine and the influence of LLPS on final crystals.

2. EXPERIMENTAL SECTION 2.1. Materials. β-Alanine, purchased from Beijing J&K Scientific Co., Ltd., Beijing, China, with a mass purity of 99.0%, was used without further purification. Anhydrous isopropanol was purchased from Tianjin Kewei Chemical Co. Ltd., China, with a mass purity of 99.0%. Deionized water was produced in our laboratory using a Millipore system. 2.2. Equipment. A cryo-compact circulator of CF41, purchased from JULABO Labortechnik GmbH, with temperature accuracy ±0.02 °C, was used to control the temperatures throughout the experiments. An optical microscope (Eclipse E200, Nikon) was applied for the morphological analysis of crystals and droplets. The scanning electron microscope (X650, HITACHI, Japan) was used as a tool to observe tridimensional morphology of the final crystals. In situ focused beam reflectance measurement (FBRM (model G400, Mettler-Toledo)) and particle video microscope (PVM (model V819, Mettler-Toledo)) were applied to monitoring the process of oiling-out crystallization. The particle size distribution was analyzed by a laser particle size analyzer of Mastersizer 3000 (British Malvern instrument co., LTD). The changes of the intensity of the transmitted laser (JD-3, Peking University Physics Department) piercing the solution were recorded to judge the oil droplets occurring during the experiments. X-ray powder diffraction (PXRD) spectra of β-alanine solids were measured by X-ray powder diffraction (type R-AXIS-RAPID, Rigaku, Japan) before and after the experiments in order to confirm that the crystal form did not change during the whole experiment. 2.3. Determination of the Ternary Phase Diagrams. 2.3.1. Determination of the Solid−Liquid Equilibrium Lines. The solubility of β-alanine at 25, 30, 35, and 40 °C was determined by a static gravimetric method as reported in the literature.27,28 Excess solid β-alanine and the corresponding binary water−isopropanol solvent mixtures (15−35 g) with different mass fractions of water were added to 50 mL flasks to obtain the suspensions. Then the suspensions were shaken at the rate of 150 rpm by a thermostatic bath shaker (CHY1015, Shanghai Sunny Hengping Scientific Instrument Co. Ltd., China) at a certain temperature under a standard uncertainty of 0.1 K for 12 h, which had been proven to be long enough to achieve solid− liquid equilibrium in our preliminary experiments. Then the suspensions were kept static at the original temperature to ensure that the undissolved solid precipitated to the bottom. Then the appropriate mass of the supernatant liquor was filtered by the precooled/heated syringes filters (0.22 μm) and moved into preweighted glass dishes as quickly as possible. Immediately, the total weight was determined. After that, the dishes were dried in a vacuum oven (DZ-2BC, Tianjin Taisite Instrument Co. Ltd., China), and their masses were periodically measured until the data remained constant, which meant that the solvent had been completely evaporated. In all of the above experiments, the masses were determined by an electronic balance (AB204-N, Mettler-Toledo, Switzerland) with an accuracy of ±0.0001 g. The experiments were repeated three times to reduce error, and the average values were reported as the final results. 2.3.2. Determination of the Liquid−Liquid Equilibrium Lines. Liquid−liquid equilibrium lines at 25, 30, 35, and 40 °C were determined by the laser monitoring method. A prepared aqueous

Table 1. Composition of the LLPS Solutions (Q1, Q2, Q3) and the Composition of the Bottom and Top Layers (g/g solution) at 25 °C (P = 0.1 MPa)a Q1 Q2 Q3 Q1-1 Q1-2 Q2-1 Q2-2 Q3-1 Q3-2

water

β-alanine

isopropanol

0.4458 0.4290 0.4488 0.5882 0.3455 0.5775 0.2775 0.5373 0.2120

0.1119 0.1433 0.2507 0.2303 0.0352 0.2804 0.0225 0.3583 0.0124

0.4423 0.4277 0.3005 0.1815 0.6193 0.1421 0.7000 0.1044 0.7756

a

Qn is the composition of the initial prepared LLPS solutions; Qn‑1 is the determined composition of the bottom layers and Qn‑2 is the determined composition of the top layers.

ternary phase diagram were prepared and stirred in a crystallizer at 25 °C. After stirring with a magnetic stirring speed at 500 rpm for 2 h, the solutions were kept static for over 5 h at 25 °C to make the solution separate into two equilibrium layers. An appropriate mass of clear liquid was taken from each layer to determine the mass fractions of βalanine in the bottom and top layers as we did in section 2.3.1. And the corresponding composition points of the two layers that are on the liquid−liquid equilibrium line were determined. Then the tie lines were plotted by connecting the initial composition points (Q1, Q2, and Q3) and the corresponding composition points of the bottom and top layers. 2.4. Determination of the Nucleation Site during Oiling-Out Crystallization. During oiling-out crystallization of β-alanine, it could be found that nucleation occurred in oil droplets as shown in the Figure 2a, and then crystals grew in oil droplets as shown in Figure 2b. To further confirm which liquid phase, the solute-rich phase or the solute-lean phase, acts as the dispersed phase (oil droplets), a cooling crystallization was carried out. In this work, a solution with the mass ratio of (β-alanine/water/isopropanol) as (4 g/4 g/4 g) was prepared at 70 °C in a crystallizer, and then transferred into a vial as quickly as possible and next the vial was sealed. The solution was allowed to selfcool under ambient conditions to the room temperature (25 °C) to observe where the nucleation occurred. B



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work, the solubility data of β-alanine in water−isopropanol solvent mixtures at temperatures ranging from 25 to 40 °C are represented in Table 3 and Figure S1. The PXRD patterns of βalanine solids of the raw materials and residual solids after stirring are shown in Figure S2, which shows that the crystal form during the entire solid−liquid equilibrium experiments did not change. It can be found that the solubility of β-alanine increases with the increasing of temperature and mass fraction of water in the binary solvent mixtures. Liquid−liquid phase separation data are represented in Table 3. The ternary phase diagrams of β-alanine−water−isopropanol system that were constructed based on all solubility data and liquid−liquid phase separation data at 25, 30, 35, and 40 °C are shown in Figure 3. There are five phase regions in the βalanine−water−isopropanol ternary system. In region 1, an isopropanol-rich liquid is saturated with the β-alanine; in region 2, β-alanine solid and two liquid phases are in equilibrium; in region 3, a water-rich liquid is saturated with the β-alanine; in region 4, two liquid phases are in equilibrium with each other, and region 5 is an unsaturated homogeneous liquid. The phase diagrams can provide useful thermodynamic information on the ternary system for crystallization. It can indicate which phase region a solution belongs to, instruct how to generate a specific supersaturation, and design the process of the crystallization of β-alanine. And it was found that the change of temperature has a little influence on the ternary phase diagrams, which reveals that cooling crystallization is not a highly efficient method for the β-alanine−water−isopropanol system when compared with evaporative crystallization and antisolvent crystallization. 3.1.2. Tie Lines in the Liquid−Liquid Region of the Ternary Phase Diagram. The compositions of the initially prepared LLPS solutions (Q1, Q2, and Q3) and the determined compositions of the bottom (Qn‑1) and top layers (Qn‑2) at 25 °C are shown in Table 1 and in Figure 4. Qn‑1 and Qn‑2 represent the two liquid layers separated from the initial solution Qn and have reached liquid−liquid phase equilibrium with each other. The dash lines Ln‑1 and Ln‑2 represent the mass fractions of β-alanine of the separated bottom and top layers, respectively, which were determined by the static gravimetric method. The intersections of the dash lines and the liquid− liquid phase separation line give the mass fractions of water and isopropanol of the two liquid layers, and then the compositions of the ternary liquids Qn‑1 and Qn‑2 can be determined. Take the solution Q1, for example: the two red dash lines L1−1 and L1−2 represent the mass fractions of β-alanine in the bottom and top layers, respectively. The intersection Q1−1 of L1−1 with the liquid−liquid phase separation line represents the composition of the bottom layer, and the intersection Q1−2 of L1−2 with the liquid−liquid phase separation line represents the composition of the top layer. The solid lines represent the tie lines which were determined by fitting to the points Qn, Qn‑1, and Qn‑2. From Figure 4, it can be found that Qn, Qn‑1, and Qn‑2 are almost at the same line, which verifies that the ternary phase diagram has high accuracy. Besides, the solute concentration of the bottom layer is much higher than that of the top layer in each LLPS solutions, which indicates that the bottom layer is the solute-rich phase and the top layer is the solute-lean phase. 3.2. The Nucleation Site during Oiling-Out Crystallization. The oiling-out phenomenon is dominated by weak interaction forces between molecules in solution:30,31 Coulombic repulsion, hard-sphere repulsion, and van der Waals attraction. From a thermodynamic perspective, the nucleation of a liquid−liquid phase separation system occurs mostly via a

Figure 2. Microphotographs of oil droplets containing crystals taken when nucleation occurs during the oiling-out crystallization. 2.5. Oiling-Out Crystallization Induced by the Change of Solvent Composition. 2.5.1. Oiling-Out Crystallization Induced by Evaporation. In this section, phase separation induced by evaporation was carried out. The macroscopically phase separation process needs several hours to complete, but the time is greatly reduced in small volume because of the large specific surface area,11 particularly in droplets. Therefore, each of the evaporation experiments in this section was carried out with a droplet of the solution. Six solutions with different compositions were prepared in sealed vials by dissolving 0.15 g of β-alanine into 12 g solvent mixtures with different water/ isopropanol mass ratios at the room temperature (25 °C). Then the solutions were stirred at 250 rpm on a magnetic plate for 2 h before the subsequent experiments. All of the six solutions (presented in Table 2) were within the liquid phase region and were clear at the

Table 2. Compositions of the Six Homogeneous Solutions Prepared for Evaporation-Induced Oiling-Out Crystallization Experiments mass fraction of homogeneous solutions (g/g solution)

mass fraction of binary solvent mixtures (g/g solvent)

sol. no.

water

β-alanine

isopropanol

water

isopropanol

Sol. Sol. Sol. Sol. Sol. Sol.

24.7 29.6 34.6 39.5 44.4 49.4

1.2 1.2 1.2 1.2 1.2 1.2

74.1 69.2 64.2 59.3 54.4 49.4

25 30 35 40 45 50

75 70 65 60 55 50

1 2 3 4 5 6

beginning. A droplet of the stirred solutions which were homogeneous was transferred immediately onto a glass microscopic slide by a syringe with a Neolus needle attached. Then the evaporation crystallization process of each droplet proceeded at the room temperature (25 °C) and was observed under an optical microscope. Each evaporation experiment of the six solutions was repeated three times. 2.5.2. Antisolvent Crystallization Experiments. In this section, four experiments based on normal crystallization, oiling-out crystallization, and quasi emulsion solvent diffusion crystallization were carried out through antisolvent crystallization in a crystallizer at 25 °C. Each experiment was observed periodically in the way of sampling solutions at different stages of the crystallization under an optical microscope. In situ FBRM and PVM were applied to analyze the process of oiling-out crystallization, and then the exact LLPS and nucleation points were confirmed by the FBRM curve and PVM images.29 The products of the four experiments were collected to conduct further characterization by SEM and Mastersizer 3000.

3. RESULTS AND DISCUSSION 3.1. Ternary Phase Diagrams of β-Alanine−Water− Isopropanol System. 3.1.1. Construction of Ternary Phase Diagrams. The ternary phase diagrams, which provide the corresponding phase regions for different ternary compositions of the β-alanine−water−isopropanol system, can be utilized to instruct the design of oiling-out crystallization process.2,9 In this C



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Table 3. Mass Fraction Solubility of β-Alanine (g solute/g solution) and Liquid−Liquid Phase Separation Data (mass fraction, g/g solution) for β-Alanine−Water−Isopropanol System at Atmospheric Pressure (P = 0.1 MPa)a 25 °C (solid−liquid equilibrium)

30 °C (solid−liquid equilibrium)

W1

W2

W3

W1

W2

W3

4.998 × 10−2 9.996 × 10−2 0.1495 0.5071 0.5224 0.5318 0.5405

3.451 × 10−4 4.488 × 10−4 3.550 × 10−3 0.4034 0.4195 0.4402 0.4595 25 °C (liquid−liquid equilibrium)

0.9497 0.8996 0.8470 8.949 × 10−2 5.805 × 10−2 2.799 × 10−2 0

4.998 × 10−2 9.986 × 10−2 0.1494 0.4945 0.5103 0.5223 0.5307


0.9496 0.8987 0.8466 8.727 × 10−2 5.650 × 10−2 2.749 × 10−2 0

W1

W2

W3

W1

W2

W3

0.5071 0.5551 0.5754 0.5928 0.5811 0.5526 0.5370 0.5041 0.4770 0.4259 0.3957 0.3710 0.3186 0.2657

0.4034 0.3331 0.2879 0.2374 0.1873 0.1442 0.1347 0.1078 0.0904 0.0632 0.0508 0.0416 0.0280 0.0211 35 °C (solid−liquid equilibrium)

0.0895 0.1118 0.1367 0.1698 0.2316 0.3032 0.3283 0.3881 0.4326 0.5109 0.5535 0.5874 0.6534 0.7132

0.5418 0.5648 0.5861 0.5887 0.5606 0.5454 0.5055 0.4710 0.4307 0.3874 0.3575 0.3240 0.2643 0.2129

0.3512 0.3108 0.2646 0.2063 0.1478 0.1364 0.1056 0.0832 0.0673 0.0467 0.0378 0.0290 0.0182 0.0156 40 °C (solid−liquid equilibrium)

0.1070 0.1244 0.1493 0.2050 0.2916 0.3182 0.3889 0.4458 0.5020 0.5659 0.6047 0.6470 0.7175 0.7715

W1

W2

W3

W1

W2

W3

4.997 × 10−2 9.985 × 10−2 0.1492 0.4756 0.4918 0.5054 0.5157


0.9494 0.8986 0.8457 8.392 × 10−2 5.465 × 10−2 2.640 × 10−2 0

4.997 × 10−2 9.982 × 10−2 0.1491 0.4627 0.4811 0.4939 0.5054


0.9494 0.8984 0.8448 8.164 × 10−2 5.346 × 10−2 2.600 × 10−2 0

W1

W2

W3

W1

W2

W3

0.5280 0.5514 0.5797 0.5946 0.5969 0.5872 0.5572 0.5181 0.4725 0.4234 0.3719 0.3186 0.2683 0.2185

0.3688 0.3331 0.2906 0.2383 0.2094 0.1769 0.1365 0.1096 0.0814 0.0571 0.0394 0.0293 0.0209 0.0195

0.1033 0.1156 0.1297 0.1671 0.1938 0.2359 0.3063 0.3723 0.4461 0.5195 0.5887 0.6521 0.7108 0.7620

0.4925 0.5437 0.5949 0.6057 0.5860 0.5336 0.5514 0.5061 0.4663 0.4235 0.3674 0.3176 0.2675 0.2197

0.4185 0.3530 0.2668 0.2123 0.1638 0.1228 0.1321 0.1018 0.0837 0.0577 0.0388 0.0282 0.0208 0.0185

0.0890 0.1033 0.1383 0.1820 0.2502 0.3436 0.3165 0.3921 0.4500 0.5188 0.5938 0.6542 0.7117 0.7618

a W1, W2, and W3 are the mass fractions of water, β-alanine, and isopropanol, respectively. The relative standard uncertainty of the solute weight measurement of solid−liquid equilibrium was estimated less than 0.09. The relative standard uncertainty of liquid−liquid equilibrium of W1, W2, and W3 were estimated less than 0.06.

two-step mechanism, in which the first step is a liquid−liquid phase separation giving rise to solute-rich droplets and over the second step, nucleation and crystal growth would proceed.32,33 To further understand the nucleation mechanism for the oiling-out crystallization of β-alanine, the nucleation site was

observed and confirmed. The result shows that the nucleation follows the two-step nucleation mechanism:34 the formation of droplets followed by nucleation within the droplets to produce crystals. Nucleation occurred in the oil droplets, i.e., dispersed liquid phase during oiling-out crystallization as shown in Figure D



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Figure 3. Ternary phase diagrams of β-alanine−water−isopropanol system at atmospheric pressure (P = 0.1 MPa): (a) at 25 °C; (b) at 30 °C; (c) at 35 °C; (d) at 40 °C. Region 1: solid−liquid phase 1; Region 2: solid−liquid−liquid phase; Region 3: solid−liquid phase 2; Region 4: liquid−liquid phase; Region 5: Liquid phase.

2. To identify which phase the dispersed oil droplets is, selfcooling crystallization was carried out.5 At the temperature of 70 °C, the solution with the mass ratio of (β-alanine/water/ isopropanol) as (4 g/4 g/4 g) existed as two liquid phases but at the temperature of 25 °C, it belonged to Region 2 (solid− liquid−liquid phase) as shown in Figure 3a. This indicated that nucleation would occur during the self-cooling process. Digital photos were taken at different times to observe the nucleation of β-alanine under oiling-out crystallization. At the beginning of the self-cooling process, the solution separated into two layers. The top layer was turbid, and the bottom layer was clear due to the change of temperature, as shown in Figure 5a. The temperature decreased with time, and then nucleation occurred in the bottom layer, i.e., solute-rich phase, which was shown in Figure 5b. After a long time, 576 min, both of the top and bottom layers were clear with crystals in the bottom layer as shown in Figure 5c. Therefore, it can be concluded that nucleation and crystal growth occur in the solute-rich phase as have been reported in the literature.5,26,35,36 And the solute-rich liquid phase acts as the dispersed phase in the ternary system during the oiling-out crystallization of β-alanine in the water− isopropanol binary solvent mixtures.

Figure 4. Tie lines (red, green, and blue solid lines) for β-alanine− water−isopropanol ternary phase diagram at 25 °C. Dash lines: mass fractions of β-alanine in the bottom and top layers. Qn‑1, Qn‑2 are the intersections of the dash lines with liquid−liquid phase separation line. E



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began, the liquid−liquid phase separation could be observed as shown in Figure 6a. Finally, crystals were obtained in the oil droplets under the evaporation of the solvent as shown in Figure 6b. Solutions from Sol. 5 to Sol. 6 experienced a normal crystallization as described in Figure 6c,d. Along with the evaporation of the solvent, there was no liquid−liquid phase separation phenomenon observed. Crystals were obtained inside the solution droplet under the evaporation of the solvent. Therefore, it can be concluded that when the mass ratios of water/isopropanol are lower than 40%, the evaporative crystallization of β-alanine experiences a liquid−liquid phase separation process. 3.3.2. Control and Optimization of Crystal Habits. In the industry of the solution crystallization process, the crystal size and shape are important factors which can affect downstream processes and crystal properties. The crystal morphology has been attracting researchers’ attention all the time.37−40 In this section, we choose antisolvent crystallization to study the crystal morphology of β-alanine because of its higher efficiency when compared with cooling crystallization. The four antisolvent crystallization experiments were carried out at the temperature of 25 °C, and the operating conditions were summarized in Table S1. For the normal crystallization (Experiment 1, point 1 → 1′ in the Figure S4), the process went through the Region 5 and turned into Region 1. At the beginning of the nucleation, oval flake crystals formed, and then another flake overlaid on the existing flake, then followed by another one. Therefore, tabular crystals were obtained at the end of the crystallization as shown in Figure 7a.

Figure 5. Digital photos of oiling-out without agitation: (a) time = 0 min; (b) time = 96 min; (c) time = 576 min.

3.3. Influence of Oiling-Out on the Crystallization Behavior and Crystal Morphology. As we have pointed out in section 3.1.1, cooling crystallization is not a highly efficient method for the β-alanine−water−isopropanol system when compared with evaporative crystallization and antisolvent crystallization. Therefore, evaporation and antisolvent operation were used to induce the oiling-out. 3.3.1. Effects of the Solvent Composition on Evaporative Crystallization. The compositions of the six prepared solutions are shown in Table 2 and represented in Figure S3 using red pentacles. All of the six solutions were homogeneous at the beginning as shown in the ternary phase diagram. Subsequently, evaporation experiments of the droplets were carried out under an optical microscope at the room temperature (25 °C). And with the evaporation operation, the six solutions appeared in different status because of the different initial positions in the ternary phase diagram. The qualitative evaporation paths are shown in Figure S3, which were marked with red dash curves. Because of the high relative volatility of the isopropanol and water, the solvent mixtures evaporated along with the red dash curves following the direction of the red arrows. Solutions from Sol. 1 to Sol. 4 experienced an oiling-out crystallization as described in Figure 6a,b. Once the evaporation of the solvent

Figure 7. SEM images of the four crystal morphology obtained by the four antisolvent crystallization: (a) Experiment 1; (b) Experiment 2; (c) Experiment 3; (d) Experiment 4.

For the oiling-out crystallization (Experiment 2, point 2 → 2′ in the Figure S4), the process went through the Region 5, Region 4, and Region 2. Before nucleation, oil droplets were formed33,5,9 as shown in Figure 8b. As the isopropanol was added into the solution, the operation point was transferred from Region 4 to Region 2, where the crystals formed in the oil droplets (Figure 8c). Octahedron shape of crystals was obtained at the end of the crystallization as shown in Figure 8d and Figure 7b.

Figure 6. Microphotographs of evaporative crystallization of the solution droplets experiencing different processes: (a, b) the evaporative crystallization of Sol. 1 which experiences oiling-out crystallization; (c, d) the evaporative crystallization of Sol. 5 which experiences normal crystallization. F



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Figure 8. Two steps nucleation process of oiling-out crystallization.

In experiments 3 and 4, the aqueous solution of the β-alanine was added into the poor solvent isopropanol which had been put into the crystallizer and kept at 25 °C. The mechanical stirring speed was kept at 400 rpm throughout the experiment. As the aqueous solution was added into the crystallizer, the composition of the solution changed along with the blue dash line following the direction of the blue arrow. Spherical crystals were obtained at the end of the crystallization as shown in Figure 7c. This method of producing spherical crystals was reported as the quasi emulsion solvent diffusion method, one of the four main methods to obtain spherical crystals.41−44 Since the interaction between the β-alanine and the good solvent (water) is stronger than that between the good (water) and poor (isopropanol) solvents, the solution containing water and β-alanine was dispersed in the isopropanol solvent under agitation, producing quasi emulsion droplets as shown in Figure 10a. Then the counter diffusion of water solvent and isopropanol solvent induced the nucleation of β-alanine. Residual water acted as bridging liquid to agglomerate the generated β-alanine crystals as shown in Figure 10b. Finally, spherical crystals were obtained as shown in Figure 10c. Many factors can influence the process of the spherical crystallization such as the mode and intensity of agitation, the temperature, the pH of the solution, and the mass ratio of the good to poor solvent.42,44 The operation of experiment 4 was the same as experiment 3 except for the additional amount of the aqueous solution of βalanine. The process went through Region 1 and Region 2. As the aqueous solution of β-alanine was added into the poor solvent isopropanol, the amount of the spherical crystals increased, which resulted in low dispersion property and agglomeration. Then under the action of LLPS, collision, and agitation, the crystals would break (Figure 7d) when compared with the crystals obtained by Experiment 3. The crystal size distribution (CSD) of the four products obtained by the four experiments was analyzed by Mastersizer 3000. And the results are depicted in Figure 11. Panels a−d are the CSD of the normal crystallization product (Experiment 1), the oiling-out crystallization product (Experiment 2), the products of the Experiment 3 and Experiment 4, respectively.

To further understand the oiling-out crystallization induced by the change of the solution composition as shown in the Experiment 2, in situ FBRM and PVM were applied to analyze the process. The solution of water and β-alanine (W1: W2 = 30 g: 10 g) was prepared and dissolved in a 200 mL crystallizer at 25 °C. Then isopropanol (100 g) was added into the crystallizer at the rate of 1.5 mL/min by a peristaltic pump, and at the same time the FBRM and PVM started to monitor the dilution process, the LLPS phenomenon and the nucleation occurred. The FBRM curve and the PVM images are shown in Figure 9. In the case of liquid−liquid phase separation, the

Figure 9. FBRM curve and PVM images of the oiling-out crystallization.

particle number increased slowly because of the formation of oil droplets, whereas in the case of nucleation, the response of the FBRM increased rapidly due to the nucleation of β-alanine and the breakup of oil droplets. The corresponding response locations were marked in Figure 9. It can be found from the PVM images that at the beginning of the liquid−liquid separation, the oil droplets were few and small, and with the dropping of the isopropanol, the oil droplets increased in number and became larger in size. Finally, crystals of β-alanine formed.

Figure 10. (a−c) Three steps involved in quasi emulsion solvent diffusion. G



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Figure 11. CSD (measured by Mastersizer 3000) of the products obtained by the four antisolvent experiments: (a) the CSD of the normal crystallization product (Experiment 1); (b) the CSD of the oiling-out crystallization product (Experiment 2); (c) the CSD of the Experiment 3 product; (d) the CSD of the Experiment 4 product.

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*Tel.: 86-22-27405754. Fax: 86-22-27314971. E-mail: junbo_ [email protected]. ORCID

Shichao Du: 0000-0002-8369-2983 Sohrab Rohani: 0000-0002-1667-1736 Junbo Gong: 0000-0002-3376-3296

It can be found that products of Experiments 1−3 have a good unimodal CSD, while a bimodal CSD of the Experiment 4 has been obtained because the spherical crystals agglomerated and broke at the end of the crystallization. Crystals at the end of the antisolvent crystallization were investigated with the SXRD and PXRD, the result of which is depicted in Figure S5. It can be concluded that the polymorphic forms did not change over the experiments. From the above results, we can see that normal crystallization, oiling-out crystallization, and quasi emulsion solvent diffusion crystallization can produce different habits of crystals. Thus, based on the ternary phase diagram, the crystal morphology of β-alanine can be controlled well. The crystal morphology would affect the bulk density, mobility and other macroscopic properties. We can also notice that the oiling-out crystallization can produce larger crystals than other crystallization methods. It may be because that in the oiling-out crystallization, the nucleation occurs in the oil droplets which resulted in less nucleation density and larger crystals than in other crystallization methods.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the financial support of National Natural Science Funds for Innovation Research Groups (21621004), National Natural Science Foundation of China (NNSFC 81361140344, NNSFC 21376164 and NNSFC 21676179), National 863 Program (2015AA021002), Major Project of Tianjin (15JCZDJC33200), and Major National Scientific Instrument Development Project (No. 21527812).

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REFERENCES

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4. CONCLUSION In this work, ternary phase diagrams of the β-alanine−water− isopropanol system were constructed at four different temperatures. Crystallization of β-alanine in different regions in the ternary phase diagram was explored. It was found that nucleation of oiling-out crystallization of β-alanine follows the two-step mechanism: the formation of oil droplets and then the nucleation of β-alanine in the oil droplets. Evaporation can induce the oiling-out crystallization when the mass ratio of the binary solvent water/isopropanol is under 40%. Furthermore, the ternary phase diagram was found playing a critical role in controlling and optimizing the crystal habits of β-alanine. The normal crystallization, oiling-out crystallization, and quasi emulsion solvent diffusion crystallization can produce different habits of crystals which have different CSDs and other different properties.

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Mass fraction solubility of β-alanine in water−isopropanol solvent mixtures (Figure S1); comparison of the PXRD pattern of raw material and the residual solid of βalanine after stirring for 12 h (Figure S2); initial positions of the six homogeneous solutions in the evaporation experiments and the schematic diagram of the evaporation processes (Figure S3); operating lines and conditions of the four antisolvent crystallization (Figure S4 and Table S1) including normal crystallization, oilingout crystallization, quasi emulsion solvent crystallization; the PXRD patterns of the raw material, and the four products obtained from the antisolvent crystallization (Figure S5) (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.7b01293. H



Article

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Oiling-Out Investigation and Morphology Control of Î²-Alanine Based

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