Article pubs.acs.org/crystal
Crystal Growth of Aspirin Using a Temperature-Controlled Microfluidic Device Takahito Tokuhisa,†,∥ Masashi Kawasaki,§,∥ David Kisailus,‡ Masamichi Yuda,§ Tadashi Matsunaga,† and Atsushi Arakaki*,† †
Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan § Analysis & Pharmacokinetics Research Labs, Department of Drug Discovery, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan ‡ Department of Chemical and Environmental Engineering, University of California, Riverside, 900 University Avenue, California 92521, United States S Supporting Information *
ABSTRACT: Identifying the most appropriate polymorph of active pharmaceutical ingredients is one of the important steps in drug development, since their bioactivities are largely dependent on their solid forms. However, the sample preparation for the characterization of crystal forms is time-consuming and requires large quantities of sample. Here, we introduce a microfluidic device-based method to prepare a sub-millimeter-sized single aspirin crystal from a small quantity of material. For the crystal preparation, a device equipped with a solution flow system and temperature controller was placed under the microscope. To use the device, concentration−temperature phase diagrams were generated, and regions where dominant nucleation or crystal growth with specific directions were clearly determined. By observing time-dependent changes of crystal number and size with solution temperature, a pathway to grow a single crystal of aspirin was determined and applied to prepare a submillimeter-sized crystal from 250 μg of aspirin. The obtained crystal was sufficiently large for single-crystal X-ray diffraction analysis, which usually requires 10 mg to 1 g of material per crystallization experiment. Thus, this method can be adapted as an efficient approach to uncovering the crystallization process to obtain required crystal forms with minimal sample consumption.
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mechanical properties (e.g., hardness, tableting, flow ability).5,6 Here, it is critical to assess the aforementioned parameters in a timely manner due to the highly competitive intellectual property arena as well as providing quick insight into new opportunities for “life-cycle management of the product”.4 During the analysis of selected crystal forms in scale-up experiments, powder X-ray diffraction (XRD) is generally used with the aid of computational predictions to determine the full structure of the formed crystals.3,5 However, the process is complicated and sometimes unsuccessful for novel or uncommon compounds. In contrast, single-crystal XRD analysis can directly determine the full crystal structure with high accuracy.7 Although single-crystal XRD analysis is useful, crystal preparation is time-consuming and usually requires a large amount of sample (i.e., 10 mg to 1 g) for identification of crystallization conditions.7,8 Thus, providing a platform to carefully examine crystallization variables, which yield suffi-
INTRODUCTION Solid form selection of active pharmaceutical ingredients (APIs) is an important issue in drug development since the bioactivities of APIs are largely dependent on the crystal polymorph or form, size, and morphology.1,2 The selection of solid form often determines the fate of commercial viability of pharmaceutical products.2 The primary mode of crystal screening is conducted via a high-throughput workflow system, which enables automation of the entire crystallization procedure.3 In order to select the best crystal form with desired physicochemical properties for drug development, a number of crystallization conditions on the basis of solvent, salt and substrate concentrations, pH, and temperature have been investigated.4 Crystal forms found using a high-throughput workflow system are then re-examined through multistep scaleup experiments in order to evaluate reproducibility and characterize specific physicochemical parameters, such as packing arrangement (e.g., molar volume, hygroscopicity), thermodynamic properties (e.g., melting point, entropy, enthalpy, solubility, vapor pressure, free energy), kinetic attributes (e.g., dissolution rate, reaction rates, stability), surface features (e.g., surface free energy, crystal habit, wettability), and © XXXX American Chemical Society
Received: June 10, 2015 Revised: July 14, 2015
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DOI: 10.1021/acs.cgd.5b00805 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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ciently large single crystalline materials for single-crystal XRD analysis while utilizing minimal material, would be very useful. A microfluidic device is a suitable tool to handle and analyze small quantities of aqueous samples. They are often integrated with functionalities such as pumping, valve separation, and heating mechanisms that allow reduction in sample consumption, provide accurate reaction conditions, and yield automated systems.9 The devices are thus widely used for various biotechnological and chemical applications, including cell separations,10 chemical gradient generators,11 and enzymatic reactors,12 all at small scales. In addition, microfluidic devices have been applied to the crystallization studies of colloidal particles,13 proteins,14 inorganic substances,15 and APIs.6,16 High-throughput crystal screening of these materials have been proposed using these devices. Another benefit to using microfluidic devices is a real-time monitoring of the changes in a targeted object under microscopic view.17 The method enables monitoring of time-dependent changes in living cells at single-cell levels in a quantitative manner18 and can be applied for quantitative evaluation of crystal formation at a single-crystal levels. The microfluidic technique could therefore be a useful approach for the systematic analysis and manipulation of crystallization processes for drug development. Here, we report a microfluidic device-based method to analyze crystallization processes from small quantities of APIs under an optical microscope. Aspirin, an antiphlogistic analgetic, was used as a model compound for this study since the crystal shapes and surfaces have been previously wellcharacterized.19−25 A concentration−temperature phase diagram of aspirin was quantitatively evaluated by observing crystal formation with the aid of a temperature-controlled microfluidic device. The analysis allows us to predict crystallization processes to obtain crystals with desired physicochemical properties. By referring to these analyses, the method was further applied for the preparation of a large single crystal of aspirin, enabling single-crystal XRD analysis.
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EXPERIMENTAL SECTION
Materials. Aspirin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Analytical reagent grade ethanol and heptane were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ultrapure water was acquired from Milli-Q (Millipore, Inc., Bedford, MA). SU-8 sheet (XP film TRIAL-50) and SU-8 developer were purchased from MicroChem Corp., MA, USA. Silicone oil (KF965) was obtained from Shin-Etsu Chemical Co., Ltd., Tokyo, Japan. Fabrication of a Microfluidic Device. A microfluidic device consisting of eight crystallization chambers (8000 μm long × 300 μm wide × 50 μm high) with channels (150 μm wide × 10 μm high) all connected to an inlet and an outlet was designed (Figure 1A). The crystallization chambers were placed within a 4 mm × 8 mm area of the device. A master mold for the microfluidic device was manufactured using a soft-lithography method.26 Briefly, SU-8 photoresist was attached to a glass wafer (with no air gaps) and was subsequently exposed to UV light through a photomask printed at 3600 dpi to create the microfluidic device. The wafers were prebaked to solidify the photoresist, exposed to UV light, and postbaked to selectively cross-link the exposed areas of the photoresist layer. Residual nonlinked resist was removed by immersion in the SU-8 developer. An upper polydimethylsiloxane (PDMS) layer was fabricated by pouring a mixture of Sylgard 184 silicone elastomer (Dow Corning Asia Ltd., Tokyo, Japan) and curing agent (10:1) onto the master mold, followed by curing for at least 20 min at 85 °C. Upon curing, the PDMS substrate was carefully peeled off the mold. Through-holes were drilled at the locations of the inlet and outlet in the PDMS layer.
Figure 1. Overview of the experimental setup of the microfluidic device used in this study. Top view of the channels (50 μm × 50 μm, width × height) and the eight crystallization chambers (8000 μm × 300 μm, length × width) in a microfluidic device (A). Side view of channels (10 μm, height) and crystallization chambers (50 μm, height) (B). The microfluidic device was placed in a temperature reservoir equipped with a Peltier module (C). The reservoir was placed on the microscope stage enabling real-time observation of crystal formation in the selected area. Silicone tubes were connected to the inlet and outlet of the device. Picture of the microfluidic device (D). The PDMS layer was then integrated onto a glass slide by activating the surfaces with plasma treatment (Basic Plasma Cleaner PDC-32G; Harrick Plasma, New York, USA) to create an irreversible bond between the PDMS layer and the glass slide. The microfluidic device contained eight crystallization chambers where aspirin crystallization was analyzed. The chambers were branched from an inlet and integrated to an outlet (Figure 1B). The volume of each crystallization chamber was calculated to be approximately 120 nL. Aspirin Crystallization Using the Temperature-Controlled Microfluidic Device. The microfluidic device was placed in the temperature reservoir equipped with a Peltier module (Figure 1C, B
DOI: 10.1021/acs.cgd.5b00805 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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Figure 2. Polarized-light microscopic images of aspirin crystals formed under various temperature and initial aspirin concentration conditions in ethanol. The crystals were formed in the crystallization chambers at 0 min (A), 30 min (B), 60 min (C), and 90 min (D). The images were taken at the same area for each concentration−temperature condition. Scale bars are 50 μm. Japan High Tech, Fukuoka, Japan). The reservoir was mounted on the stage of the microscope. The temperature of the aqueous solution in the crystallization chamber of the microfluidic device was measured using a thin thermocouple probe (Testo735; Testo K. K., Kanagawa, Japan). Aspirin solutions were prepared by dissolving aspirin powders in water, ethanol, or heptane at 60 °C in a water bath. Approximately 120 nanoliters of aspirin solution were loaded into the crystallization chamber using a syringe pump (KDS 200 Two-Syringe Infusion Pump; Muromachi Kikai Co., Ltd., Tokyo, Japan) equipped with a 10 μL syringe (Hamilton Company USA, Nevada, USA) through a silicon tube connected to an inlet port. The flow rate of the syringe pump was 1 μL/min, and the sample consumption was approximately 1 μL/trial. Inlet and outlet ports of the microfluidic device were sealed with silicon oil to prevent evaporation of aspirin solutions. The temperature of the device was initially held at 60 °C for 10 min and then cooled to the desired crystallization temperature with a temperature gradient of 20 °C/min. Images of crystals in the crystallization chambers were obtained using polarization microscopy (BX51; Olympus Corporation, Tokyo, Japan) with a 10× objective lens, a computer-controllable motorized stage, and a cooled digital camera (DP-70, Olympus Corporation). Lumina Vision acquisition software (Mitani Corporation, Tokyo, Japan) was used to acquire the images and movies. A phase diagram analysis was performed using aspirin dissolved in ethanol at initial concentrations of 50, 100, 150, 200, and 250 mg/mL. Crystallization experiments were performed at temperatures of 0, 10, 20, 30, 40, 50, and 60 °C. Optical micrographs of crystals in the crystallization chambers were obtained after incubation for 0, 30, and 60 min (from the point of temperature equilibration) using a polarization microscope. Crystal modification over time was analyzed by periodically observing microchambers with a microscope equipped with a CCD camera. To investigate the aspirin crystal growth, the size
and shape changes of individual crystals were evaluated every 10 min. Crystal growth rates of each crystallization condition were calculated from the measured size changes of the crystals. Preparation of a Large Single-Crystal of Aspirin for X-ray Diffraction Analysis. A large aspirin crystal (crystal size > 200 μm × 100 μm) was formed using the temperature-controlled microfluidic device. An aspirin solution (250 mg/mL in ethanol) was flowed through the device at 1 μL/min. The chamber temperature, initially held at 60 °C, was cooled to 10 °C at −20 °C/min, and then held for 5 min to induce nucleation. The temperature was then raised to 30 °C at +2 °C/min, and held for 60 min in order to enable growth. Newly formed crystals were extracted from the chambers using a CryoLoop (Hampton Research, Aliso Viejo, CA), flash-frozen in liquid nitrogen, and subsequently used for XRD analysis. XRD diffraction patterns were collected at 293 K with a 5 s exposure and 1.0° oscillation using an XtaLAB P200 diffractometer (Rigaku Corp., Tokyo, Japan) with multilayer mirror monochromated Cu-Ka radiation (λ = 1.54184 Å).
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RESULTS AND DISCUSSION
Aspirin Crystal Formation in the TemperatureControlled Microfluidic Device. Prior to the crystal formation studies, the accuracy of the temperature control for this device system was verified. The temperature was automatically controlled by a Peltier module set at the desired temperature and heating/cooling rates. Solution temperatures within the device were checked at different regions along the microchannels by inserting a thin thermocouple probe into the microfluidic device. The differences between the preset and measured temperatures were within ±1.5 °C (Figure S1). C
DOI: 10.1021/acs.cgd.5b00805 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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Aspirin crystallization in the microfluidic device was investigated with various solvents at a crystallization temperature of 30 °C. The method enabled direct enumeration of individual crystals under the microscope. In order to analyze the entire area of the microchamber, we selected a 10× objective lens (although a higher resolution crystal analysis can be achieved using a higher magnification lens). The smallest detectable crystal size was approximately 5 μm × 5 μm when the lens was used. Thus, crystals larger than this size were evaluated in this study. When water was used as a solvent, thin plate- or leaflet-like crystals approximately 25−166 μm (average length) were obtained (Figure S2A). For ethanol and heptane solvents, either square-shaped plates measuring 89−232 μm (Figure S2B) or elongated needle-like crystals (55−163 μm along their long axis, Figure S2C) were formed, respectively. These results were in good agreement with previous bulk crystallization studies using milliliter-volume containers,25 suggesting the reduction to nanoliter-volume did not affect crystal morphology. Since crystals having a well-defined morphology with clear surfaces and edges were formed in ethanol, it was selected as the solvent for the following investigations. On-Chip Phase Diagram Analysis of Aspirin. Aspirin has been reported to have several crystal habits including square-shaped plates and elongated needle-like crystals with {001} and {100} dominating faces when ethanol was used as solvent.25 Although the crystal habits and the surface structures of aspirin have been well-characterized,20,21,25 its nucleation and growth behavior, with the exception of work on dissolution behavior in solutions,21−23 is poorly understood. In this study, we demonstrated aspirin crystallization using the microfluidic device under 35 different crystallization conditions at various temperatures (0−60 °C) and aspirin concentrations (50−250 mg/mL) in ethanol. Crystals formed in the crystallization chambers were recorded at four time points (0, 30, 60, and 90 min) (Figure 2A−D). On the basis of the time-course observations, changes in crystal number and size were observed mainly within 30 min, yet remained constant at 60 and 90 min for all examined aspirin concentrations. This result suggests crystal nucleation and growth occurred primarily within the first 30 min. After 30 min, aspirin in solution was depleted, and thus no significant changes in crystal number and size were observed. Crystal formation, which generated crystals greater than 5 μm × 5 μm, was observed in 15 of the 35 conditions by 90 min. Plate-like morphologies were observed in 3 of the 15 chambers, whereas the other 12 exhibited needle-like structures. Typical examples of plate-like and needle-like crystal formation processes are presented in supplementary movies (Movies S1 and S2). Larger aspirin crystals (>200 μm × 100 μm) were obtained using higher initial concentrations (i.e., from 200−250 mg/mL). No visible crystals were formed below 100 mg/mL or when the crystallization temperature was higher than 50 °C, suggesting that these conditions were considered to be within unsaturated regions of ethanolic solutions of aspirin. A two-dimensional concentration−temperature phase diagram depicts the result of crystallization tests at 60 min (Figure 3), since no significant changes in crystal size were observed beyond this time. The concentration−temperature conditions forming visible crystals under microscopic observation were identified as a labile region where nucleation events predominantly occur. A supersolubility curve was then drawn as a boundary between the crystal forming (aspirin
Figure 3. Concentration−temperature phase diagram of aspirin obtained from the analysis of the temperature-controlled microfluidic device. Supersolubility and solubility curves were drawn based on the results from the crystallization condition at 90 °C using ethanol as a solvent. The gray curves with arrow heads represent a predicted route for aspirin crystal formation at the aspirin concentration of 250 mg/ mL under each temperature condition. Black solid line: supersolubility curve, dashed black line: solubility curve, and gray solid line: predicted crystal formation route.
concentration 150 mg/mL, 0−40 °C) and nonforming regions (aspirin concentration 100 mg/mL, 0−40 °C) (Figure 3, solid line). We also tested an aspirin concentration of 125 mg/mL. Since the crystal formation was determined at 0 °C, whereas no crystal was observed at 10 °C, the supersolubility curve was set between 100 and 250 mg/mL. A solubility curve (Figure 3, dotted line) was constructed by dissolving crystals by heating to 90 °C at 1 °C/min increments. The temperatures at which the crystals in the microchamber became invisible (under microscopic observation) were subsequently plotted. Crystals formed at the initial aspirin concentration of 150 mg/mL dissolved at 66 °C, whereas those obtained using aspirin concentrations of 200 and 250 mg/mL dissolved at 75 and 82 °C, respectively. The supersolubility and solubility curves separate the diagram into three regions, including a labile region where the nucleation occurs, a metastable region where the crystal growth is predominant, and an unsaturated region where both crystal nucleation and growth are inhibited. A predicted route for aspirin crystal formation at the initial aspirin concentration of 250 mg/mL was described in the phase diagram (gray line, Figure 3). Phase diagrams are often generated using microtiter plates or other several micro- to milliliter volume containers.4 On the other hand, the microfluidic device-based method of this study consumed approximately 1 μL (120 nL per chamber ×8), which corresponds to 6−30 μg. This small sample volume reduces the sample consumption by approximately 10−100 times less than traditional crystallization tests.4 In addition, this volume size enabled accurate and quick temperature control of the fluid, which provided reproducible results in crystal size, morphology, and number for each crystallization test. Although our method is practical with limited amounts of APIs, it will not be applicable for the crystallization experiments involving salting-out and pH changes.4 Quantitative Evaluation of Crystal Number and Size. In order to verify the obtained phase diagram, crystal size and number in a single crystallization chamber were quantitatively evaluated (Table 1). The analysis was performed for an initial aspirin concentration of 250 mg/mL at crystallization times of 0, 5, and 30 min. No visible crystals were observed at 0 min (Figure 2A) for all examined temperature conditions. It should D
DOI: 10.1021/acs.cgd.5b00805 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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Table 1. Quantitative Evaluation of Aspirin Crystals Formed in the Microchambersa crystal number
major axis (μm)
minor axis (μm)
aspect ratio
temperature (°C)
T=0 min
T=5 min
T = 30 min
T=0 min
T=5 min
T = 30 min
T=0 min
T=5 min
T = 30 min
T=0 min
T=5 min
T = 30 min
60 50 40 30 20 10 0
0 0 0 0 0 0 0
0 0 61 43 13 0 0
0 0 92 44 16 30 78
− − − − − − −
− − 482 1168 765 − −
− − 455 1276 847 1013 766
− − − − − − −
− − 41 21 114 − −
− − 28 27 128 57 22
− − − − − − −
− − 11.8 55.6 6.7 − −
− − 16.3 47.3 6.6 17.8 34.8
a
Initial aspirin concentration was 250 mg/mL. Crystal numbers and sizes were evaluated at crystallization times of 0, 5, and 30 min. The values were indicated as the average of five microchambers. −: Not detected.
dissolution rate from specific crystal surfaces in solution, were considered to be involved in the definition of crystal habits. At lower temperatures (e.g., 0 and 10 °C), diffusion of aspirin molecules was relatively low, and the molecules were likely incorporated onto the crystal surfaces that have higher surface energies. Specific surface energies of the {110} and {011} faces of aspirin in solution were calculated to have higher surface energies than {100} faces,23 and thus the crystal growth along the and directions resulted in the formation of elongated needle-like crystals. In contrast, under higher temperature conditions (i.e., 20 °C, 30 °C, and 40 °C), diffusion of aspirin molecules in solution increased. This facilitated the incorporation of aspirin molecules onto the long and short crystallographic axes, allowing the formation of platelike crystals.24,25 This was likely due to a reduction in the magnitudes of crystal facet growth velocities. However, the higher growth temperature also resulted in dissolution of aspirin crystals. Dissolution rates depend on the extent of exposure of different crystal planes.21 Apparent dissolution of aspirin crystals was observed in our real-time observation over 40 °C. The observed crystal habits were defined by the magnitudes of substrate diffusion and dissolution of crystals under specific temperatures as well as the substrate concentrations. Thus, the crystal growth direction in aspirin was sensitive to the crystallization temperature. The crystal habit of pharmaceuticals (and other products) affects their biocompatibility and manufacturability.4 We demonstrated the ability to modulate and identify trends in crystal habit using our method. On-Chip Crystal Growth Control of Aspirin Using a Temperature-Controlled Microfluidic Device. In order to form a sub-micrometer-sized aspirin crystal, which could be used for conventional XRD analysis, we estimated an appropriate formation route from the obtained temperature− concentration phase diagram (Figure 3) and quantitative crystal data (Table 1). We selected temperature and concentration conditions for nucleation (i.e., to produce fewer crystals) and crystal growth (sufficient nutrient for growth) in order to produce a sub-micrometer-sized aspirin crystal. Thus, the primary microchamber temperature was set at 60 °C and decreased to 10 °C at a rate of 20 °C/min to induce nucleation. Using a fast cooling process of −20 °C/min, the initial solution condition in the metastable region immediately shifted to the labile region. The condition change primarily facilitated nucleation events without visible crystal growth. After a 5 min incubation time at 10 °C, the microchamber temperature was then increased to 20 °C, 30 °C, or 40 °C at a rate of 2 °C/ min for crystal growth. As a control, the temperature was held
be noted that crystal nucleation may have occurred during the temperature change from 60 to 10 °C, but nuclei would be too small to observe by optical microscopy. Crystal formation was confirmed after 5 min at temperatures of 20 °C, 30 °C, and 40 °C. Since nucleation of crystals occurred, the aspirin concentration in solution decreased, shifting the solution conditions from the metastable region to labile region (Figure 3). In contrast, visible crystals were not observed at 0 and 10 °C, where the solution should be within the labile region, and thus, sluggish kinetics prevented significant growth (i.e., only nuclei are present, Figure 3). The highest number of crystals were obtained at 40 °C (after 5 min), while the largest crystal sizes were obtained at 30 °C (Table 1). However, at 30 min, slight changes in the crystal number and size at these conditions were observed. For example, at 40 °C, the crystal size decreased but the crystal number increased. This suggests an occurrence of simultaneous crystal nucleation and formation between 5− 30 min (at this temperature condition), and thus, this specific condition may be within the boundary between labile and metastable regions (Figure 3). Crystal formation was also observed at lower temperatures (i.e., 0 and 10 °C) at 30 min (Table 1). A relatively higher number of crystals were formed at 0 and 40 °C. In addition, the crystal aspect ratios changed at different crystallization temperatures. Needle-shaped crystals with aspect ratios between approximately 6 and 60 were formed at crystallization temperatures of 0 °C, 10 °C, 30 °C, and 40 °C (Table 1). The largest average major axis (approximately 1300 μm) was observed when the crystallization temperature was set at 30 °C, and the highest average aspect ratio crystals (ca. 55.6) were obtained under this condition. Rectangular-shaped crystals with an aspect ratio less than 10 (i.e., ca. 6.6, Table 1) were exclusively obtained at the crystallization temperature of 20 °C. The fewest crystal numbers (16 crystals/microchamber) were detected at this condition. Here, it is likely that the growth rate of crystals along the short axis (i.e., perpendicular to the long axis) where {110} and {011} crystal faces dominate was highest at this temperature.19,25 At lower temperatures, growth of crystals was diffusion limited, while at temperatures above 20 °C, the solubility increased, yielding larger aspect ratio crystals (Table 1), but with an overall lower mass yield due to the higher solubility. Similar tendencies were also observed at aspirin concentrations of 200 mg/mL (Table S1). Varieties in aspirin crystal habits were observed when different solvents were used for crystallization, although the formation mechanisms have yet to be thoroughly described.19,25 The quantitative crystal evaluation of this study showed that the crystal habits of aspirin were also highly dependent on the crystallization temperature. Two factors, substrate diffusion and E
DOI: 10.1021/acs.cgd.5b00805 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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at 10 °C for the same duration. Consequently, needle-shaped crystals were observed in Figure 2C,D. When the temperature was increased to 30 °C, a small crystal appeared at 5 min and grew to approximately 150 μm × 400 μm after 30 min (Figure 4), whereas at 20 and 40 °C, rectangular-shaped crystals with
suggested formation of single-crystalline particles (based on measured lattice parameters showing good agreement with reference values, Figure 5).
Figure 5. X-ray diffraction pattern of a large aspirin crystal grown by using the temperature controlled microfluidic device.
Sample preparation for single-crystal XRD usually utilizes 10 mg to 1 g of material per crystallization experiment.7,8 Powder XRD also requires similar sample amounts for preparation.4 On the other hand, the crystal preparation of this study consumed approximately 250 μg of aspirin per experiment, approximately 100−1000 times less than previous methods. In early phases of drug development, the amount of API available for the crystal characterization is usually limited.4 However, to use the microfluidic device-based method, structural information by single-crystal XRD can be obtained in the early stages of drug development after the crystal screening. This will shorten the time scale of drug development and may change the development strategy.
Figure 4. Polarized-light microscopic images of sub-micrometer-sized aspirin crystals formed within the microfluidic device (A). Schematic plot describing the time−temperature changes within crystallization chambers of the microfluidic device juxtaposed with representative images of aspirin crystals at different stages (B). The temperature was held at 60 °C for 5 min and changed to 10 °C by cooling at −20 °C/ min. After 5 min incubation time at 10 °C, the temperature was increased to 30 °C at a rate of 2 °C/min and held for 30 min. Ethanol was used as a solvent for the crystal growth. Scale bars are 50 μm.
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relatively smaller sizes were obtained. At a hold temperature of 20 °C, a crystal appeared after 10 min and grew to approximately 100 μm × 200 μm after 30 min (Figure S3), while at a hold temperature of 40 °C, a significantly larger crystal (i.e., 200 μm × 300 μm) grew after only 20 min (Figure S4). The polarized-light images suggested that the large crystals were indeed single-crystalline aspirin. However, after 30 min at 40 °C, the crystal was reduced in size (ca. 100 μm × 300 μm) due to dissolution. Therefore, 30 °C was chosen to be a suitable condition for larger crystal growth. The heating rate between the first and second anneal was further investigated at rates of 2 °C/min and 20 °C/min. Using heating rates of 20 °C/min, many (>100) visible crystals appeared during the temperature change (Figure S5), whereas few crystals (
