Langmuir 2006, 22, 6477-6480
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Screening of C60 Crystallization Using a Microfluidic System Kyosuke Shinohara, Takeshi Fukui, Hiroaki Abe, Naoto Sekimura, and Koji Okamoto* Department of Quantum Engineering and Systems Science and Department of Nuclear Engineering and Management, UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ReceiVed May 19, 2006 We have carried out screening of C60 crystallization using a simple liquid/liquid interfacial precipitation method in a microfluidic device. By controlling the time, temperature, and concentration, various metastable phases of C60 crystals were found, including tubes, spheres, open-ended hollow columns, stars, branches, and trees. The obtained C60 crystal shapes are similar to those of snow crystals. These findings suggest an urgent need to screen C60 crystallization for the development of fullerene C60 drugs.
Introduction Since their initial discovery in 1985,1 fullerene C60’s have attracted significant attention for their unique physical and chemical properties and have resulted in the creation of a new research field.2 Fullerene C60 is also an excellent candidate for new drugs.3-6 Because the metastable crystals have different solubility and bioavailability from stable crystals, screening metastable or stable phases of crystals is critical to the development of new drugs.7 In terms of protein crystals, efficient screening techniques using microfluidic chips have already been presented.8,9 However, on fullerene C60, any polymorph and screening technique findings for crystals have not been reported. Here, we report on the screening of metastable phases of C60 crystals, including tubes, spheres, trees, branches, hollow-ended columns, and multiple pods using a microfluidic system. Our screening method utilized an organic/alcohol interface in a Y-shaped microchannel. Such a microfluidic device allows for the control of space, time, temperature, and concentration during the growth and precipitation of C60 crystals. This screening method is based on the liquid/liquid interfacial precipitation (LLIP) method, which was recently reported for the synthesis of needlelike C60 nanowhiskers (C60 NWs).10,11 One of our major motivations for this study is the idea that because microfluidic devices have a very large interfacial area per unit volume and extremely short diffusion distances12 we could confirm new metastable phases of C60 crystals, which cannot be observed in flask experiments because of very short lifetimes following Ostwald’s rule of step. * Corresponding author. E-mail:
[email protected]. Tel: +814-7136-4597. Fax: +81-4-7136-4598. (1) Kroto, H. W.; Heath, J. R.; O’Brien, S. C.; Curl, R. F.; Smalley, R. E. Nature 1985, 318, 162. (2) Hebard, A. F.; Rosseinsky, M. J.; Haddon, R. C.; Murphy, D. W.; Glarum, S. H.; Palstra, T. T. M.; Ramirez, A. P.; Kortan, A. R. Nature 1991, 350, 600. (3) Jensen, H. W.; Wilson, S. R.; Scuster, D. I. Bioorg. Med. Chem. 1996, 4, 767. (4) Ros, T. D.; Prato, M. Chem. Commun. 1999, 663. (5) Nakamura, E.; Isobe, H.; Tomita, N.; Sawamura, M.; Jinno, S.; Okayama, H. Angew. Chem., Int. Ed. 2000, 39, 4254. (6) Isobe, H.; Sugiyama, S.; Fukui, K.; Iwasawa, Y.; Nakamura, E. Angew. Chem., Int. Ed. 2001, 40, 3364. (7) Morissette, S. L.; Soukasene, S.; Levinson, D.; Cima, M. J.; Almarsson, O. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 2180. (8) Zheng, B.; Roach, S.; Ismagilov, R. F. J. Am. Chem. Soc. 2003, 125, 11170. (9) Hansen, C. L.; Classen, S.; Berger, J. M.; Quake, S. R.; J. Am. Chem. Soc. 2006, 128, 3142. (10) Miyazawa, K.; Kuwasaki, Y.; Obayashi, A.; Kuwabara, M. J. Mater. Res. 2002, 17, 83. (11) Lee, S. H.; Miyazawa, K.; Maeda, R. Carbon 2005, 43, 887 (12) Squires, T. M.; Quake, S. R. ReV. Mod. Phys. 2005, 77, 977
Figure 1. Schematic of the experimental setup. (a) Microfluidic system for C60 crystal synthesis. (b) Test section of the system. The glass microchip was immersed in the water bath. The atmospheric temperature was kept at 20 °C. The starting water bath temperature was set at 5 °C.
Experimental Section Raw Material. The raw material used was a commercial product of 99.5% C60 powder formed by the electric-arc technique (Lot. No.020715 made by the Honjo Chemical Co., Tokyo, Japan). Synthesis of C60 Crystals in the Microchannel Reactor. The test solutions were toluene with C60 and alcohol, including 2-propanol, ethanol, and methanol. In all experiments, the concentration of C60 dissolved in toluene was constant at 0.58 mg/mL (0.13 mol %), which was a quarter of the saturation concentration at 25 °C.13 Two solutions were kept at a low temperature of 0 °C by a temperature controller until just before introduction into the channels. Toluene and alcohol were introduced into a Y-shaped microchannel with 100 µm width, 40 µm depth, and 40 mm length (ICC-SY05, Institute of Microchemical Technology Co., Kawasaki, Japan) independently with a flow rate of 1.0 µL/min (Figure 1a). The microchannel was (13) Ruoff, R. S.; Malhotra, R.; Huestis, D. L.; Tse, D. S.; Lorents, D. C. Nature 1993, 362, 140.
10.1021/la0614177 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/20/2006
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Figure 2. Optical micrographs of C60 crystals synthesized in the microchannel. The two parallel lines are channel walls. (a) Liquid/ liquid interface between toluene and alcohol. The width of the microchannel is 100 µm. Toluene and alcohol flowed in the upper channel and the lower channel, respectively. (b) Hollow-center crystals of ∼30 µm length. The two insets show close-ups of the left (tube) and right arrows (crosswise edge). (c) Treelike crystals of ∼50 µm length. The two insets show close-ups of the left and right arrow locations. (d) Open-side hollow columns of 30 µm length. (e) Particles, sprouts, and branches. The left inset is a close-up of the left arrow location. The right inset is a TEM image of a sprout. (f) Acute multiplex pod crystal of 30 µm size. fabricated on the glass substrate by a wet etching technique.14-17 The microchannel was immersed in a water bath, and a waterimmersion objective lens (Olympus LUMPlanFl M ) 100×, NA ) 1.0, Olympus Co., Tokyo, Japan) was used for in situ observations (Figure 1b). The room temperature was kept at 20 °C using an air conditioner. The starting water temperature was 5 °C as set by the temperature controller until just before the introduction of the test solutions. Subsequently, the water bath was left out at room temperature. Therefore, the temperature of the water bath increased and approached 20 °C gradually during the experiments. The water bath temperature was constantly checked by a thermometer. We did not measure the temperature inside the microchannel. The liquid volumes of the bath and the microchannel were 300 mL and 200 nL, respectively. In addition, the cover glass between the water and the microchannel was thin (0.7 mm). Therefore, we can assume that the temperature inside the microchannel was similar to that of the surrounding water and that the temperature of the test solutions was close to the bath temperature as soon as they entered the channel. In the microchannel, two solutions formed a liquid/liquid interface and a multistream laminar flow. With flow, the two solutions did not mix because of an extremely low Reynolds number (Re ) 0.23). After 30 s, the formation of the liquid/liquid interface was confirmed (Figure 2a), and the fluid flow was stopped in order to mix the two solutions. If any crystallization was confirmed, then we noted the mixing time and the water bath temperature. To capture the crystals onto a microgrid for SEM and TEM observations, the test solutions (14) Shinohara, K.; Sugii, Y.; Aota, A.; Hibara, A.; Tokeshi, M.; Kitamori, T.; Okamoto, K. Meas. Sci. Technol. 2004, 15, 1965. (15) Hisamoto, H.; Saito, T.; Tokeshi, M.; Hibara, A.; Kitamori, T. Chem. Commun. 2001, 2662. (16) Ueno, M.; Hisamoto, H.; Kitamori, T.; Kobayashi, S. Chem. Commun. 2003, 936. (17) Kobayashi, J.; Mori, Y.; Okamoto, K.; Akiyama, R.; Ueno, M.; Kitamori, T.; Kobayashi, S. Science 2004, 304, 1305.
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Figure 3. Scanning electron micrographs (SEM) of the C60 crystals synthesized by the toluene/isopropyl alcohol (IPA) combination. (a) Turned up “macaroni”-like crystals of 5 µm diameter. (b) “Penne”like crystals of 5 µm diameter. (c) Close-up of the cross section of the tube crystal. The cross section of the void was also hexagonal. (d) Spheres of 5 µm diameter (e) Short rhombus crystals of 10 µm length. (f) Needlelike crystals (NWs) of 30 µm length. (a-d) Observed on the same microgrid. were soon introduced again at 1.0 µL/min for 30 s. The mixing time was then reset. After 30 s, the fluid flow was stopped again, and fresh test solutions started to mix. We repeated this process: introduce, stop, mix, observe, check mixing time and temperature, wash away, and stop.
Results and Discussion In the first experiment, we introduced toluene with C60 and IPA, which is the usual combination for NW synthesis, into the channels. Within 5 min after the fluid flow was stopped, at a bath temperature of 6 °C, C60 crystals were formed (Figure 2b). The length of the crystals was approximately 30 µm. The center axis areas of the crystals were semitransparent (left inset of Figure 2b). In addition, several crystals had crosswise cut edges (right inset of Figure 2b). Scanning electron microscopy (SEM) revealed the reason for the formation of the semitransparent NWs in Figure 3a. Several “macaroni”-like crystals of 7 µm diameter were observed. Their cross sections were hexagonal, corresponding to the normal C60 NWs.10,11 Another type of tube that was cut crosswise at the edge was also observed in Figure 3b. The edge outline was not parallel to the center axis, indicating that the cross sections of these “penne”-like crystals were also hexagonal. The characteristics of the cross sections of these crystals were emphasized in another magnified SEM image in Figure 3c. Within the void area, multilayers of the crystal were observed. The cross section of the void was not circular but was hexagonal. Some spherical crystals shown in Figure 3d were also observed on the same microgrid. Within 15 min after the flow was stopped, at a bath temperature of 9 °C, treelike NWs appeared in Figure 2c. A number of branches were observed to sprout from the main trunk. The main trunks and the branches were approximately 50 and 10 µm in length,
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Figure 4. (a) Transmission electron micrograph and (b-f) scanning electron micrographs of the C60 crystals synthesized by the toluene/ ethanol combination. (a) TEM image of the hollow-ended crystal of 5 µm diameter. (b) Two bifurcations branches of 20 µm length. (c) Three bifurcations branches of 15 µm length. (d) Two-dimensional multiplex pod crystal of 20 µm size. (e) Three-dimensional multiplex pod of 20 µm size. (f) Multiplex pod crystal with acute edges.
respectively. The main trunks originated at the upper channel wall, indicating that nucleation of the crystals occurred at the wall on the toluene side. Thirty-two minutes after the flow was stopped, at a bath temperature of 16 °C, short, rhombus-shaped columns of 10 µm length were formed (Figure 3e). Their aspect ratio was smaller than that of normal NWs. Forty minutes after the flow was stopped, at a bath temperature of 18 °C, the typical C60 NWs with high aspect ratios appeared in Figure 3f. Their lengths and diameters were more than 20 and less than 5 µm, respectively. These corresponded well with the properties of the well-known C60 NWs synthesized via the toluene-IPA combination.10,11 In the flask experiments, the usable alcohol solution (poor solvent) was limited to IPA for the synthesis of C60 NWs crystals. To change the solvated state and the supersaturation of C60 from the conditions of the previous studies, in the second experiment we attempted to use ethanol instead of IPA. One minute after the flow was stopped, at a bath temperature of 5 °C, crystals with hollow ends appeared on the toluene side (Figure 2d). The size was about 4 µm in diameter and 30 µm in length. A transmission electron microscope (TEM) micrograph showed the outline of the open-side void in Figure 4a. Five minutes after the flow was stopped, at a bath temperature of 7 °C, many particles of a few micrometers in size were formed on the toluene side (Figure 2e). Several sprouts from the particles were also observed (right arrow in Figure 2e). The sprouts grew in one direction. However, a Y-shaped branch crystal was also observed (left arrow). The Y-shaped crystal had two (Figure 4b) or three (Figure 4c) of the small separation branches. Seven minutes after the flow was stopped, at a bath temperature of 8 °C, several crystals with sharp edges appeared in Figure 2f. The crystals radiated in all directions and became multiple pods. The crystals had six to eight columns with 2D (Figure 4d) or 3D
Figure 5. SAED patterns of (a) needle crystals and (b) hollowended crystals. (c) Phase diagram of the C60 crystals synthesized in the microfluidic device. The horizontal and vertical axes represent the water bath temperature and the nondimensional time, respectively. The nondimensional time is defined as t′ ) t/tD where t is the mixing time and tD is the characteristic time by molecular diffusion. tD ) L2/Dalc where L is the width of the microchannel and Dalc is the diffusion coefficient of alcohols to toluene from ref 20. In all cases, the concentration of C60 in toluene was constant at 0.58 mg/mL. Tubes, dendrites I (trees), spheres, prisms, columns, and needles were synthesized by the toluene/IPA combination. Hollow columns, dendrites II (multiplex pod), and dendrites III (branched) were synthesized by the toluene/ethanol combination. Plates and particles were synthesized by the toluene/methanol combination.
structures (Figure 4e). Some crystals had acute edges in Figure 4f. The size of each crystal was about 20 µm, and interestingly, the other columns were positioned at a regular angle of 60°. When methanol was used as the solvent alcohol solution, no NWs were formed. Only particles or clusters were formed in the microchannel. Eighteen minutes after the flow was stopped, at a bath temperature of 12 °C, several particles of 3-5 µm size appeared. After 22 min, larger hexagonal plates appeared at a bath temperature of 14 °C. Microscopic analysis using selected area electron diffraction (SAED) revealed that the C60 crystals synthesized in the microfluidic device had a hexagonal close-packed structure (hcp) or a face-centered cubic (fcc) structure (Figure 5a and b). The lattice constants were a ) 19.2 Å and c ) 9.7Å (Figure 5a) and a ) 23.3 Å (Figure 5b). They were found to be single-crystalline. Overall, the lattice constants of these crystals were larger than those of pristine C60: a ) 14.2 Å. This indicated that the C60 crystals synthesized in the microfluidic device may be van der Waals crystals including the solvent molecules in the structure.
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Summarizing the bulk structures described above, we noticed a certain theme. The bulk structures of the C60 crystals synthesized in the microfluidic device were very similar to those of snow crystals. Snow crystals have several types of structures, including plate, prism, column, end-hollow column, dendrite, and needle. Each structure type has been categorized by temperature and supersaturation of water.18 By utilizing the bath temperature and the mixing time after the flow is stopped, a phase diagram of the C60 crystals synthesized in the microchannel was constructed. Also, for the C60 crystals, the temperature and solvent mainly determine the shape as seen in Figure 5c. Normal C60 NWs with high aspect ratios,10,11 which were usually synthesized at room temperature using saturated C60 solutions, may correspond to the “needles” in this diagram. In inorganic crystallography, snow crystal-like shapes are commonly observed.19 Conversely, in (18) Libbrecht, K. Rep. Prog. Phys. 2005, 68, 855.
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carbon crystallography, to our knowledge, the above complex phases are quite rare.
Conclusions By exploiting the microfluidic environment, various metastable phases of C60 crystals were confirmed. If additional new phases of C60 crystals were found after the C60 drugs were placed on the market, then the social impact would be serious. The present method developed here and current findings suggest an urgent need to screen metastable C60 crystals for the development of fullerene drugs. Acknowledgment. We thank Dr. Li of the University of Tokyo for TEM and SAED analyses. This work was partially supported by a grant-in-aid from the Japan Society for the Promotion of Science, Japan. LA0614177 (19) Kanaras, A. G.; Sonnichsen, C.; Liu, H.; Alivisatos, A. P. Nano. Lett. 2005, 5, 2164. (20) Bosse, D.; Bart, H. J. J. Chem. Eng. Data 2005, 50, 1525.
