CRYSTALLIZATION OF GRISEOFULVIN BY BATCH ANTI-SOLVENT PROCESS B. De Gioannis, P. Jestin, P. Subra * Laboratoire d Ingénierie des Matériaux et des Hautes Pressions, CNRS, Institut Galilée, Université Paris XIII, 99 Avenue Jean Baptiste Clément, 93430, Villetaneuse, FRANCE. Tel: +33 149403436 Fax: +33 149403414 E-mail: subra@limhp.univ-paris13.fr Griseofulvin was crystallized by CO 2 addition in a stirred batch reactor. Based on phase equilibria behaviour, acetone was selected as dissolution medium. The influence of the stirring rate was investigated to control crystals size. The increase of the stirring rate from 33 to 500 rpm leads to a change of morphology from needle form to bipyramid form, beside to a reduction of size. INTRODUCTION Supercritical fluids are of great interest especially in the pharmaceutical field, to improve the bioavailability of pharmaceutical agents. Others materials like cosmetics, ceramics or explosives although, less studied, are potential fields. Numerous papers [1-4] have shown the CO 2 feasibility to produce particles of micro or submicro size, with narrow particle size distribution. Nevertheless, extensive studies of the processes and identification of mechanisms that control it [5-7] have still to carried out, specially to lead to the implementation of the supercritical technology on an industrial scale. The aim of our research is to identify mechanisms that control the precipitation using CO 2 as antisolvent. In the semi-continuous process, that involves a spray of the solution into a continuous flow of CO 2, the quality of the spray is crucial for the process performances. Quality of spray refers to size of droplets generated, or micro- macro-mixing of the solution with CO 2, that will influence the time scales for nucleation and growth. In the batch antisolvent process, it is the CO 2 that is introduced within the liquid solution. Upon diffusion of CO 2, sursaturation and further precipitation occur in the bulk of the solution. In a batch, one has at disposal an efficient way to obtain a quicker homogenisation of the solution, that is the stirring device. In the semi-continuous mode, the homogenisation is dependent of the nozzle type, of the solution and CO 2 flows, that is more complicated to control than a stirring device. In this work, griseofulvin was chosen as model compound. Griseofulvin is an antifungal compound, non soluble in CO 2 (6.16 E-7 mole/mole at 40 C and 100 bar), and quite soluble in organic solvent as methylene chloride, acetone and ethanol. The specific aim is to study the influence of the stirring rate upon the morphology and/or the size of griseofulvin crystals. MATERIALS Carbon dioxide was supplied from Air Liquide (99.5% purity). Acetone and griseofulvin were purchased from Sigma Aldrich.
METHODS A scheme of the apparatus used for batch crystallization is showed in Figure 1. The reactor is a cylindrical vessel (TOP INDUSTRIE) of inner diameter of 5 cm and a length of 25 cm. Its free volume is 0.490 L. The vessel is equipped with a electric motorized magnetic stirrer, an eight-bladed disc turbine of 2.5 cm in diameter, and three sapphire windows. An stainless steel frit of 2 µm porosity is located at the vessel bottom. P T T P Figure 1 Experimental apparatus for gas crystallization. A solution of griseofulvin in acetone is introduced in the reactor and the reactor is closed. Once the operative temperature is reached, the experiment is started. The agitator is switched on and the CO 2 is added to the solution through the disc turbine until a pressure of 100 bar. To prevent further growth of crystals, the solution is then immediately removed by opening the valve located at the vessel bottom. To maintain the pressure during the filtration step, fresh CO 2 is added. When all the liquid is evacuated, the stirring is stopped and a flow of pure CO 2 is maintained for 30 min to dry the crystals. The pressure is then reduced gently to atmospheric pressure by venting the CO 2 from the bottom. Crystals are recovered on filter and walls and are further analyzed. The morphology of crystals was determined by light and scanning electron microscopy (SEM) and x-ray powder diffraction. The Figure 2 shows the pressure profile during the experiment. The pressure profile summarizes the crystallization steps that correspond to: pressurization up to 60 bar, pressurization from 60 bar to 100 bar, liquid filtration, crystals drying and vessel depressurization, respectively.
100 80 pressure (bar) 60 40 20 0 I II III IV V 0 20 40 60 80 100 120 time (min) Figure 2 - Pressure profiles during the crystallization process. RESULTS AND DISCUSSION Phase behaviour of griseofulvin in solvent mixtures has been previously studied [8] together with expansion of several solvents upon addition of CO 2 [9]. The addition of CO 2 to a solution of griseofulvin in acetone leads to the solute precipitation. Figure 3 shows the decreasing solubility of griseofulvin when composition of the acetone-co 2 mixture increases in CO 2. The behaviour was completely different with ethanol instead of acetone, since CO 2 promoted the solubilization of griseofulvin in the solvent. Therefore, acetone was selected as dissolution medium for precipitation process at 40 C. 0,010 0,008 experimental 40 C/100b griseofulvin (mol/mol) 0,006 0,004 0,002 0,000 0,0 0,2 0,4 0,6 0,8 1,0 χ CO2 (mol/mol) Figure 3 - Solubility curve of griseofulvin in CO 2 -acetone mixture at 100 bar and 40 C. Crystallization from acetone solution was carried out at various stirring rates ranging from 33 rpm to 500 rpm. The CO 2 introduction rate was kept constant to 1.3 bar/min. Table 1 reports morphologies and sizes obtained in such conditions.
CO 2 introduction rate stirring rate (rpm) morphology 1.3±0.1 bar/min 33 long needles length 12-20mm width 100-250µm 1.3±0.1 bar/min 98 long needles length 13mm width 50-200µm 1.3±0.1 bar/min 250 bipyramid length 400µm 800µm width 300-400µm 1.3±0.1 bar/min 500 bipyramid length 360µm -550µm width 171µm -180µm Table 1- Morphology obtained for different stirring rates. Two different morphologies were obtained. At low stirring rate, particles were elongated crystals, that exhibited well defined faces, as seen in Figure 4. The points of needles were also well defined pyramid. When the highest stirring rate of 500 rpm was used, a morphology of bipyramid was obtained. But particles can be not so well defined, as shown in Figure 5, for 250 rpm. Partial interpenetrant crystals were frequently observed, due to two of more intergrown individuals. Particles generated at this stirring rate also showed a large distribution of sizes. The bipyramidal crystals indicate that growth occurred in a volume whereas the needle type morphology suggested a growth in a privileged direction. As mentioned in the experimental section, the CO 2 is introduced in the solution by the stirring device. When the stirring rate increases, the mixing of CO 2 with the solution is quicker. The slurry of crystals in solution is also better suspended in the crystalliser. Both phenomena seem to combine together to produce smaller and more compact crystals. Figure 4- SEM image of griseofulvin crystals at 33rpm and 1.3 bar/min
Figure 5 - SEM image of griseofulvin crystals at 250rpm and 1.3 bar/min CONCLUSION The CO 2 addition to a solution of griseofulvin in acetone decreases the griseofulvin solubility, with consequent crystallization. In a batch crystallizer, the stirring rate was found to influence markdly the morphology and size of crystals. The increase of the stirring rate leads to a switch from a needle to bipyramid morphology with a reduction of particles size distribution. Influence of the CO 2 introduction rate is currently investigated, but this early work has already demonstrated that the GAS crystallization process permits to control size and morphology of crystals, by manipulation of the process parameters. ACKNOWLEDGEMENT The authors thank Patrick Portes for the SEM images and Patrick Boissinot for the technical support. REFERENCES [1] REVERCHON,E., J. Supercrit. Fluids, Vol. 15, 1999, p.1. [2] REVERCHON, E., DELLA PORTA, G., Powder Technology, Vol. 106, 1999, p.23. [3] THIERING, R., DEHGHANI, F., DILLOW, A., FOSTER, N. R., J. Chem. Technol. Biotechnol., Vol.75, 2000, p.42. [4] RANDOLPH, T. W., RANDOLPH, A. D., MEBES, M., YEUNG, S., Biotechnol. Prog., Vol. 9, 1993, p.429. [5] WUBBOLTS, F.E., Supercritical crystallization. Volatile Components as Anti-solvents; Universal Press Science Publisher: The Netherlands, 2000. [6] BERENDS, E. M., BRUINSMA, O.S.L., DE GRAAUW, J., VAN ROSMALEN, G.M., AIChE J., Vol. 42, 1996, p.431.
[7] COCERO, M.J., FERRERO S., J. Supercrit. Fluids, Vol. 22, 2002, p.237. [8] DE GIOANNIS, B., VEGA GONZALEZ, A., SUBRA P., accepted in J. Supercrit. Fluids. [9] SUBRA, P., PASSARELLO, J.P., Proceeding of 7 th Meeting on Supercritical Fluids Particle Design Materials and Natural Products Processing, Vol. 2, 2000, p.343.