Use of Urea as Habit Modifier in the Supercritical Antisolvent

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MATERIALS AND INTERFACES Use of Urea as Habit Modifier in the Supercritical Antisolvent Micronization of Sulfathiazole Giuseppe Caputo and Ernesto Reverchon* Dipartimento di Ingegneria Chimica e Alimentare, UniVersita` di Salerno, Via Ponte don Melillo 1, Fisciano (SA), I-84084, Italy

The purpose of this work is to study the effect of urea as an additive for the habit modification of sulfathiazole (STZ) crystals formed using supercritical antisolvent (SAS) precipitation. The process was performed using acetone and carbon dioxide as solvent and antisolvent, respectively. The effect of STZ concentration, urea concentration, temperature, and pressure on the crystals habit was investigated, and the obtained products were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). Urea was found to be very effective in the modification of the crystal habit and size of STZ crystals. The habit changed from a plate shape to spherical particles with a very narrow particle size distribution at a urea/STZ mass ratio of 1%. Mean particle size ranged between 0.5 and 1 µm depending on the STZ concentration in acetone. XRD and DSC analysis indicated the formation of a form I polymorph among the five known polymorphisms of STZ. Because of rapid precipitation, only partly crystalline particles were observed with respect to the raw material. Despite the use of urea as an additive, no detectable urea residue was found in the crystals, because urea is soluble in carbon dioxide and is effectively removed during the washing step with CO2. It is, thus, concluded that urea is mainly adsorbed on the crystal surface rather than being incorporated into the lattice of the STZ crystals. Introduction The manipulation of polymorphism, morphology, and size of crystals at micro- and nanoscale levels is a very relevant challenge from an application point of view because of their strong influence on materials properties. The selection of a specific polymorph means to change the packing of molecules from one assembly to another by the proper selection of solvent, additives, and crystallization process. Solvent selection enables the solute-solvent interaction to be tuned to promote the desired conformation, the mode of molecular assembly, the modification of the growth kinetics as reflected in the crystal habit, and the transformation of one phase to another. Additives are chosen to mimic and promote specific motifs of molecular geometry or to block the growth of unwanted crystal faces. Researchers have tested different additives as habit modifiers such as tailormade additives (i.e., structurally related compounds), surfactants, and homopolymers.1,2 Because of the structural similarities, the use of additives often results in their irreversible incorporation into the lattice of the host, forming solid solutions. This leads to a contamination of the crystals that is not desirable, particularly in the case of pharmaceutical compounds. Additives can also be used as size-reduction agents of crystals. The discovery of the phenomenon of “size reduction” induced by specific additives was made first by Sarig and Mullin3 in the case of common salts growth from solution up to about 100 µm. However, generally speaking, it is difficult to produce a pure polymorph and, at the same time, to control the crystal size * Corresponding author. Tel.: +39 089964116. Fax: +39 089964057. E-mail: [email protected].

and crystal-size distribution (CSD), particularly at micrometric levels. Moreover, the desired polymorph often contains impurities from at least one other form. A well-known example is that of sulfathiazole (STZ), an antimicrobial drug belonging to the sulfonamide compounds. Four polymorphs of sulfathiazole exist and are well-characterized.4 The rank order of stability of the four forms under ambient conditions is form III > form IV > form II ≈ form I. Recently, a fifth polymorph was discovered,5 and an amorphous form is also known.6 Each polymorph is crystallized from a different solvent or solvents mixture, and a variety of solvents have been studied.7,8 Stabilization of form I over the other forms is due to the ability of a solvent to inhibit the association of a dimeric ring. In the cases of forms II, III, and IV, the effect of the solvents is to stabilize a particular mode of ring-to-ring association.8,9 Various crystallization processes have been used to control polymorphism, crystal size and distribution, and crystal habit.1 Conventional methods (crushing, grinding, and milling) used for micrometric particle generation could not allow an efficient control of the particle quality, because they sometimes produce thermal degradation, contamination, and differences among product batches. Alternative efficient processes that use supercritical fluids (SCFs) as the processing medium for the production of microand nanosized particles have emerged.10,11 These processes try to take advantage of some specific properties of gases at supercritical conditions, such as enhanced solvent power, large diffusivities, and tunability of process conditions. SCFs are characterized by a continuous adjustable solvent power obtained by varying pressure and temperature. Moreover, their diffusivities can be about 2 orders of magnitude greater than those of

10.1021/ie061629z CCC: $37.00 © 2007 American Chemical Society Published on Web 05/11/2007

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liquids. As a result, SCF-based processes are characterized by fast mass transfer and performances that cannot be obtained by conventional solvents. Various SCF-based processes for microparticles generation have been developed to exploit these properties. They can be classified into three groups according to the solvating behavior of the SCF: as a solvent, as an antisolvent, and as a solute. They consist of several processes, leading to very different particles in terms of size, shape, and morphology.10,11 Among these, semicontinous supercritical antisolvent (SAS) precipitation has been successfully applied to many organic and inorganic substances,10,12,13 and the results have been quite different, depending on the process mode adopted and, of course, on the nature of the material and the properties of the fluid phases. In the SAS process, supercritical carbon dioxide and a liquid solution are continuously delivered to a high-pressure precipitator in which the SCF forms a solution with the liquid, inducing the precipitation of the dissolved solid. As a general rule, SAS has to be carried out at pressures that ensure the complete miscibility between the liquid solvent and CO2, which usually occurs in the range 100-150 bar and 40-60 °C, depending on the solvent. The solute concentration can significantly affect the final particle size and is one of the main operating parameters.14,15 The mean particle size that can be obtained ranges from 0.1 µm to tens of microns, and different morphologies can be obtained depending on the processed material. It is a very rapid process if compared with the traditional liquidantisolvent crystallization, with a time scale of the particle formation of about 10-5 s.16 For this reason, amorphous spherical particles are a common habit, and, frequently, they are the scope of the process. However, some substances, characterized by a fast growing rate with respect to the precipitation rate, can form crystals, particularly when a liquid phase is developed during precipitation. Unfortunately, in these cases, the size of the product is hard to control, and large crystals and CSDs are usually obtained. A processing strategy that can be used to modify crystal habit and CSD is the use of habit modifiers to selectively change crystal growth kinetics during crystallization.1,17 This method is advantageous because crystal habit can be modified without onerous postprocessing operations. Crystallization of STZ induced by supercritical carbon dioxide as an antisolvent has been investigated by Kitamura et al.18 using a batch antisolvent process (GAS) at different values of temperature and pressurization modes. Depending on the pressurization rate, crystal sizes from some millimeters down to tens of microns were obtained. Kordikowski et al.19 investigated polymorphs control of sulfathiazole in liquid and supercritical CO2 using a semicontinuous process. Using methanol as solvent, forms I, III, and IV and their mixtures were obtained, depending on the temperature and the composition of the supercritical mixture. Using acetone, form I and mixtures of forms I-IV and I-amorphous were obtained, depending on the composition of the supercritical solution. The fluid composition was used as a control parameter for the production of pure forms or mixtures of different form. No size control of crystals was obtained. Yeo et al.20 studied the effect of CO2 injection rate, kind of solvent, and temperature on the crystal habit of STZ crystallized using the GAS process. They observed the formation of only one pure form of sulfathiazole from acetone when they crystallized at high injection rates of the antisolvent, whereas at low injection rates, the presence of a second polymorph was observed. However, the control of crystal size was not obtained; indeed, at all operating conditions,

crystals up to 750 µm were produced, probably because the GAS process is not able to ensure a completely controlled environment during crystallization. Until now, the use of additives as habit modifiers during supercritical fluid crystallization has been studied in a very reduced number of works. One of these works is the crystallization of chlorpropamide in the presence of urea as a polymorph conversion additive using the rapid expansion of SCsolution (RESS).21 Recently, Jarmer et al.22 proposed the use of poly(sebacic anhydride) as a growth inhibitor of griseofulvin using a supercritical antisolvent process. They obtained a change of habit from acicular to bipyramidal due to a selective inhibition of crystal growth, but bimodal CSDs and griseofulvin crystals of several microns in mean size were obtained. Since particle size control during STZ micronization by SAS has been, until now, substantially unsuccessful, the use of a crystallization modifier is an intriguing strategy to perform this process. Therefore, in this work, STZ has been precipitated from acetone by the SAS process in the presence of urea as an additive to control polymorphism, crystal habit, and dimensions of STZ particles. Urea was selected for its molecular structure, selective interaction with STZ molecules, and solubility in acetone. It possesses several features that make it an attractive pharmaceutical additive: it is nontoxic, biodegradable, and odorless and can be generally considered to be safe for the human body. The effect of the main process parameters on the powder properties was investigated. Materials and Analytical Methods Sulfathiazole (STZ, C9H9N3O2S2) and urea (CO(NH2)2) of purity 99.5% were bought from Sigma-Aldrich (Italy). Acetone (purity 99.8%) was bought from Carlo Erba Reagenti (Italy). CO2 (purity 98%) was purchased from SON (Napoli, Italy). The solubility of STZ and urea in acetone at 20 °C are 1.9 wt % and 0.4 wt %, respectively. All products have been used as received. SAS precipitation experiments have been repeated twice. The experiment at 1 wt % urea was repeated three times. Samples of the processed powder have been observed by scanning electron microscopy (SEM) (LEO 420, U.S.A.). Powders have been dispersed on a carbon tab previously stuck to an aluminum stub. Samples have been coated with goldpalladium (layer thickness ) 250 Å) using a sputter coater (model 108A, Agar Scientific, U.K.). Several SEM images from different parts of the precipitation vessel have been taken for each run to verify the powder uniformity. Particle size and particle-size distribution (PS, PSD) were evaluated from SEM images using the Sigma Scan Pro 5 software (Systat Software, Inc., U.S.A.); approximately 1000 particles have been considered in each particle-size distribution calculation. Histograms representing the particle-size distribution have been best fitted using Microcal Origin 7.0 software (Microcal Software, Inc., U.S.A.). Log-normal curves giving a reasonably good representation of the nonsymmetrical distributions have been obtained. Solid-state analysis of the samples has been performed using an X-ray powder diffractometer (model D8 Discover, Bruker, U.S.A.) with a Cu sealed tube source. Samples were placed in the holder and flattened with a glass slide to ensure a good surface texture. The measuring conditions were as follows: Ni filtered Cu K_ radiation, λ ) 1.54 A, 2θ angle ranging from 20 to 70° with a scan rate of 3 s/step and a step size of 0.2°.

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Calorimetric analysis has been performed using a DSC TC11 (Mettler, U.S.A.). Temperature and enthalpy of fusion were calibrated with pure indium standard (melting point ) 156.6 °C). Powder samples (5 ( 0.5 mg) were accurately weighed, crimped in an aluminum pan, and heated from 25 to 220 °C at 10 °C/min under a nitrogen purge. Melting points have been calculated using the software provided with the instrument. Differential scanning calorimetry (DSC) was also used to evaluate the urea residue in the processed STZ. Five blends of STZ and urea in the composition range 0.01-1 wt % of urea were prepared. Mixing was carried out by manual and mechanical shaking for 10 min. Before withdrawing samples for analysis, the powders were shaken for an additional 2 min to reverse the effects of possible powder packing and segregation. The blends of STZ and urea were analyzed by DSC, and the area under the urea melting peak was calculated. Experimental Apparatus The configuration of the SAS apparatus consists of a modified high-performance liquid chromatography (HPLC) pump equipped with a pulse dampener used to feed the liquid solution, and a diaphragm high-pressure pump used to deliver carbon dioxide. A cylindrical vessel of 0.5 dm3 inner volume (i.v.) (i.d. ) 5 cm) was used as the precipitation chamber. The liquid mixture was sprayed in the precipitator through a thin-wall stainless steel nozzle (60 µm in diameter, 800 µm in length), and SC-CO2 was pumped through another inlet port located on the top of the chamber. CO2 was heated to the process temperature before entering the precipitator. A stainless steel sintered metal disk was put at the bottom of the chamber to collect the solid product, allowing the CO2-organic solvent solution to pass through. A SAS experiment begins by delivering supercritical CO2 to the precipitation chamber until the desired pressure is reached. When antisolvent steady flow is established, pure liquid solvent is sent through the nozzle to the precipitation chamber at a flow rate of about 1 mL/min in cocurrent mode with supercritical CO2, the flow rate of which is regulated to obtain a CO2 mole fraction of 0.98 inside the precipitator. Then, the flow of the liquid solvent is stopped and liquid solution is delivered through the nozzle at 1 mL/min flow rate. The experiment ends when the delivery of the liquid solution to the chamber is interrupted. However, supercritical CO2 continues to flow for further 60 min to wash out the residual content of acetone solubilized in the supercritical antisolvent contained in the chamber. If the final purge with pure CO2 is not done, acetone condenses during the depressurization and partly solubilizes the precipitated powder, modifying its morphology. A more detailed description of this apparatus and procedures can be found in previous papers.12,23 View-Cell Apparatus. A windowed precipitator was also used to visualize the kind and number of fluid phases of the mixture CO2/acetone/STZ formed during SAS precipitation. The vessel is a stainless steel cylinder of 0.375 dm3 with two quartz windows put along the longitudinal section of the chamber. A detailed description of the vessel and the apparatus can be found elsewhere.24 Experiments were performed following the same procedures described above. The direct observation through large windows allowed us to follow the macroscopic evolution of the process from the liquid jet breakup to the deposition of precipitated particles. A video camera placed in front of one window allowed us to record the experiments. Results and Discussion The micronization of sulfathiazole from acetone solutions was performed by changing temperature in the range 40-80 °C,

Table 1. STZ Crystal Habits Produced by SAS Experiments at Various Pressures and Temperatures (Precipitation Conditions: Solvent ) Acetone, STZ Concentration ) 1.9 wt %; Yield ) Recovered STZ from Crystallizer with Respect to Fed STZ) pressure, bar

temperature, °C

yield, wt %

crystal habit

120 100 80 80 80

40 40 40 50 60

94 95 62.5 82 80

near-spherical and agglomerated near-spherical and agglomerated platelike and agglomerated expanded particles expanded particles

pressure in the range 80-120 bar, and STZ concentration in the acetone from 0.5 to 1.9 wt %. Moreover, the effect of urea as habit modifier has been studied at various concentrations in the range 0.5-5 wt % with respect to STZ. In all the experiments, CO2 and solution flow rate were regulated to obtain a CO2 mole fraction of 0.98. Effect of Pressure. The experiments performed at different pressures are reported in Table 1. Experiments have been carried out using the standard and the windowed apparatus. Because pressure is isothermally varied between 120 and 80 bar, the resulting crystal habit changes significantly. This can be readily seen by comparing the SEM images reported in Figure 1. At 120 and 100 bar, the SAS process produces particles arranged in near-spherical agglomerates of about 0.5-1 µm diameter, the surface of which is composed of smaller faceted crystals. The precipitation has also been observed using the windowed apparatus. The binary system CO2/acetone formed a clear supercritical homogeneous mixture as expected on the basis of the phase diagram of this system. The liquid jet was very short and visible only very close to the injector. When the acetone solution containing STZ was fed to the precipitator, a homogeneous phase with orange color was observed. This is a known phenomenon due to Rayleigh scattering induced by highfrequency density fluctuation in the proximity of a critical point. Thus, the presence of STZ induces the approach to a phase transition point of the ternary system. It has been observed for several compounds that SAS precipitation performed at conditions relatively near the mixture critical point can produce micronic and submicronic spherical particles.11,15 They are probably the result of droplets formation at the injector exit and their subsequent drying due to SC-CO2 dissolution in the solvent. As a rule, amorphous spherical particles are obtained.11,15,24 In the case of STZ processing, microdroplets are also formed (Figure 1a), but crystallization kinetics is very fast and its rate is comparable with that of droplets drying; as a result, the original shape of the droplet is partly maintained, but crystallization superimposes on it and multicrystalline particles are produced. When the pressure is decreased to 80 bar, a different crystal habit is observed. As shown in Figure 1b, crystals have an irregular shape composed of not well-defined plate crystals of a few microns. Visual observation of the SAS process operated at 80 bar disclosed a new situation. When only CO2 and acetone were injected, a liquid and a vapor phase were formed in the precipitator. The liquid phase accumulated at the bottom of the precipitator separated by a meniscus from the upper vapor phase. The amount of liquid phase was very small compared to the vapor phase because the system is very rich in CO2. The liquid jet was long (several cm) and entered deeply in the vapor phase. When acetone solution containing STZ was fed, no evident changes of the phase behavior were observed, but during the precipitation, the jet became progressively darker until crystals were observed on the vessel walls. Thus, precipitation takes

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Figure 1. SEM images of STZ crystallized from acetone at 40 °C, CSTZ ) 1.9 wt %, XCO2 ) 0.98, and (a) P ) 100 bar, (b) P ) 80 bar; 20 KX magnification.

Figure 2. Acetone/STZ solution injected at 80 bar and 40 °C. The spreading of a long jet into the vapor clear phase and the formation of a liquid phase at the bottom are noteworthy.

Figure 3. X-ray diffraction patterns for raw and SAS-processed STZ at different pressures.

place mainly from the vapor phase. At the end of the experiments, the powder was collected only from the walls of the vessel to avoid sampling of particles that had been in contact with the liquid phase. In Figure 2, a frame of the video recorded during the precipitation is reported. The long solution jet and the liquid meniscus on the bottom of the precipitator can be observed. The observed crystal habit could be a further evolution of the original droplets: the superimposed crystallization process produced the flacking of the multicrystalline particles observed at 100 and 120 bar. The X-ray diffraction (XRD) patterns of the unprocessed and SAS-processed STZ are totally different (Figure 3), whereas SAS-processed samples obtained at various pressures are similar. The XRD pattern of the unprocessed STZ corresponds to the form III polymorph as classified in the literature.4 This polymorph is characterized by a hexagon truncated shape,8 as we

Figure 4. DSC thermograms of raw and SAS-processed STZ at different pressures.

verified in the SEM images of the raw material. In the case of SAS-processed STZ, the XRD patterns correspond to form I. The degree of crystallinity can be evaluated using the relative integrated intensity of a selected peak (θ ) 22° in the case of STZ25). As shown in Figure 3, the 22° peak in the case of the SAS-processed samples is smaller than that of the unprocessed STZ. Assuming that unprocessed STZ was 100% crystalline, we have calculated a 43% crystalline content of SAS-processed STZ by comparing the area under the 22° peak of processed and raw STZ. In Figure 4, DSC curves of unprocessed and SAS-processed STZ are reported. The curve of unprocessed STZ shows two endothermic transitions at about T ) 175 °C and T ) 202 °C, in which the first one corresponds to the solid-solid transition between form III and form I19 and the second one corresponds to the melting from solid to liquid state, confirming that the raw STZ consisted solely of polymorph III. DSC endothermic curves of SAS-processed STZ are characterized by only one melting peak at T ) 202 °C at all pressures investigated. This result indicates that the crystals formed by SAS are stable with respect to temperature changes until the melting point is reached. Thus, DSC analysis confirms that STZ obtained by the SAS process consisted only of the polymorph I and the amorphous material. Therefore, the pressure variation affects the crystal habit and not the polymorphism. According to Blagden,8 each STZ polymorph is characterized by a definite crystal shape and, particularly, form I STZ possesses a needlelike morphology. In the case of SAS-processed STZ, we have obtained a polymorph I with a different morphology. It is likely that the fast crystallization that takes place in this process forces the crystal structure toward a less ordered polymorph and that the final particle habit partly maintains the shape of the starting droplets as shown in Figure 1a.


Figure 5. SEM images of STZ crystallized from acetone at CSTZ ) 1.9 wt %, XCO2 ) 0.98, and P ) 80 bar: (a) spherical particles up to 5 µm obtained at 50 °C and (b) balloons up to 20 µm obtained at 60 °C.

Effect of Temperature. In this set of experiments, STZ was precipitated from acetone at 40, 50, and 60 °C, taking as constant the pressure at 80 bar (Table 1). Figure 5 shows two SEM images of STZ obtained at 50 and 60 °C that can be compared to that reported in Figure 1 (T ) 40 °C). STZ morphologies obtained at 50 and 60 °C are completely different in shape and size from that obtained at 40 °C. During operation at 50 °C, STZ precipitates as spherical particles of about 1-5 µm whose surface is made of very small crystal nuclei. However, the sample is not homogeneous, and a different morphology similar to that obtained at 40 °C was also observed. During operation at 60 °C, STZ spherical particles are again obtained, but with larger diameters up to about 20 µm, and the morphology is uniform in the sample. Experiments carried out with the windowed precipitator showed that, during operation at both 80 bar/50 °C and 80 bar/60 °C, an opalescent single phase was formed during the precipitation. These expanded spherical particles (called balloons) are not new for the SAS process; they are the result of the expansion of the liquid droplet due to the diffusion of CO2 and the following precipitation of the solute on the droplet surface.12,15 This morphology can be explained by considering the formation of liquid droplets and their drying; in this case, hollow expanded droplets are produced. This difference can be understood considering the vapor-liquid equilibrium of the mixture solvent/ antisolvent/solute on a plane pressure-composition. Indeed, it has been shown in previous works that a relationship exists between the particles morphology and the position of the operating point with respect to the phase equilibrium diagram.15 A temperature modification at constant pressure modifies the phase envelope of the ternary system STZ/acetone/CO2. Therefore, even if the operating point in the plane pressurecomposition is the same, its position with respect to the phase envelope is different: expanded particles are formed in the single-phase vapor side of the phase diagram when the operating pressure is below the mixture critical point of the ternary system.11,15,24 In this case, the superimposition of the crystallization process modified the STZ particles, though in a lower extent: the original shape of the spherical droplet was maintained, and small crystals formed its surface. XRD and DSC analyses on STZ obtained at 50 and 60 °C yielded basically the same results as that at 40 °C, and form I and amorphous STZ were produced again. Effect of the Presence of Urea. To summarize the results obtained until now: the micronization of STZ has been only partly successful since droplets have been generated, but the

Table 2. STZ Particle Habits and Size Produced by SAS Experiments at Various Pressures and STZ and Urea Concentrations; T ) 40 °C pressure, bar

STZ wt %

urea wt %

particle habit

particle size, µm

80 100 100 100 100 100 100 100 100

1.9 0.25 0.5 1 1.9 1.9 1.9 1.9 1.9

1.0 1.0 1.0 1.0 0.5 1.0 1.5 2.0 5.0

spherical particles spherical particles spherical particles spherical particles agglomerated plates spherical particles agglomerated plates agglomerated plates agglomerated plates

1.0 0.45 0.45 0.9 0.86 -

corresponding dried microparticles have irregular shapes and are partly coalescing because of the very fast crystallization of the compound. This phenomenon is particularly relevant at 80 bar and 40 °C. A possible solution to this problem is to add a crystallization modifier; i.e., an additive with the scope of slowing the STZ crystallization process. A good candidate for this role is urea. Indeed, it is possible to consider a mechanism in which urea molecules might interact with the molecular aggregation process of STZ to form hydrogen bonds able to compete with hydrogen-bonded motifs that characterize STZ crystals. Therefore, STZ was SAS precipitated from acetone at different values of urea concentration, as shown in Table 2. Five values of urea concentration ranging from 0.5 to 5 wt % with respect to STZ were investigated. Also, the effect of STZ concentration on particle size and habit was investigated. The presence of urea has a dramatic effect on STZ particles. During operation at 100 bar and 40 °C, platelike crystals were produced at all concentrations of urea, except at 1.0 wt %. As shown in Figure 6, when working at 1 wt % of urea, spherical particles were obtained, maintaining the droplet shape. Particles have a mode of 0.86 µm, and the particle population is unimodal with a standard deviation of 0.5 µm. Practically no agglomeration is observed. Also, when operating at 80 bar, spherical particles were obtained with a mode of about 1 µm (Figure 6). Figure 7 shows two example of STZ particles obtained at 0.5 and 2 wt % of urea. In both cases, crystals exhibit a platelike morphology with a slight tendency to maintain the form of nearspherical agglomerates. The size distribution is relatively large, with plate size up to about 5 µm. Visual observation of the precipitation in the presence of urea showed the same behavior observed at the same pressure and temperature without urea. Therefore, also in the presence of urea,

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Figure 6. SEM images of STZ crystallized in the presence of 1 wt % of urea at 40 °C and (left) 100 bar, (right) 80 bar.

Figure 7. SEM images of STZ crystallized in the presence of (left) 0.5 wt % and (right) 2 wt % of urea.

Figure 8. XRD diffraction patterns for SAS-processed STZ at different concentrations of urea.

at 100 bar and 40 °C, precipitation takes place into a homogeneous gaseous phase near the critical transition. The effect of urea on the STZ particles is basically due to an interaction between STZ and urea molecules within liquid droplets traveling the surrounding gaseous phase. XRD analysis (Figure 8) indicates that (a) STZ crystals of form I are again obtained, (b) characteristic peaks of urea are not present in the STZ samples, and (c) the degree of crystallinity increases slightly with urea concentration. Particularly, the sample obtained at 5 wt % of urea has the highest crystallinity. The presence of form I is also confirmed by DSC thermograms reported in Figure 9 at different values of urea; in addition, the thermogram of untreated urea and STZ are

Figure 9. DSC thermograms of raw and SAS-processed STZ at different concentrations of urea.

reported. The pattern of SAS-processed STZ in the presence of urea is similar to that obtained without this compound. Moreover, DSC patterns do not present the melting peak of urea. In Figure 9, a slight shift of the STZ melting peak can be observed by increasing the urea concentration. However, a similar shift can be detected when comparing unprocessed and SASprocessed STZ without urea. Therefore, it is likely due to experimental uncertainty more than to the presence of urea. In conclusion, urea is not present in a detectable quantity in STZ crystals after SAS processing, because it is probably extracted by CO2 during the process and the final washing step. Indeed, urea is soluble in SC-CO2 and its molar solubility at 80 bar and 40 °C is in the order of 1 × 10-6.


Figure 10. SEM images of STZ crystallized in the presence of urea at 1 wt % at various STZ concentrations in acetone: (a) 1 wt % 10, KX mag.; (b) 0.5 wt %, 10 KX mag.; (c) 0.25 wt %, 10 KX mag.; and (d) 0.25 wt %, 80 KX mag.

The amount of urea cannot be easily and effectively quantified by liquid chromatography, because the absence of chromophores in urea limited its detection in both the UV and visible ranges. Therefore, the residual content of urea was evaluated according to the DSC method described in the analytical section. Five STZ/urea blends at urea concentrations of 1, 0.5, 0.05, 0.02, and 0.01 wt % were prepared and analyzed. When the urea concentration of the blends decreases, the area under the melting peak of urea decreases near proportionally, until at a urea concentration of 0.01 wt % the peak cannot be revealed. We have assumed 0.02 wt % as the detection limit of the method. Because the DSC patterns of Figure 9 do not present the melting peak of urea at any initial concentration of urea, we have concluded that the urea concentration of processed STZ is below 0.02 wt %. No monotonic trend of the effect of urea concentration on STZ particles habit has been found: 1 wt % of urea is an optimum value below and above which the modification effect is largely reduced. This behavior is not new and unexpected, since some studies report growth enhancement occurring at low impurity levels followed by a reversal at higher levels when the blocking effect becomes dominant.1 To summarize these results, at 1% of urea with respect to STZ, we obtained an overall reduction of the crystal growth with the possibility of producing more regular and noncoalescing microparticles; urea was completely eliminated at the end of the process. It is now important to discuss the mechanism of habit modification played by urea. Crystallization is a molecular selfassembly process, initiated in solution with the onset of supersaturation, and the mode of assembly in solution is

Figure 11. PSD of STZ as a function of its concentration in acetone: Curea ) 1 wt %, T ) 40 °C, P ) 100 bar, XCO2 ) 0.98.

reflected in the mode packing in the resulting solid phase. Additives can interact with this process that can be tuned to modify the growth kinetic of a desired face or of the crystal. In our case, the effect of urea is likely due to the formation of hydrogen bonds between urea and STZ able to modify the hydrogen-bonded motifs that characterize the self-assembly of the pure STZ crystal network. Therefore, the mechanism responsible for the habit modification can be a nonselective interaction of urea on all the faces of STZ crystals promoted by hydrogen bonds that inhibit the growth and the regular assembly of the lattice, producing a spherical habit with any sharp edges (Figure 6). Effect of STZ Concentration in the Presence of Urea. The effect of the STZ concentration in the liquid solution has been studied at 40 °C, 100 bar, and a urea concentration of 1 wt %. In this set of experiments, STZ concentration in acetone has been varied between 0.25 and 1.9 wt %; the latter value is the

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saturation concentration (Table 2). SEM images taken at 10 KX magnification are reported in Figure 6 and parts a, b, and c of Figure 10 for 1.9, 1.0, 0.5, and 0.25 STZ wt % mixtures, respectively. A marked enlargement of particle size is obtained with concentration increase. This result is expected since the influence of this parameter in SAS experiments has been studied several times, and this trend confirm those studies.15,26 It is, however, relevant that the same concentration of urea maintains the effect of producing spherical noncoalescing particles irrespective of STZ concentration in the starting solution. As shown in Table 2 and Figure 11, the particle population is unimodal with a mode of about 0.5 µm for particles obtained at 0.25 and 0.5 wt % and with a mode of about 1 µm for particles obtained at 1.0 and 1.9 wt %. Figure 10d is an 80 KX enlargement of particles obtained at 0.25 wt % and shows the polycrystalline nature of the STZ microparticles: the surface of each microparticle is formed by several sub-microcrystals evolved with the constraints of the original spherical shape of the solution droplets.

(5) Apperley, D. C.; Fletton, R. A.; Harris, R. K.; Lancaster, R. W.; Tavener, S.; Threlfall, T. L. Sulfathiazole Polymorphism Studied by MagicAngle Spinning NMR. J. Pharm. Sci. 1999, 88 (12), 1275-1280. (6) Lagas, M.; Lerk, C. F. Polymorphism of Sulfathiazole. Int. J. Pharm. 1981, 8, 11-24. (7) Khoshkhoo, S.; Anwar, J. Crystallization of Polymorphs: The Effect of Solvent. J. Phys. D: Appl. Phys. 1993, 26, B90-B93. (8) Blagden, N. Crystal Engineering of Polymorph Appearance: The Case of Sulfathiazole. Powder Technol. 2001, 121, 46-52. (9) Blagden, N.; Davey, R, J.; Lieberman, H. F.; Williams, L.; Payne, R.; Roberts, R.; Rowe, R.; Docherty, R. J. Crystal Chemistry and Solvent Effects in Polymorphic Systems Sulfathiazole. J. Chem. Soc., Faraday Trans. 1998, 94 (8), 1035. (10) Jung, J.; Perrut, M. Particle Design Using Supercritical Fluids: Literature and Patent Survey. J. Supercrit. Fluids 2001, 20, 1. (11) Reverchon, E.; Caputo, G. Fine Particle Generation Techniques Using Supercritical Fluids. Trends Chem. Eng. 2003, 8, 183-198. (12) Reverchon, E. Supercritical Antisolvent Precipitation of Micro- and Nano-particles. J. Supercrit. Fluids 1999, 15, 1.

Conclusions

(13) Reverchon, E.; Adami, R. Nanomaterials and Supercritical Fluids. J. Supercrit. Fluids 2006, 37 (1), 1-22.

The crystallization of sulfathiazole by supercritical antisolvent has been successfully performed using urea as the crystallization modifier. Indeed, urea at 1 wt % controlled the crystallization process and, thus, preserved the spherical shape of the particles originated from the droplets produced by the injector and eliminated the phenomenon of their agglomeration. STZ particles obtained in this work show a lower degree of crystallinity with respect to raw STZ, and its polymorph corresponds to form I, which is the less stable among the five known polymorphisms of STZ. For this reason, a short-term stability study was conducted on pure STZ and urea-processed STZ to examine if changes occur during storage at room conditions. Indeed, because of the amorphous content of the samples, there was the possibility of altering the habit and dimensions of particles. STZ samples were stored at room temperature in a sealed vial and analyzed after 120 days by SEM and XRD analyses. No detectable changes were observed in the morphology and in the XRD peaks height and area, proving a good stability of SAS-processed materials. The obtained experimental results motivate further work and provide directions for future investigations about the potential of the use of habit modifiers in the supercritical antisolvent precipitation. The authors believe that coupling the well-known tunability of SCF-based processed with the use of “tailor-made” additives can greatly enhance the control of crystal habit and polymorphism of a large variety of products.

(14) Reverchon, E.; Della Porta, G.; Falivene, M. G. Process Parameters and Morphology in Amoxicillin Micro and Submicro Particles Generation by Supercritical Antisolvent Precipitation J. Supercrit. Fluids 2000, 17 (3), 239.

Acknowledgment This work was financially supported by the Italian Ministery of University and Research (MIUR- PRIN 2004 project). The authors gratefully acknowledge Dr. Domenico Ercolino for running the SAS experiments and samples analysis. Literature Cited (1) Mullin, J. W. Crystallization, 3rd ed.; Butterworth Heinemann: London, 1993; p 264-364. (2) Mersman, A., Ed. Crystallization Technology Handbook, 2nd ed.; Marcel Dekker: New York, 2001; p 563. (3) Sarig, S.; Mullin, J. W. Size Reduction of Crystals in Slurries by the Use of Crystal Habit Modifiers. Ind. Eng. Chem. Process Des. DeV. 1980, 19, 494-497. (4) Anwar, J.; Tarling, S.; Barnes, P. Polymorphism of Sulfathiazole. J. Pharm. Sci. 1989, 78, 337-342.

(15) Reverchon, E.; Caputo, G.; De Marco, I. Role of Phase Behavior and Atomization in the Supercritical Antisolvent Precipitation. Ind. Eng. Chem. Res. 2003, 42 (25), 6406-6414. (16) Mawson, S.; Kanakia, S.; Johnston, K. P. Nozzle for Control of Particle Morphology in Precipitation with Compressed Fluid Antisolvent. J. Appl. Polym. Sci. 1997, 64, 2105. (17) Myerson, A. S., Ed. Handbook of Industrial Crystallization, 2nd ed.; Butterworth Heinemann: Boston, 2002; p 67. (18) Kitamura, M.; Yamamoto, M.; Yoshinaga, Y.; Masuoka, H. J. Crystal Size Control of Sulfathiazole Using High Pressure Carbon Dioxide. J. Cryst. Growth 1997, 178, 378-386. (19) Kordikowski, A.; Shekunov, T.; York, P. Polymorph Control of Sulfathiazole in Supercritical CO2. Pharm. Res. 2001, 18 (5), 682-688. (20) Yeo, S.; Kim, M.; Lee, J. Recrystallization of Sulfathiazole and Chlorpropamide Using the Supercritical Fluid Antisolvent Process. J. Supercrit. Fluids 2003, 25, 143-154. (21) Vemavarapu, C.; Mollan, M. J.; Needham, T. E. Crystal Doping Aided by Rapid Expansion of Supercritical Solutions. AAPS Pharm. Sci. Tech. 2002, 3 (4), 1-15. (22) Jarmer, D. J.; Lengsfeld, C. S.; Anseth, K. S.; Randolph, T. W. Supercritical Fluid Crystallization of Griseofulvin: Crystal Habit Modification with a Selective Growth Inhibitor. J. Pharm. Sci. 2005, 94 (12), 2688. (23) Reverchon, E.; Della Porta, G.; Pace, S.; Di Trolio, A. Supercritical Antisolvent Precipitation of Submicronic Particles of Superconductor Precursors. Ind. Eng. Chem. Res. 1998, 37 (3), 952. (24) Reverchon, E.; De Marco, I.; Della Porta, G. Rifampicin Microparticles Production by Supercritical Antisolvent Precipitation. Int. J. Pharm. 2002, 243 (1-2), 83. (25) Kishita, A.; Kishimoto, S.; Nagashima, N. Characterization of Organic Crystal Products. J. Cryst. Growth 1996, 167, 729. (26) Reverchon, E.; De Marco, I.; Caputo, G.; Della Porta, G. Pilot Scale Micronization of Amoxicillin by Supercritical Antisolvent Precipitation. J. Supercrit. Fluids 2003, 26, 1-7.

ReceiVed for reView December 18, 2006 ReVised manuscript receiVed March 22, 2007 Accepted April 2, 2007 IE061629Z

Use of Urea as Habit Modifier in the Supercritical Antisolvent

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