Supercritical antisolvent precipitation of Cephalosporins

Powder Technology 164 (2006) 139 – 146 www.elsevier.com/locate/powtec

Supercritical antisolvent precipitation of Cephalosporins Ernesto Reverchon ⁎, Iolanda De Marco Università di Salerno, Dipartimento di Ingegneria Chimica ed Alimentare Via Ponte don Melillo, 84084 Fisciano (SA), Italy Received 5 April 2005; received in revised form 10 November 2005; accepted 22 March 2006 Available online 15 May 2006

Abstract We successfully performed the micronization of some Cephalosporinic antibiotics by Supercritical AntiSolvent (SAS) precipitation from Dimethylsulfoxide (DMSO) and evaluated the effect of temperature, pressure, concentration of the liquid solution and carbon dioxide molar fraction on the precipitation process. In particular, we varied temperature from 40 to 60 °C, pressure from 90 to 180 bar, concentration of the liquid solution from 10 to 90 mg/mL and CO2 molar fraction from 0.5 to 0.98. We obtained different morphologies of precipitates: sub-microparticles, microparticles, balloons (micrometric empty shells) and large crystals. We tried to explain how the presence of solute modifies the vapor–liquid equilibria (VLEs) of the system DMSO–CO2 and to relate the different morphologies observed to the position of the process operating point with respect to the ternary system mixture critical point (MCP). Particle dimensions range from 0.1 to 14 μm for spherical particles and from 3 to 50 μm for balloons. © 2006 Elsevier B.V. All rights reserved. Keywords: Supercritical antisolvent precipitation; Cephalosporins; Vapor–liquid equilibria; Micronization; Microparticles

1. Introduction Supercritical antisolvent precipitation (SAS) has been used to micronize several kinds of compounds. Some reviews have been published, in which the results obtained on different compounds have been widely illustrated [1–6]. The results can be quite different, depending on the process mode (batch or semi-continuous), on the nature of the material and on the highpressure vapor liquid equilibria (VLEs) characterizing the ternary system solvent–solute–supercritical antisolvent. Crystals, spherical nano, sub-micro and microparticles with mean diameter ranging from 0.1 μm to several microns and empty shells are the morphologies that have been most frequently observed [1–6]. Fundamental aspects of the SAS process have been relatively less studied, such as the VLEs of the ternary system involved in the process and the correlation between the process parameters and morphologies and the dimensions of the precipitated powders [7,8]. Some studies on the interplay among spray

⁎ Corresponding author. Tel.: +39 089 964116; fax: +39 089 964057. E-mail address: [email protected] (E. Reverchon). 0032-5910/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2006.03.018

formation, mass transfer and SAS performance have also been proposed [5,9,10]. The solubility behavior of a binary mixture containing supercritical CO2 can be modified by the addition of a low volatile third component. If the ternary system shows poorer solubility compared with the binary systems antisolvent + solvent and antisolvent + solute, it is called non-cosolvency (antisolvent) system [11,12]. In some recent works [13,14], we attempted to relate high pressure VLEs with the morphology of SAS precipitated materials. Correlations between the observed morphologies and the position of the process operating point with respect to the mixture critical point (MCP) (i.e., the pressure at which the ternary mixture is supercritical) were proposed. We studied the modifications of the binary system Dimethylsulfoxide (DMSO)–CO2 induced by the presence of a third compound [13,14]. This binary system is particularly interesting since it has been used in several cases for SAS micronization [1,5,13,14]. The classification proposed by van Konynenburg and Scott [15,16] for fluid phase behaviors gives account of six basic types of binary systems, starting from largely symmetrical mixtures and continuing with increasing differences in the

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molecular size, volatility and polarity of both components. Even if this classification has been thought for binary systems, it can be used also for ternary mixtures. The analogy with the binary behavior becomes evident in quasi-binary plots. Phase equilibria in binary systems are usually depicted graphically in pressure, temperature, composition (P,T,x) space or in (P,x), (T,x) and (P,T) sections. In the case of SAS precipitation of Yttrium Acetate from DMSO [13], VLE behavior seems to be substantially the same of the corresponding binary system and to differ only for the movement of the MCP towards higher pressures. In the case of Cefonicid precipitation [14], a substantial modification of the binary VLE behavior was observed. Indeed, increasing the temperature or the concentration of the liquid solution, balloonlike particles, rather than spherical micro and sub-microparticles, were obtained. We hypothesized that the SAS operating point was shifted in a gaseous subcritical region, located on the right side of the P,x diagram. The aims of this work are to ascertain the feasibility of SAS processing of Cefuroxime and Cefoperazone from DMSO and to understand how VLEs are influenced by temperature and liquid solution concentration. Moreover, we want to study the effect of pressure and of carbon dioxide molar fraction on Cefonicid micronization. From the thermodynamic point of view, we want to evaluate the role of the high pressure VLEs in the two systems formed by Cefuroxime and Cefoperazone in DMSO and to extend our knowledge on the precipitation of the system Cefonicid– DMSO–CO2. 2. Experimental apparatuses, materials, procedures and methods 2.1. Apparatuses The SAS laboratory apparatus consists of an HPLC pump equipped with a pulse dampener (Gilson, mod. 805) used to deliver the liquid solution, and a diaphragm high-pressure pump (Milton Roy, mod. Milroyal B) to deliver supercritical CO2. A cylindrical vessel with an internal volume of 500 cm3 is used as the precipitation chamber. The liquid mixture is delivered to the precipitator through a 200-μm stainless steel nozzle. Nozzles with other diameters are also available but we have demonstrated in a previous work [13] that particle size (PS) and particle size distribution (PSD) of SAS precipitates is only slightly influenced by nozzle diameter when the proper micronization conditions are selected. A second collection chamber located downstream the precipitator is used to recover the liquid solvent. Further information and a schematic representation of the apparatus have been given elsewhere [1,14,17]. The view cell SAS apparatus is similar to the previously described one and differs only for the precipitator, that consists of a stainless steel cylindrical vessel (375 cm3 I.V.) with two quartz windows put along two longitudinal sections (NWA, Germany). It is possible to visually observe the formation of different phases and the macroscopic evolution of the

precipitation process from the liquid jet break-up to the deposition of precipitated particles [14]. 2.2. Materials and methods Cefonicid, Cefoperazone and Cefuroxime sodium salts with purities of 99.9% were kindly supplied by Farmabios Spa (Italy); DMSO with purity of 99.5% was supplied by SigmaAldrich (Italy). CO2 (purity 99%) was purchased from SON (Naples, Italy). The approximate solubilities of the Cephalosporins in DMSO were measured at room temperature: for Cefoperazone the solubility is equal to 125 mg/mL, whereas Cefuroxime and Cefonicid show solubilities larger than 150 mg/ mL. The untreated materials were formed by irregular crystals with particle size ranging between 20 and 200 μm. All materials were used as received. Samples of the precipitated powder were observed using a Scanning Electron Microscope (SEM) (Assing, mod. LEO 420). SEM samples were covered with 250 Å of gold using a sputter coater (Agar, mod. 108A). The particle size (PS) and the particle size distributions (PSDs) were measured using an image analysis performed using Sigma Scan Pro software (Jandel Scientific), that is an image processing program to count, measure and analyze digital images; about 1000 particles were considered in each PSD calculation. The number of particles was chosen in agreement with the criteria used in the image analyses of pharmaceutical powders [18]: 500 to 1500 measured particles represent a good compromise between the time spent for the analysis and the accuracy of results. 2.3. Experimental procedures A SAS experiment begins by delivering supercritical CO2 to the precipitation chamber until the desired pressure is reached. Antisolvent steady flow is then established. Then, pure solvent is sent through the nozzle to the chamber with the aim of obtaining steady state composition conditions during the solute precipitation. At this point, the flow of the liquid solvent is stopped and the liquid solution is delivered through the nozzle. The experiment ends when the delivery of the liquid solution to the chamber is interrupted. However, supercritical CO2 continues to flow to wash the chamber for the residual content of liquid solubilized in the supercritical antisolvent. If the final purge with pure CO2 is not performed, solvent condenses during the depressurization and can solubilize or modify the powder. More details were given elsewhere [19]. 3. Results The range of operating conditions used in SAS experiments was initially selected on the basis of our previous experience on this process [13,17]. Therefore, we performed a first set of experiments on Cefoperazone and Cefuroxime at a pressure of 150 bar and a temperature of 40 °C, varying the concentration of the antibiotic in the liquid solution. The liquid solution flow rate was fixed at 1.2 mL/min and the ratio between CO2 flow rate and liquid flow rate (R) was set at 22 on a mass

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an increase of the mean PS and an enlargement of the PSD. However, in the case of Cefonicid [14], the increase of concentration produced a change in the morphology of precipitates: sub-microparticles were obtained at low concentrations; whereas, balloons (empty shells with a smooth surface) were obtained at concentrations higher than 75 mg/mL. We explained these results hypothesizing that, in this case, the higher was the concentration of the liquid solution, the higher was the elevation of the MCP pressure. Therefore, in correspondence of high concentrations, the solute precipitated from a subcritical gaseous phase, located on the right side of the two-phase liquid–vapor (LV) region, producing balloons [14]. To find a confirmation or a denial of this behavior on another compound of the same kind, we studied the effect of the concentration of the liquid solution on Cefoperazone, fixing

(a)

60

10 mg/mL

50

Particles, %

40

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10

90

0 0

1

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3

Diameter, µm

(b) 10 mg/mL

100

25

50

90

Fig. 1. SEM images taken at the same enlargement of Cefoperazone precipitated from DMSO at 150 bar, 40 °C: (a) 25 mg/mL; (b) 50 mg/mL; (c) 90 mg/mL.

basis (corresponding to a CO2 molar fraction equal to 0.98). Then, series of experiments were performed varying one parameter at a time, using the SAS and the windowed SAS apparatuses. 3.1. Effect of liquid solution concentration In previous SAS works [14,17,20–23], it was observed that an increase of the concentration of the liquid solution produces

Volume, %

80

60

40

20

0 0

1

2

3

Diameter, µm Fig. 2. PSDs of Cefoperazone powders precipitated from DMSO at 150 bar, 40 °C; (a) calculations in terms of particle number percentages; (b) calculations in terms of volume cumulative percentages.

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pressure at 150 bar and temperature at 40 °C and varying the concentration of the liquid solution from 10 to 90 mg/mL DMSO. We always obtained spherical micro and sub-microparticles as shown in Fig. 1, where three SEM images obtained at the same enlargement, but at different concentrations, are reported. Elaborating SEM images information, we obtained the PSDs of Cefoperazone powders, as reported in Fig. 2. PSDs were calculated in terms of particle number percentage (Fig. 2a) and of volume cumulative percentage (Fig. 2b). Increasing Cefoperazone concentration from 10 to 90 mg/mL in DMSO, the mode of the distributions (calculated in terms of number percentage) moves from 0.25 μm at 10 mg/mL to 0.5 μm at 90 mg/mL and the particle size distribution enlarges. Operating at 90 mg/mL, particles as large as 3 μm were obtained. We evaluated the effect of concentration on particles morphology also in the case of Cefuroxime, fixing again pressure at 150 bar and temperature at 40 °C and varying the concentration of the liquid solution from 25 to 75 mg/mL in DMSO. Cefuroxime precipitated in form of sub-microparticles, ranging from 0.1 to 0.9 μm, in correspondence of liquid concentrations from 25 to 50 mg/mL (Fig. 3a). At high concentrations, mixed wrinkled microparticles (ranging from 1 to 3 μm) and balloons (ranging from 5 to 20 μm) were obtained

Table 1 List of experiments performed on Cefoperazone, Cefuroxime and Cefonicid at different operating conditions Experiment # P (bar) T (°C) C (mg/mL) xCO2 SEM results Cefoperazone 1 150 2 3 4 5 6 7 8 9 Cefuroxime 10 11 12

150

13 14 15 Cefonicid 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

150

40

10 25 50 75 90 10 15 25 50

0.98 Sub-micrometric particles Micrometric particles

40

25 50 75

60

25 50 75

0.98 Sub-micrometric particles Wrinkled microparticles and balloons Wrinkled balloons

40

10 25 50 75 90 10 15 25 10 50 50 25 50

60

60

120 90 120 180

50 40 60 40

Two phases: micrometric particles and large crystals

0.98 Sub-micrometric particles Micrometric particles and balloons Two phases: micrometric particles and balloons

Micrometric particles No material collected in the precipitation chamber 0.98 Sub-micrometric particles 0.95 Micrometric particles 0.92 0.9 0.8 0.7 0.6 0.5

(Fig. 3b). Therefore, a bimodal particle distribution is obtained when precipitated powder is analyzed. It is possible to note that the tested Cephalosporins, at low concentrations, precipitated in form of sub-microparticles. A summary of all the experiments performed is reported in Table 1. 3.2. Effect of temperature

Fig. 3. SEM images of Cefuroxime precipitated from DMSO at 150 bar, 40 °C; (a) sub-microparticles at 50 mg/mL; (b) wrinkled microparticles and shells at 75 mg/mL.

Some experiments were performed on the selected Cephalosporins at 60 °C, varying again the concentration of the liquid solution. In the case of the system Cefoperazone–DMSO, at all the liquid concentrations tested, we observed a sharp separation of the particles along the precipitator; the powders recovered in the upper and the lower part of the chamber were different from a macroscopic point of view. SEM analysis confirmed the macroscopic observation: two different morphologies were obtained. In the upper part of the precipitator the powder was

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Fig. 5. Wrinkled empty shells of Cefuroxime precipitated from DMSO at 150 bar, 60 °C, 75 mg/mL.

liquid solution at 50 mg/mL. At 90 bar, a liquid phase formed inside the precipitator and no powder was recovered at the end of the experiment; i.e., all solute was extracted. In correspondence of all the other values of pressure tested, micro and submicroparticles were obtained. In Fig. 6, the particle size distributions are shown in terms of the particles number percentage at different pressures; increasing the pressure, the mean particle size decreases and the particle size distribution sharpens. The mode of the distributions shifts from 0.27 μm at 180 bar to 0.84 μm at 120 bar; the PSDs cover the range 0.1– 0.8 μm at 180 bar and 0.25–2.5 μm at 120 bar. 3.4. Effect of carbon dioxide molar fraction

formed by spherical micro and sub-microparticles (Fig. 4a); whereas, in the lower part it was formed by large crystals (Fig. 4b). Increasing the liquid solution concentration from 10 to 50 mg/mL, we observed an increase of the mean PS of the particles in the upper part of the precipitator from 0.7 to 2.1 μm, corresponding to and enlargement of the PSD. At 10 mg/mL, the particles ranged from about 0.3 to 6.3 μm; at 50 mg/mL, they cover the interval from 0.5 to 14 μm. In the case of the system Cefuroxime–DMSO, one phase filled the precipitation chamber at all the liquid solution concentrations. SEM analysis revealed that the powder obtained was formed by wrinkled empty shells, ranging from 3 to 20 μm, as shown in Fig. 5. This morphology is similar to the one obtained for the same compound at 150 bar, 40 °C and concentrations larger than 50 mg/mL.

To perform this set of experiments, we fixed the pressure at 180 bar. This value of pressure, according to previous experiments, assures full developed supercritical conditions in the precipitation vessel. Temperature was set at 40 °C and 14 12

180 bar

10

Pa r t ic le s , %

Fig. 4. SEM images of micronized Cefoperazone precipitated from DMSO at 150 bar, 60 °C, 25 mg/mL; (a) micro and sub-microparticles from the upper part; (b) large crystals from the lower part of the precipitator.

8 6

150 bar

120 bar

4 2

3.3. Effect of pressure A series of experiments was performed on Cefonicid to obtain more indications on the influence of pressure on the powder morphology, particle size and particle size distribution. These experiments were performed between 90 and 180 bar, fixing the temperature at 40 °C and the concentration of the

0 0 .0

0 .5

1.0

Diameter,

1.5

2 .02

.5

m

Fig. 6. PSDs of Cefonicid precipitated from DMSO at 40 °C, 50 mg/mL. Effect of the operating pressure.

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MCP2

Pressure, bar

MCP1

Fig. 7. SEM image of Cefonicid obtained at 180 bar, 40 °C, 50 mg/mL, xCO2 = 0.5.

concentration of the liquid solution at 50 mg/mL Cefonicid in DMSO. Then, we varied the ratio (R) between the antisolvent and the liquid solution flow rates. In particular, we performed the experiments using R values in the range from 0.43 to 22 that correspond to CO2 molar fractions ranging from 0.5 to 0.98. The effect of this process parameter on the SAS precipitates has been rarely analyzed, but the study of the powder morphology and dimensions in correspondence of lower values of R (and lower xCO2) could be very interesting in terms of SAS process productivity. Decreasing R, the productivity of the process may be remarkably increased: indeed, using the same quantity of CO2, a larger quantity of micronized product can be obtained. In all the experiments, we obtained spherical micro and submicroparticles. As an example, in Fig. 7 a SEM image of the particles obtained at the lowest CO2 molar fraction tested (xCO2 = 0.5) is reported. The PSDs in terms of number of particle percentage of Cefonicid, at different CO2 molar fractions, are reported in Fig. 8. We can observe that, decreasing CO2 molar 60

50

x CO2= 0.98

Particles, %

40

30

20

0.8 10

0.5

0 0

1

2

3

4

Diameter, µm Fig. 8. PSDs of Cefonicid powders precipitated from DMSO at 180 bar, 40 °C, 50 mg/mL. Effect of CO2 molar fraction.

DMSO

MCP

x

CO2

Fig. 9. Modification of DMSO–CO2 (solid line) equilibria due to the presence of solute at 40 °C; MCP is the mixture critical point of the binary system; MCP1 is referred to the possible mixture critical point when Cefoperazone has been added and MCP2 to the possible mixture critical point when Cefonicid or Cefuroxime at high concentrations have been added.

fraction, the mode of the distributions increases and the PSD enlarges. The decrease of R also influences the yield of the process: decreasing R, lower percentages of the injected material were recovered in the precipitator, due to an increase of Cefonicid solubility in the fluid phase. 4. Discussion 4.1. Experiments performed at 40 °C. Effect of liquid solution concentration The experiments performed on Cefoperazone at 40 °C show a continuous increase of the mean particle size and an enlargement of the PSD with the increase of concentration. These results are similar to those observed several times in literature [14,17,20–23] and can be explained in terms of nucleation and growth. At all Cefoperazone concentrations and in correspondence of the SAS operating conditions selected, the ternary system Cefoperazone–DMSO–CO2 is in supercritical conditions and according to some authors [9,24], the liquid surface tension of the droplet goes almost instantaneously to zero at the exit of the injection device, before the jet break-up. The liquid solution and supercritical CO2 almost instantaneously form a supercritical phase and the solute starts to precipitate. When the concentration increases, the solute is present in larger quantities and the particle growth process is favoured. The experiments performed on Cefonicid and Cefuroxime in correspondence of the same operating conditions led to a variation of morphology: at lower concentrations, sub-microparticles are obtained; whereas, at higher concentrations, microparticles and balloon-like particles are produced. A possible explanation is that, when we start the injection of the liquid solution, the concentration of the solute in the precipitation vessel increases gradually; therefore, in the first

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part of the experiment, a slight shift of the MCP pressure is obtained and the operating point lies in the supercritical region: the solute precipitates in form of microparticles. As the injection continues, the concentration increases and a more marked shift of the MCP pressure can be obtained; droplets are formed at the exit of the injector and CO2 diffuses in them producing balloons. The operating point position, thus, lies in a subcritical region in the right side of the two-phase LV region, from which the shells are produced [7,25]. In Fig. 9, the possible modification of the DMSO–CO2 VLEs due to the presence of the third component (solute) is reported at 40 °C in the pseudo-binary equilibrium diagram P,x that allows the VLEs representation on a solute-free basis. It is an isothermal section (at 40 °C) of a type I or a type II system, according to the classification made by van Konynenburg and Scott [15,16]. The binary system DMSO–CO2, considering the thermodynamic data published by Kordikowski et al. [26], can be a type II system, since at 303.15 K (30 °C) a liquid–liquid immiscibility appears. The solid line in Fig. 9 represents the VLEs of the binary system DMSO–CO2, the dashed line the hypothesized VLEs of the system Cefoperazone–DMSO–CO2 and the dashed-dotted line the hypothesized VLEs of the systems Cefonicid–DMSO– CO2 and Cefuroxime–DMSO–CO2 at high concentrations. The operating point is always the same and lies in the supercritical region with respect to the MCP of the system Cefoperazone– DMSO–CO2. In the case of the other two systems, the operating point lies in a subcritical gaseous phase, located on the right side of the two-phase LV region. 4.2. Experiments performed at 60 °C. Effect of liquid solution concentration Increasing the temperature at 60 °C, a different behavior has been observed for the three Cephalosporins. In the case of the SAS tests performed on Cefoperazone and Cefonicid, two phases formed inside the precipitator: in the case of Cefoperazone, microparticles and crystals were obtained; whereas, in the case of Cefonicid, microparticles and balloon-like particles were obtained. Probably, in the case of Cefoperazone, the increase of the temperature from 40 to 60 °C induces a deformation (enlargement) of the two-phase LV region and the shift of the SAS operating point inside it. Two phases are formed and the solute splits in the liquid phase (formation of crystals) and in the gaseous phase (formation of spherical microparticles). In the case of Cefonicid, both the morphologies, obtained in the upper and lower part of the precipitator, (microparticles and balloons) are typical of the precipitation from gaseous phase. A possible way to explain this observation is that, in this case, the presence of solute induces not an enlargement or a distortion of the liquid–vapor region, but the transition to a different phase behavior. In the case of Cefuroxime, one phase is formed in the precipitation chamber. Wrinkled balloons are observed, revealing a precipitation from a subcritical gaseous phase. In this case, probably, the operating point lies on the right side of the twophase LV region.

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4.3. Experiments performed at different pressures and CO2 molar fractions Experiments performed on Cefonicid varying the operating pressure and CO2 molar fraction always generated micrometric spherical particles. The increase of pressure induces a decrease of the PS and produces sharper PS distributions, as observed in Fig. 6. This can be easily explained: increasing the pressure, an acceleration of the precipitation process is induced and the solute will be more rapidly released from the supercritical phase. The trend of the particle size and distribution observed varying CO2 molar fraction (see Fig. 8) can be justified considering that, when we reduce the ratio between CO2 and liquid solution flow rates (R); i.e., CO2 molar fraction, the fluid phase formed in the precipitator contains larger quantities of DMSO and the processes of solubilization and solute precipitation are slower. Therefore, microparticles formation process is shifted towards the growth process and larger particles are produced. The decrease of R also induced a Cefonicid yield reduction. Therefore, a higher productivity of the process was contrasted by a lower yield. In conclusion, the variation of CO2 molar fraction may have advantages and disadvantages: decreasing R, the productivity of the process can be remarkably increased; indeed, in correspondence of the same quantity of CO2 used, a larger quantity of micronized product can be obtained. However, in the case of Cefonicid, working at CO2 molar fractions larger or equal to 0.7 is preferable, to avoid the partial extraction of the product. 5. Conclusions The micronization of three Cephalosporins was successfully performed. In all the cases studied, we found that concentration of solute in the liquid phase and temperature influence the ternary system VLEs. Depending on the compound chemical structure, at the same pressure, temperature and CO2 molar fraction, modifications of fluid phase equilibria produced different powder morphologies. Acknowledgments The authors thank Farmabios Spa (Italy) for providing the Cephalosporins. We also acknowledge Dr. Francesco Broda for his help in performing the experiments reported in this work and MiUR (Italian Ministry of Scientific Research) for the financial support. References [1] E. Reverchon, Supercritical antisolvent precipitation of micro- and nanoparticles, J. Supercrit. Fluids 15 (1) (1999) 1–21. [2] J. Jung, M. Perrut, Particle design using supercritical fluids: literature and patent survey, J. Supercrit. Fluids 20 (3) (2001) 179–219. [3] R. Thiering, F. Dehghani, N.R. Foster, Current issues relating to antisolvent micronisation techniques and their extension to industrial scales, J. Supercrit. Fluids 21 (2) (2001) 159–177.

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