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Supercritical Antisolvent Precipitation of Ampicillin in Complete Miscibility Conditions lvaro Tenorio, María D. Gordillo, Clara M. Pereyra, and Enrique J. Martínez de la Ossa Antonio Montes,* A Department of Chemical Engineering and Food Technology, Faculty of Sciences, University of Cadiz, 11510 Puerto Real (Cadiz), Spain ABSTRACT: Microparticles of ampicillin have been precipitated by a supercritical antisolvent process (SAS) using carbon dioxide and N-methylpyrrolidone as the antisolvent and solvent, respectively. The mean particle size (PS) and particle size distribution (PSD) of the processed antibiotic were chosen as responses to evaluate the process performance. The levels for a screening design of experiments were chosen to allow the process to take place in a single supercritical phase. Under these conditions, all of the experiments led to the successful precipitation of ampicillin. Concentration was a key factor as this had the most marked effect on both PS and PSD.
1. INTRODUCTION The supercritical antisolvent process (SAS) is a semicontinuous precipitation technique that produces micrometric and submicrometric particles using a supercritical fluid (SCF) as an antisolvent. The drawbacks of the conventional process to produce micro- or nanoparticles include the difficulty in controlling the particle size and particle size distribution of precipitates and the removal of liquid solvent residues. These limitations are particularly relevant for the production of pharmaceutical compounds.1 From the point of view of thermodynamics, the SAS process must satisfy the requirements outlined below. The solute must be soluble in the organic solvent but insoluble in the SCF. The solvent must also be completely miscible with the SCF, or two fluid phases would form and the solute would remain dissolved or partly dissolved in the liquid-rich phase.2 Thus, the SAS process exploits both the high power of supercritical fluids to dissolve organic solvents and the low solubility of pharmaceutical compounds in supercritical fluids3 to cause the precipitation of these materials once they are dissolved in an organic solvent, and thus spherical microparticles can be obtained.4 SAS has also been used in the two-phase region or vapor region to obtain particles with morphologies other than spherical. For example, balloon-like particles of yttrium acetate were obtained by precipitation from a homogeneous subcritical gas-rich phase. Morphologies frequently observed in SAS processing include crystals with various habits and sizes, long rods, butterfly-like particles, snowballs, and starbursts, and these depend on the nucleation and growth kinetics of the material and on the degree of supersaturation of the liquid-rich phase.2 In a previous investigation,4 the importance of the operation conditions of the SAS process and their influence on particle size (PS) and particle size distribution (PSD) were studied. Four trials of the design matrix were unsuccessful, and powder was not generated. As a result, the calculations concerning the main effects were questionable from a statistical point of view. A new design matrix with higher values was developed to ensure successful precipitation and to analyze the main effects again. In discussing the effect of operating temperature and pressure for r 2011 American Chemical Society
a given application, it is usual to refer simply to the solvent/ antisolvent system. The appropriate operating temperature and pressure must be selected; it is possible to increase the pressure up to the mixture critical point. Above these conditions, the two components are fully miscible, and an interface is not formed, thus making mass transfer faster. According to experimental data reported by Rajasingnam et al.,5 the CO2-NMP system is miscible at a pressure of 180 bar and at both temperatures studied in the previous work (308 and 328 K).1 On the other hand, the pressure required could be greater than 180 bar because the presence of the third component (the solute) shifts the mixture critical point to higher pressures.6 Another factor that contributes to the critical pressure increase is the heat evolved on mixing the carbon dioxide and the organic solution. Close to the MCP, the enthalpy of mixing causes jet stability for exothermic mixing due to an increase in the critical temperature (Tc) and pressure (Pc), whereas the jet stability can be reduced due to heat absorption, that is, endothermic mixing, due to a decrease in these critical parameters.7 Thus, CO2 and NMP are completely miscible close to (or above) Tc and Pc, but an increase in the local temperature would lead to immiscibility. Furthermore, this heat plays an important role in the observed coalescence of particles. Even in the case where complete miscibility of CO2 and NMP solutions is achieved, this heat can contribute to the particle fusion.8 In previous studies, we noted that the experiments carried out below the critical point of the mixture at the selected operating temperature took place in the presence of two phases, and two types of precipitate were observed. In the work described here, the aim was to elucidate the influence that the experimental conditions, chosen with respect to the mixture critical point, had on the results of the trial. The levels for each variable were changed in such a way that all experiments took place in a single supercritical phase, as described Received: June 22, 2010 Accepted: December 21, 2010 Revised: November 11, 2010 Published: January 13, 2011 2343
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Table 1. Two-Level Assessment for Each Factor factors C (mg/mL)
Figure 1. SEM image of unprocessed ampicillin.
below. The operating conditions under which the experiments were conducted were above the mixture critical point (MCP) value; however, in some experiments, the presence of traces of solvent and different precipitates in the filter could be observed, and this finding can be explained by the modification of the phase equilibrium of the binary NMP-CO2 system due to the presence of the ampicillin. Reverchon affirms that the scale up of the process is possible because, when the operating conditions are above the MCP, very similar results can be obtained for the precipitation of the solute in the laboratory and pilot plant, and parameters such as nozzle design have a limited influence on the process.9
2. EXPERIMENTAL SECTION 2.1. Materials and Analytical Methods. N-Methylpyrrolidone (purity 99.5%) was purchased from Sigma-Aldrich Chemicals (Spain). CO2 with a minimum purity of 99.8% was supplied by Carburos Metalicos S.A. (Spain). Ampicillin (APC) (purity g97%) was purchased from Sigma-Aldrich Chemicals (Spain). SEM pictures of APC as received (Figure 1) and samples of the powder precipitated on the wall of the vessel were obtained using a SIRION FEG scanning electron microscope. Prior to analysis, the samples were placed on carbon tape and then covered with a coating of gold using a sputter coater. The SEM images were processed using Scion image analysis software (Scion Corp.) to obtain the particle sizes. The mean particle size (PS) and coefficient of variation (CV) as measurement of the distribution width were calculated using Statgraphics plus 5.1 software. Around 500 particles were counted to perform the analysis in each experiment. 2.2. Design of Experiment (DOE). A pilot plant, developed by Thar Technologies (model SAS 200), was used to carry out all experiments. The equipment and procedure were described in detail in a previous publication.4 All factors that could have an effect on the performance of the SAS process were considered: the initial concentration of the solution (C), the temperature (T), the pressure (P), the liquid solution flow rate (QL), the supercritical CO2 flow rate (QCO2), the washing time (tw), and the nozzle diameter (θn). This DOE was extensively explained by Subra et al.10 and in our previous publication.4 The coding scheme used to describe the factor levels is based on the “þ” and “-” signs, where “þ” and “-”, respectively, denote the high and low levels of a factor. These two levels for each factor are shown in Table 1, and the values were
low level 10
high level 60
T (K)
328.15
338.15
P (bar)
180
275
QL (mL/min)
2
5
QCO2 (g/min)
32
66
tw (min)
120
180
θn (μm)
100
200
chosen mainly on the basis of previous studies on SAS ampicillin precipitation.4 The low and high concentration levels were 10 and 100 mg/ mL in our previous work. In the study described here, the lower limit was 10 mg/mL to obtain a sufficient quantity of ampicillin for subsequent analysis, and the upper limit was 60 mg/mL to avoid saturation of the ampicillin solution at room temperature. The low and high temperature levels were 308 and 328 K, respectively, in the previous work, and the low and high pressure levels were 90 and 180 bar. In the current study, the low and high temperature levels were increased to 328 and 338 K and the low and high pressure levels to 180 and 275 bar. It was confirmed that all experiments were above the critical point of the mixture for the new pressure and temperature ranges.4,11 The lower liquid flow rate was increased from 1 to 2 mL/min because it was observed that the use of a 200 μm diameter nozzle with a liquid flow rate of 1 mL/min did not lead to atomization of the solution and a precipitate was not obtained.12 The CO2 flow rate, nozzle diameter, and washing times are the same as those used in the previous work.4 Among the most important solid-state properties defined by the crystallization process are the dimensional properties of the final product (PS, PSD, and particle morphology) because these describe some of the key specific characteristics such as processing behavior, particle permeability, and bioavailability.3 The PS and PSD of the processed ampicillin in this study were chosen as responses to evaluate the process performance. The factorial design used in this work is shown in Table 2.
3. RESULTS AND DISCUSSION All experiments in the design matrix shown in Table 2 were carried out according to the same experimental procedure, with the operating conditions indicated by the coding scheme (high and low levels for each factor). SEM images of the ampicillin samples micronized in these experiments show the formation of spherical nanoparticles with a mean particle size in the range 218-363 nm. The sizes are uniformly distributed, and the coefficients of variation are in the range 0.279-0.480, as reported in Figure 2. The ampicillin nanoparticle distribution can be fitted to lognormal distributions, and these are compared in Figure 3, in which the mean particle sizes (PS) and standard deviation (SD) are also shown. It can be seen in Figure 3 that the smallest particle sizes with the narrowest particle size distribution were obtained in experiment 57, followed by those from experiments 39, 77, and 83, respectively. The experiments all led to the successful precipitation of ampicillin. All of the precipitate was recovered from the single phase on the inner wall of the precipitator vessel, and images of 2344
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Table 2. Design Matrix, Responses, and Contrasta responses runs
T(2)
P(3)
QL (4=12)
QCO2 (5=13)
tw (6=23)
θn (7=123)
I
means PS (nm)
C.V.
12
þ
þ
-
þ
-
-
-
þ
363
0.388
22
þ
-
þ
-
þ
-
-
þ
289
0.480
39
-
þ
þ
-
-
þ
-
þ
227
0.357
57
-
-
-
þ
þ
þ
-
þ
218
0.279
77
-
-
þ
þ
-
-
þ
þ
287
0.289
83
-
þ
-
-
þ
-
þ
þ
281
0.302
98
þ
-
-
-
-
þ
þ
þ
333
0.450
128 means PS (nm)
þ 72.0
þ 15.0
þ -19.0
þ 13.5
þ -26.5
þ -31.5
þ 30.0
þ 289.25
316
0.348
C.V. a
C(1)
0.11
-0.026
0.013
-0.071
-0.019
-0.006
-0.029
0.361
C.V.: Coefficient of variation calculated as the ratio of the standard deviation to the mean.
Figure 2. SEM images of ampicillin precipitated from (a) run 12, (b) run 22, (c) run 39, (d) run 57, (e) run 77, (f) run 83, (g) run 98, and (h) run 128.
the powder precipitated on the precipitator vessel wall and on the nozzle are shown in Figure 4. The effects on the selected responses for each column were calculated as described in our previous work,4 and the results are represented in Figure 5. Concentration is the factor that had the most marked influence on the PS and PSD. Once again, an increase in the initial concentration of the solution led to larger particles with a wider size distribution; that is, the higher is the initial concentration of the solution, the faster is the condensation rate and thus the bigger is the particle size. This result is consistent with that obtained by Reverchon et al.,13 a finding that was also explained in terms of competition between nucleation
and growth processes. Liquid flow rates, CO2 flow rates, temperature, pressure, nozzle diameter, and washing time did not have much influence on the particle size. The volumetric expansion of NMP with CO2 at various temperatures was reported by Rajasingam et al.5 Reverchon noted that for pressures larger than the asymptotic volume, there was no marked effect of pressure on particle size, and an increase in temperature produced a nonnegligible increase in particle size,14 as observed in this work. It was observed (Figure 2) that experiment (d) gave the lowest particle size in comparison to experiments (a) and (g) due to the lower initial concentration in (d). We can confirm this because the initial concentration of the solution is the factor that has the 2345
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Figure 3. Probability density function of particle sizes for the experiments of the design matrix.
Figure 4. Photographs of wall precipitator (a) and nozzle (b) with ampicillin precipitated by SAS process.
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most marked effect. Furthermore, for the same pressure and temperature conditions, experiments (d) and (g), the effect of an increase in the jet velocity is to direct the process toward the formation of spherical nanoparticles of ampicillin (d) rather than solid aggregates (g).12 It was noted that when experiments were carried out in the complete miscibility region, the operating conditions did not have a marked influence on particle size and particle size distribution. However, in the partial miscibility regions (near the MCP), the operating conditions did influence these parameters.4 Martin et al.15 affirmed that the distinction between the two operating regimes, complete or partial miscibility, must also be considered for the scale up of SAS processes. Reverchon et al.,9 under complete miscibility conditions, obtained very similar results for the precipitation of amoxicillin in both the laboratory and the pilot plant, with the initial concentration of the solution identified as the most influential parameter. Reverchon et al.2 also found, on working at pressures above the mixture critical point, that the parameters that affect the mixing between the two streams, such as nozzle design and precipitator, or the Reynolds number of the nozzle, have a negligible effect on the precipitation. In this study, the nozzle size did not affect the precipitation. Furthermore, these results are consistent with those obtained in a previous study in which an empirical hydrodynamics model was applied. This model allowed the disintegration regimes to be estimated for some ampicillin precipitation experiments. The model considered the operating conditions that influence the hydrodynamics of the system, particularly the pressure, temperature, and the liquid solution flow rates.12 It was found that there are limiting hydrodynamic conditions that must be overcome to obtain a dispersion of the liquid solution in the dense medium; this dispersion must be sufficiently fine and homogeneous to direct the process toward the formation of uniform spherical nanoparticles. At pressures above the MCP, the so-called “gaseous plume” or “gas-like jet” is produced, and this is characteristic of the states of complete miscibility of mixtures (above their MCP). However, under supercritical conditions that are just above the MCP, there is a transition within the jet from multiphase mixing to single-phase mixing. This transition occurs after jet break up, as soon as the transient interfacial tension between the liquid phase and the bulk CO2 has dropped to zero. In this case, the nucleation kinetics dictate whether precipitation takes place in the multiphase jet region with the generation of spherical microparticles or in the single phase jet region with the formation of nanoparticles.16
Figure 5. Main effect plot of the factors on (a) particles size and (b) particle size distribution. 2346
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4. CONCLUSION Microparticles of ampicillin were obtained by an SAS process under complete miscibility conditions. All experiments were successful, and spherical ampicillin nanoparticles were obtained with a similar mean particle size in the range 218-360 nm. The particles were uniformly distributed in a single supercritical phase and were recovered from the phase at the inner wall of the precipitator vessel. For the particle response, the liquid solution concentration was identified as the most important factor, and the other variables had only a minor influence on the PS and PSD. An increase in the initial concentration of the solution led to larger particle sizes with a wider distribution.
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(14) Reverchon, E. Supercritical antisolvent precipitation of microand nanoparticles. J. Supercrit. Fluids 1999, 15, 1. (15) Martin, A.; Cocero, M. J. Numerical modeling of jet hydrodynamics, mass transfer, and crystallization kinetics in the supercritical antisolvent (SAS) process. J. Supercrit. Fluids 2004, 32, 203. (16) Reverchon, E.; Torino, E.; Dowy, S.; Braeuer, A.; Leipertz, A. Interactions of phase equilibria, jet dynamics and mass transfer durinf supercritical antisolvent micronization. Chem. Eng. J. 2010, 156, 446.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT We gratefully acknowledge the Spanish Ministry of Science and Technology (Project CTQ2007-67622) for financial support. ’ REFERENCES (1) Reverchon, E.; Della Porta, G. Production of antibiotic microand nano-particles by supercritical antisolvent precipitation. Powder Technol. 1999, 106, 23. (2) Reverchon, E.; Caputo, G.; De Marco, I. Role of phase behaviour and atomization in the supercritical antisolvent precipitation. Ind. Eng. Chem. Res. 2003, 42, 6406. (3) Shekunov, B. Y.; York, P. Crystallization processes in pharmaceutical technology and drug delivery design. J. Cryst. Growth 2000, 211, 122. (4) Tenorio, A.; Gordillo, M. D.; Pereyra, C. M.; Martínez de la Ossa, E. M. Relative importance of the operating conditions involved in the formation of nanoparticles of ampicillin by supercritical antisolvent precipitation. Ind. Eng. Chem. Res. 2007, 46, 114. (5) Rajasingam, R.; Lioe, L.; Pham, Q. T.; Lucien, F. P. Solubility of carbon dioxide in dimethylsulfoxide and N-methyl-2-pyrrolidone at elevated pressure. J. Supercrit. Fluids 2004, 31, 227. (6) Lora, M.; Bertucco, A.; Kikic, I. Simulation of the semicontinuous supercritical antisolvent recrystallization process. Ind. Eng. Chem. Res. 2000, 39, 1487. (7) Dukhin, S. S.; Zhu, C.; Dave, R.; Pfeffer, R.; Luo, J. J.; Chavez, F.; Shen, Y. Dynamic interfacial tension near critical point of a solvent-antisolvent mixture and laminar jet stabilization. Colloids Surf., A 2003, 229, 181. (8) Davila, M. J.; Caba~nas, A.; Pando, C. Excess molar enthalpies for binary mixtures related to supercritical antisolvent precipitation: Carbon dioxide þ N-methyl-2-pyrrolidone. J. Supercrit. Fluids 2007, 42, 172. (9) 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. (10) Subra, P.; Jestin, P. Screening design of experiment (DOE) applied to supercritical antisolvent process. Ind. Eng. Chem. Res. 2000, 39, 4178. (11) Lee, C. W.; Jung, C. Y.; Byun, H. S. High pressure phase behavior of carbon dioxide þ 1-methyl-2-pyrrolidinone and carbon dioxide þ 1-ethyl2-pyrrolidinone systems. J. Chem. Eng. Data 2004, 49, 53. (12) Tenorio, A.; Jaeger, P.; Gordillo, M. D.; Pereyra, C. M.; Martínez de la Ossa, E. M. On the selection of limiting hydrodynamic conditions for the SAS process. Ind. Eng. Chem. Res. 2009, 48, 9224. (13) Reverchon, E.; Della Porta, G.; Falivene, M. G. Process parameters and morphology in amoxicillin micro and submicron particles generation by supercritical antisolvent precipitation. J. Supercrit. Fluids 2000, 17, 239. 2347
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