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Ind. Eng. Chem. Res. 2008, 47, 3900–3906
Co-Precipitation of β-Carotene and Polyethylene Glycol with Compressed CO2 as an Antisolvent: Effect of Temperature and Concentration Facundo Mattea, Angel Martin,* and Maria J. Cocero High Pressure Processes Research Group, Department of Chemical Engineering and EnVironmental Technology, UniVersity of Valladolid, 47011 Valladolid, Spain
In this work, the coprecipitation of β-carotene and polyethylene glycol (PEG) is studied. First, the effect of pressure and temperature was studied (pressures of 8-12 MPa, temperatures of 288-313 K), Afterward, the effect of the initial concentration of the different substances on the morphologies of the particles was studied. X-ray diffraction (XRD), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) analysis were used to observe the nature of the particles that were obtained. The results indicated the sensitivity of the precipitation to temperature, making impossible to obtain particles at temperatures above 288 K. With regard to the concentration effect on the morphology of the particles, it was possible to obtain different morphologies just by changing the concentration ratio between the substances. Also, the analysis of the coprecipitates indicated that the β-carotene inside the polymer matrix must be in an amorphous form. Introduction There are almost 600 different carotenoids in nature. But only a few of them are interesting for industrial or pharmaceutical applications that are related to human health and diet. The most common and used ones are β-carotene, lutein, and lycopene. The chemical structure of these carotenoids (presented in Figure 1) gives a clear idea of why these substances can be used as colorants and antioxidants. The conjugated double bonds in their chemical structure absorb the blue light, so the molecules show colors from pale yellow to vivid red, and they are responsible for the color of vegetables and fruits such as tomatoes and carrots. Also, because of their chemical structure, they can behave as natural antioxidants. Although these substances present such interesting applications as use as colorants or antioxidants, they also have some important drawbacks when they are used in industrial processes. For example, they suffer degradation with light, heat, and the presence of oxygen. Besides, their applications are restrained to processes with oil or organic solvents, because the solubility of most of the carotenoids in water is negligible, compared to those solvents. Furthermore, because the pure carotenoids present a powdery form, their handling is not very suitable for industrial processes in which small quantities of them are used. To overcome most of these drawbacks, the carotenoids are usually mixed with an edible and hydrophilic polymer, which not only enhances the dissolution rate in water by this way,1 but also provides better protection against degradation reactions2 and improves their handling in industrial or pharmaceutical processes. Coprecipitation is an excellent alternative to obtain the mixture of carotenoids and polymers; coprecipitates have been used by several authors in controlled release applications,3,4 agricultural applications,5 and as adsorbents for chromatographic techniques, crystallizer supports, or aerosols.6 Several techniques can be used to obtain the coprecipitation of microparticles. For example, wet milling, spray drying, evaporative recrystallization, or liquid antisolvent crystallization are some of the most common ones; however, almost all of them present a series of weaknesses when they are applied to carotenoids. The most important ones are degradation of the * Tel. +34 983 42 31 74. Fax +34 983 42 30 13. e-mail: mamaan@ iq.uva.es.
substances, contamination of the final product with organic solvents, and difficulty to obtain small-sized particles with a narrow particle size distribution (PSD). Because of these drawbacks, the use of supercritical fluids in the precipitation processes is a good alternative to the conventional ones.7–9 The precipitation due to the addition of a supercritical fluids that acts as an antisolvent is an excellent
Figure 1. Chemical structure of some carotenoids.
Figure 2. SAS process scheme. Legend: (1) solution pump, (2) carbon dioxide pump, (3) heat exchanger, (4) precipitation vessel, (5) external frit, (6) back-pressure regulation valve, (7) separation vessel, (F) flow meter, (P) pressure meter, and (T) thermocouple.
10.1021/ie071326k CCC: $40.75 2008 American Chemical Society Published on Web 04/30/2008
Ind. Eng. Chem. Res., Vol. 47, No. 11, 2008 3901 Table 1. Precipitation Experiments Used To Study the Influence of Temperature on the Coprecipitation of β-Carotene and polyethylene Glycol Experiment
temperature, T (K)
P (MPa)
CPEGa (g/L)
1 2 3 4 5 6 7 8 9
288 288 285 287 289 291 285 287 289
8 10 10 10 10 10 10 10 10
11 11 22 22 22 22 22 22 22
CBCARb (g/L)
0.3 0.3 0.3 0.,3 0.6 0.6 0.6
Figure 3a 3b 4a 4b 4c 4d 4e
a CPEG ) initial concentration of polyethylene glycol.b CBCAR ) initial concentration of β-carotene.
of the substances were the variables with the most influence on the effectiveness of the precipitation. The main goals of this work were to study the effect of the temperature in the coprecipitation of β-carotene and polyethylene glycol with supercritical carbon dioxide as the antisolvent in a SAS process, and especially at temperatures near the maximum temperature in which the particles can be obtained. The thermodynamic behavior of the system was already studied in a previous work,16 and the conclusions of that work are used to explain the results that are obtained. In addition, the possibility to obtain particles with different morphologies by changes in the initial concentration of the substances is studied. Materials Crystalline β-carotene with a minimum purity of 99% was kindly provided by VITATENE Leo´n (Spain). PEG 20000 was purchased from Sigma-Aldrich. Dichloromethane (DCM, 99.5% pure) that was purchased from Panreac Quı´mica (Spain) was used to prepare the solutions. Carbon dioxide (CO2), at 99.95% purity, was delivered by Carburos Meta´licos S.A. (Spain). Methods
Figure 3. SEM picture of PEG particles obtained at 288 K and pressures of (a) 8 MPa (b) 10 MPa.
option to treat substances that are highly sensitive to degradation such as the carotenoids, and if supercritical carbon dioxide is used, relative low temperatures are needed and the entire process can be performed in an inert atmosphere, which avoids the degradation reactions already mentioned. What is more, thanks to the high solubility of organic solvents in supercritical carbon dioxide, it is easy to obtain solvent-free products without any additional process steps. High supersaturations;and, thus, smaller particles with narrower PSD;are obtained with supercritical fluids, compared to those obtained using the liquid antisolvent process. Even more, it is possible to vary the supersaturation achieved by changes in the process variables, such as pressure or temperature. The use of supercritical fluids to obtain polymers, particles, and microparticles has been widely studied by several researchers, as reviewed by Yeo and Kiran.10 Most of the research works that have been reviewed are related to the precipitation of biological polymers, or biologically accepted polymers that are mainly involved in pharmaceutical applications or in the food industry. The coprecipitation of polymers and drugs is the subject of study of a large quantity of research works. In most of these studies, the objective was to obtain homogeneous properties of the final product and to achieve controlled release systems.11 In previous works, microparticles of pure β-carotene,12 lycopene,13 and lutein14 were successfully obtained by the SAS process, using supercritical carbon dioxide as the antisolvent and dichloromethane or ethyl acetate to dissolve the carotenoids. Particles with mean sizes in the range of 10-80 µm and with different crystalline morphologies were obtained and the overall yield of the process was 100%. Also, in a previous work,15 lutein and polylactic acid were coprecipitated with successful results using the SAS process with carbon dioxide as the antisolvent. Moreover, the feasibility of the process applied to β-carotene and polyethylene glycol with a mean molecular weight of 20000 g/mol (PEG 20000) was proved in the work of Martin et al.,16 which showed that the temperature and the initial concentration
A schematic diagram of the pilot plant used for the SAS precipitation is shown in Figure 2. Two diaphragm pumps (Dosapro, Spain) are used to feed the supercritical carbon dioxide (SC-CO2) and the organic solution to the vessel. The precipitator is an isolated and jacketed AISI 316 stainless steel vessel with a volume of 1.5 L. This precipitator is equipped with a concentric tube nozzle for the injection of the solution and CO2, and with a porous metallic frit for the collection of particles at its outlet. The pressure in the precipitator is controlled by two back-pressure regulator valves placed in parallel for safety reasons. A vessel is used to achieve the separation of solvent and CO2 after pressure release. Other elements are the heat exchangers (which are required to cool CO2 before pumping it and for achieving the operating conditions), safety devices (safety valve and rupture disk), and instrumentation. A typical experiment starts by pumping pure CO2 into the precipitator. When the desired operating conditions (temperature, pressure, and flow rate) are achieved and remain stable, the solution is fed to the precipitator. When the desired amount of solution has been injected, the liquid pump is stopped and only pure CO2 is fed. The flow of CO2 is maintained during a period long enough for the complete removal of solvent from the precipitator. After the decompression, a sample of the particles retained in the frit is collected. All the samples are stored in a nitrogen atmosphere, protected from light, and maintained at temperatures of 289 K, and in the experiments with a temperature of 289 K, a polymer film is obtained, as shown in Figure 4C. When a higher polyethylene glycol/β-carotene ratio is used particles with a higher mean diameter are obtained. However, the agglomeration in the products exhibited the same trend. The phase behavior of this system was studied in a previous work16 and modeled with a Perturbed Hard Sphere Chain equation-of-state-based (PHSC EoS) model; a complete description of the PHSC EoS model can be found in Song et al.18 The phase diagram obtained with the PHSC EoS model of the system DCM-CO2-PEG 20000 is presented in Figure 5. Although the figure is for the system at 311 K, the same behavior is present at lower temperatures. The binary DCM-CO2 system has a one-phase region for pressures of >8 MPa at almost any carbon dioxide concentration; however, when polyethylene glycol is added to the system, a liquid-liquid region appears and higher pressures are required to achieve a one-phase region. At lower temperatures, the liquid-liquid region is smaller and the precipitation can be made at a pressure of 10 MPa. Although the precipitation is possible at temperatures below 288 K, the homogeneous phase that is attained under these
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Figure 10. Optical microscopy photomicrographs of β-carotene particles encapsulated in a polyethylene glycol matrix: (A, B) β-carotene inside the polymer matrix, and (C) typical product of the precipitation process.
Figure 11. Summary of the particles obtained while increasing the relative molar concentration of polyethylene glycol to β-carotene (MPEG/MBCAR) from 0.1 to 0.4.
conditions is not a supercritical phase but rather a liquid phase; thus, the supersaturating and precipitation kinetics are slower, which leads to bigger and more-agglomerated particles. The concentration of the substances has an important effect on the morphologies of the co-precipitates, as has been already reported by several authors.2,5 Therefore, experiments that vary the relationship between the molar concentration of the polymer and carotenoids were conducted, while keeping a constant pressure of 10 MPa and temperature of 288 K. A summary of the experimental conditions is presented in Table 2. In the first three experiments of Table 2, the β-carotene concentration (CBCAR) was kept constant, at 1.2 g/L, while the concentration of the polyethylene glycol (CPEG) was increased from 6 g/L to 22 g/L. The results of these experiments were as expected. As the CPEG/CBCAR ratio was increased, the coating of the carotenoids was better. Figure 6 shows a SEM image of the resulting particles obtained with the lowest CPEG/ CBCAR ratio. The coating of the carotenoid particle is poor. In fact, only very small quantities of polyethylene glycol particles are present in the surface of the carotenoid particles, as indicated in Figure 5 by the white borders of the relatively big dark particles. When the MPEG/MBCAR ratio is increased to 0.5, a partial covering with small polyethylene spheres of the carotenoid particles was obtained, as can be seen in Figure 7. The high concentration of β-carotene leads to a faster precipitation and to the growth of the particles; the polyethylene glycol particles then precipitate over the carotenoid particles. It is remarkable that the mean size of the polyethylene glycol particles is 1 µm, as is shown in Figure 7B, which is almost 20 times smaller than the particles that were obtained with pure polyethylene glycol under the same conditions. When even higher MPEG/MBCAR ratios are used, complete covered particles are obtained, as shown in Figure 8 and 9. Also, the morphologies of the particles can be altered by changes in the concentration ratios of the substances; Figure 8 shows hollow
spheres of polyethylene glycol with a mean size of 20 µm. Finally, when the concentration ratios was doubled the particles have the form and shape presented in Figure 9, a sphere like particle of polyethylene glycol containing the β-carotene inside the polymer matrix. Although the presence of the β-carotene inside the polymer particles is not very clear from the SEM images, optic microscopy images were taken of the same samples as those presented in Figure 10, where the vivid red color of β-carotene inside the polymer matrix is a clear evidence of their presence; also, an image of the particles collected from a regular experiment is presented in Figure 10C. The agglomeration shown in almost all the particles obtained in the experiments could be explained because of the low temperature needed to produce the precipitation of the polyethylene glycol particles. Moreover, at the temperature and pressure conditions necessary for a successful precipitation (288 K and 10 MPa), the process is not really a supercritical antisolvent process, because the CO2 is a high-pressure liquid, instead of a supercritical fluid. To summarize the effect of the concentration over the morphology of the coprecipitates, Figure 11 shows the evolution of the particles obtained with a relative concentration from pure β-carotene to pure polyethylene glycol. As the concentration of polyethylene glycol is increased, relative to the β-carotene concentration, better coatings are obtained, and after the complete encapsulation of the carotenoid particles is achieved, a change in the polymer concentration could lead to different morphologies. The obtained products were analyzed by differential scanning calorimetry (DSC) and X-ray diffraction (XRD) and compared to the unprocessed materials, as well as with a physical mixture of them. Figure 12 shows the DSC analysis of the pure substances and the particles obtained in experiments 3, 5 and 6 of Table 2. The β-carotene calorimetric curve exhibits a steep peak at 455 K, and the shape of the curve after that temperature
Ind. Eng. Chem. Res., Vol. 47, No. 11, 2008 3905
Figure 12. DSC thermographs of (A) unprocessed β-carotene,; (B) unprocessed PEG 20000, (C) a MPEG/MBCAR molar concentration ratio of 0.5, (D) a MPEG/MBCAR molar concentration ratio of 2, and (E) a MPEG/MBCAR molar concentration ratio of 4.
Figure 13. XRD patterns of (A) unprocessed β-carotene, (B) unprocessed PEG 20000, (C) the physical mixture of polyethylene glycol and β-carotene (MPEG/MBCAR ) 0.5); (D) a MPEG/MBCAR molar concentration ratio of 0.5, (E) a MPEG/MBCAR molar concentration ratio of 2, and (F) a MPEG/ MBCAR molar concentration ratio of 4.
indicates degradation of the carotenoid. The pure polyethylene glycol curve presents a peak at 340 K. The DSC thermographs of the different products obtained from the experiments in Table 2 do not have the characteristic peak of β-carotene but, rather, only the one of pure polyethylene glycol. Only curve C from the particles obtained from experiment 3 of Table 2, with the solution with the lower polyethylene glycol:carotene concentration relationship, exhibits a very small peak, indicating the degradation of the carotenoid at 448 K. The absence of the characteristic peak of β-carotene indicates that the carotene is in an amorphous state, instead of the original crystalline form of the substance before the precipitation. The effect of the polymer matrix during the precipitation could stabilize the amorphous state, preventing the encapsulated substance to obtain the crystalline form, as has been reported for several pharmaceutical drugs.19,20 The same results were observed in the XRD analysis; the different XRD patterns are presented in Figure 13. In this case, a physical mixture of polyethylene glycol and β-carotene, which also was measured, is represented by curve C. The same results as those observed in the DSC analysis were observed, except that only in the pure
β-carotene and in the physical mixture, the peak of crystalline β-carotene is present. The results presented in the XRD analysis confirm the absence of crystalline β-carotene in the final precipitated products. However, the vivid red to orange color of the products, together with the fact that the precipitation process of the pure carotenoids has a yield of precipitation near 100%,8 are clear indication of the amorphous form of the β-carotene particles inside the precipitates. Conclusions The coprecipitation of β-carotene and polyethylene glycol with a molecular mass of 20000 by the SAS process has been studied; the effect of the operating variables that could change the morphology of the precipitates has been especially studied. The effect of temperature is crucial on the precipitation of the polymer. A small change in the temperature of the process could lead to a polymer film instead of particles. The concentration of the substances in the solution to be precipitated has significant influence on the degree of coating and the morphology of the coprecipitates. Different morphol-
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ogies of coprecipitates can be obtained as hollow spheres, carotenoid particles that are partially covered with relatively small polyethylene glycol spheres, or smooth surface spherical particles, only by changes in the concentration ratio between the polymer and the carotenoid. The absence of β-carotene in crystalline form, as it was before being dissolved in dichloromethane, in the coprecipitated particles could be due to the inhibition effect of the polymer over the crystallization process of the carotenoid. All of the XRD and DSC results of the coprecipitated do not contain any evidence of crystalline β-carotene, but the product exhibited a vivid red to orange color, and the optical pictures reveal small red particles trapped in gray transparent polymer particles. Thus, the β-carotene must be in an amorphous state. Acknowledgment Financed by the Spanish Ministry of Science and Technology, PPQ 2003-07209, and by the Marie Curie ERT EPSS 007767 PROBIOMAT project. The technical and financial support from VITATENE Leo´n is gratefully acknowledged. Literature Cited (1) Perrut, M.; Jung, J.; Leboeuf, F.; Tarret, A. Dissolution rate improvement of a poorly-soluble drug by particle collection on an excipient bed. In Proceedings of ISSF 2005 International Symposium on Supercritical Fluids, Orlando, FL, 2005. (2) He, W.; Suo, Q.; Hong, H.; Shan, A.; Li, Ch.; Huang, Y.; Li, Y.; Zhu, M. Production of natural carotene-dispersed polymer microparticles by SEDS-PA co-precipitation. J. Mater. Sci. 2007, 42, 3495. (3) Elvassore, N.; Bertucco, A.; Calceti, P. Production of protein-loaded polymeric microcapsules by compressed CO2 in a mixed solvent. Ind. Eng. Chem. Res. 2001, 40, 795. (4) Tu, L. S.; Dehgani, F.; Foster, N. R. Micronization and microencapsulation of pharmaceuticals using carbon dioxide antisolvent. Powder Technol. 2002, 126, 134. (5) Taki, S.; Badens, E.; Charbit, G. Controlled release systems formed by supercritical anti-solvent coprecipitation of herbicide and biodegradable polymer. J. Supercrit. Fluids 2001, 21, 61. (6) Cook, R. O.; Pannu, R. K.; Kellaway, I. W. Novel Sustained release microspheres for pulmonary drug delivery. J. Controlled Release 2005, 104 (1), 79. (7) Jung, J.; Perrut, M. Particle design using supercritical fluids: literature and patent survey. J. Supercrit. Fluids 2001, 20, 179. (8) Shariati, A.; Peters, C. J. Recent developments in particle design using supercritical fluids. Curr. Opin. Solid State Mater. Sci. 2003, 7, 371.
(9) Martin, A.; Cocero, M. J. Fundamentals and mechanisms of precipitation processes with supercritical fluids. AdV. Drug. DeliVery ReV. 2008, 60 (3), 339. (10) Yeo, S.-D.; Kiran, E. Formation of polymer particles with supercritical fluids: a review. J. Supercrit. Fluids 2005, 34, 287. (11) Bahrami, M.; Ranjbarian, S. Production of micro and nanocomposite particles by supercritical carbon dioxide. J. Supercrit. Fluids 2007, 40, 263. (12) Cocero, M. J.; Ferrero, S.; Miguel, F. Crystallization of β-carotene by continuous gas process. Effect of the mixer on crystal formation. In Proceedings of the Fourth International Symposium on High Pressure Process Technology and Chemical Engineering, Venice, Italy, 2002. (CDROM.) (13) Miguel, F.; Martin, A.; Gamse, T.; Cocero, M. J. Supercritical anti solvent precipitation of lycopene. Effect of the operation parameters. J. Supercrit. Fluids 2006, 36, 225. (14) Martin, A.; Mattea, F.; Miguel, F.; Cocero, M. J. Supercritical antisolvent precipitation of lutein: influence of process parameters. In Proceedings of the Eighth Conference on Supercritical Fluids and Their Application, Ischia, Italy, 2006; p 401. (15) Miguel, F.; Martin, A.; Mattea, F.; Cocero, M. J. Precipitation of lutein and co-precipitation of lutein and poly-lactic acid with the supercritical anti-solvent process. Chem. Eng. Process. 2007, doi:10.1016/j.cep.2007.07.008. (16) Martin, A.; Mattea, F.; Gutierrez, L.; Miguel, F.; Cocero, M. J. Co-precipitation of carotenoids and biopolymers with the supercritical antisolvent process. J. Supercrit. Fluids 2007, 41, 138. (17) Nalawade, S. P.; Picchioni, F.; Janssen, L.P.B.M. Supercritical carbon dioxide as a green solvent for processing polymer melts: Processing aspects and applications. Prog. Polym. Sci. 2006, 31, 19. (18) Song, Y.; Lambert, S. M.; Prausnitz, J. M. A Perturbed hard-spherechain-equation of state for normal fluids and polymers. Ind. Eng. Chem. Res. 1994, 32, 1047. (19) Guilbaud, J.-B.; Cummings, L.; Khymyak, Y. Z. Encapsulation of Indomethachin in PVP: solid-state NMR studies. Macromol. Symp. 2007, 251, 41. (20) Khougaz, K.; Clas, S.-D. Crystallization inhibition in solid dispersions of MK-0591 and poly(vinylpyrrolidone) Polymers. J. Pharm. Sci. 2000, 89, 1325. (21) Stievano, M.; Elvassore, N. High-pressure density and vaporliquid equilibrium for the binary system carbon dioxide-ethanol, carbon dioxide-acetone and carbon dioxide-dichloromethane. J. Supercrit. Fluids 2005, 33, 7. (22) Gonzalez, A. V.; Tufuru, R.; Subra, P. High pressure vapor-liquid equilibrium for the binary systems carbon dioxide + dimethyl sulfoxide and carbon dioxide + dichloromethane. J. Chem. Eng. Data 2002, 47, 492.
ReceiVed for reView October 2, 2007 ReVised manuscript receiVed February 13, 2008 Accepted February 27, 2008 IE071326K
