Supercritical Micronization of β Carotene

Transcription

1 Sofia Antunes Master Dissertation in Biological Engineering September 2007 Supercritical Micronization of β Carotene Sofia Inês de Matos Antunes Instituto Superior Técnico, Universidade Técnica de Lisboa The aim of the present work was the micronization of β carotene, an important carotenoid, due to its properties as colorant, antioxidant and as a precursor in the synthesis of vitamin A. The micronization of β carotene has as main goal the increase of its dissolution rate in water, allowing, simultaneously, the preparation of aqueous suspensions containing this carotenoid. The process was developed in a supercritical micronization apparatus type SAS, using carbon dioxide as antisolvent and tetrahydrofuran as solvent. The effect of operating pressure in the particle size and morphology of the final product has been studied. The runs of micronization have also permitted to study the solubility of β carotene in the supercritical mixture under several conditions of pressure, temperature and flow rate. The micronization of an extract of natural β carotene has also been made, in order to evaluate the purification ability of the process. Some relationships between experimental variables studied and solubility of β carotene in the supercritical mixture were recognized. It has been concluded that solubility strongly depends on temperature and on THF fraction and does not depend much on pressure. It has been shown that particle morphology is changed when the operating pressure changes and it also depends on the position of the operating point relatively to the mixture s critical point. Additionally, it s been shown that particle size increases with the operating pressure, when one operates over the critical point, and a relationship between the particle size and the density of the binary mixture has been established. The supercritical antisolvent micronization was well done in the processing of β carotene and the control of particle size and morphology through the manipulation of the operating conditions is possible. Moreover, this process allows the micronization of the natural extract of β carotene, although purification is not achievable. Keywords: β carotene; supercritical fluids; micronization; carbon dioxide; solubility; tetrahydrofuran Supercritical Micronization of β Carotene 1

2 1. INTRODUCTION Particle size plays an important role in several applications among pharmaceutical, chemical and food industries. Combustion processes of solid explosives, chromatography and drug administration may be strongly influenced by particle size and size distribution. Coloring efficiency, catalyst activity and superconducting properties are also enhanced by reducing particle size [1]. Micronization refers to all the processes that aim to reduce the particle size of solid matter. High pressure homogenization and jet and ball milling are examples of traditional processes of micronization. However, some disadvantages have been pointed to these techniques, such as product heterogeneity, instability and decomposition, due to high pressures and friction practiced. More recently, some particle size engineering techniques have been developed, such as spray drying and precipitation with supercritical fluids. This kind of processes allow the incorporation of desirable attributes in the product, for instance, control of particle size and size distribution, stability and bioavailability. Otherwise, these processes require less handling, that leads to higher yields and simplifies cleaning and sterilization [2]. Most of the biological products are insoluble in water and, consequently, the use of organic solvents is essential, giving way to product contamination with undesirable substances. Supercritical fluids are an important solution to this problem, as the micronization processes that employ this technology allow to obtain a product that is solvent free. Supercritical antisolvent (SAS) micronization stands for the precipitation process that makes use of an antisolvent supercritical carbon dioxide that mixes with the solvent that contains the substance to micronize. Thanks to ability of the supercritical fluid to diffuse rapidly into the organic solvent, supersaturation emerges within a thin gap of time and precipitation takes place. For the micronization to succeed, two main conditions must be satisfied: first, the solute must be soluble in the organic solvent and insoluble in the supercritical fluid at the operating conditions of pressure and temperature; second, the solvent and the antisolvent must be miscible [3]. Many authors have already studied the SAS micronization of several substances [4 29]. However, modeling this process is still to be done, despite the effort of several authors in the study of thermodynamics, hydrodynamics and kinetics of such systems [12, 25, 20, 21, 29]. β carotene is a compound of high molecular weight that belongs to the family of carotenoids and is highly insoluble in water. It is composed of a hydrocarbon chain (C 40 H 56 ) containing eleven conjugated double bounds, which form the chromophore. β carotene is easily degradable by light, heat and oxygen, and its color ranges from yellow to dark red, according to its purity, source and location. The main natural sources of this carotenoid are several plants, algae and fungi, but the bulk of its production is by synthetic means. β carotene is one of the most important carotenoids, thanks to its properties as an antioxidant, colorant and precursor of vitamin A. Its efficiency has already been proved in the prevention of some heart and eye diseases [30]. Some studies also report its importance against several types of cancer [30, 31], but there is still some controversy around this matter [31]. Supercritical Micronization of β Carotene 2

3 The aim of this work is to produce microparticles of β carotene, through supercritical antisolvent micronization. The use of microparticles should enhance β carotene dissolution rate in water and the formulation of aqueous suspensions of this compound might be possible too. Solubility data of β carotene in the supercritical system will also be collected, due to its importance in so many technological fields of supercritical fluids, such as chromatography, extraction and precipitation. 2. MATERIALS AND METHODS 2.1. Materials Synthetic β carotene was obtained from Sigma ( 95% UV purity) and natural β carotene was produced and provided by BioTrend. Tetrahydrofuran (THF) was the solvent used and was obtained from Merck ( 99,8% purity stabilized with BHT). Carbon dioxide was supplied by Air Liquide Apparatus Supercritical antisolvent micronization was performed using an apparatus already described [24]. This is made of a high pressure vessel made of stainless steel, which is immerged in a thermostatic water bath. Two streams feed the vessel at its top, the gas stream and the solution stream containing the compound to micronize. The bottom of the vessel contains a frit that allows the recovery of the dry powder, the supercritical solution passing through it and being conducted to a microvalve that regulates the flow rate and, consequently, the system s pressure. The expansion takes place in an expansion vessel, where the solvent that still contains a small amount of solute is recovered. The gas phase leaves this vessel and passes through a flow meter and a gas counter. All the measures have been made according to a similar procedure. The first step is to pump CO 2 until the desired pressure is reached. Simultaneously, pressure in the expansion vessel is also established. A previously calculated amount of solvent is then injected into the system, in order to reach its steady state, and the flow rate at the exit is established. After that, the solution containing the solute is added and micronization takes place. Finally, CO 2 passes through the vessel for about 75 minutes, what should reduce the solvent concentration in the vessel to 1% of its initial value [10]. Dry powder and liquid solvent are recovered at the end and put to analyze Analytical methods The solution of β carotene in THF collected in the expansion vessel was analyzed by spectrophotometry (Shimadzu UV PharmaSpec 1700). The spectrum was traced and Lambert Beer law at 458 nm for this system was determined before the measures. The absorbance measured allowed to calculate solubilities of β carotene in the supercritical phase and spectra traced permitted to evaluate to some extent the powder s and solution s β carotene purity. Degradation of β carotene was analyzed by high Supercritical Micronization of β Carotene 3

4 performance liquid chromatography (Shimadzu LC.2010CHT with UV/visible detector type and a reverse phase column Chromolith Performance RP 18). The samples of powder were observed by a scanning electron microscope (Phillips XL 30 FEG). Samples were covered with 250 Å of gold, using a sputter coater (Jeol, model JFC 1100). 3. RESULTS AND DISCUSSION 3.1. Solubility results Solubilities of β carotene in the supercritical mixture were measured at several conditions of temperature (35, 40 and 50 C), pressure (75, 100 and 130 bar) and liquid flow rate (0,3, 1, 2 and 3 ml/min). Table 1 presents these results. Table 1 Experimental conditions and solubilities measured. Trial 0 P1 P2 T1 T2 Q1 Q2 Q3 Pressure (bar) Temperature ( C) x CO2 (mole fraction) 0,956 0,956 0,956 0,956 0,956 0,986 0,916 0,879 x THF (mole fraction) 0,044 0,044 0,044 0,044 0,044 0,014 0,084 0,121 x β caroteno (mole fraction) ,29 3,35 1,95 3,25 1,76 0,40 7,34 16,57 Figure 1 shows the mole fraction of β carotene as a function of temperature, at constant pressure and liquid flow rate. It can be observed that solubility decreases as temperature increases and an exponential relationship has been established, due to the density increase as temperature rises at constant pressure. Similar conclusions have been reached by other authors [32]. Figure 1 Mole fraction of β carotene in supercritical mixture of CO 2 /THF as a function of temperature. Supercritical Micronization of β Carotene 4

5 Figure 2 shows the solubility of β carotene as a function of pressure. No relationship has been established for two reasons: firstly, at 75 bar system is below its critical point and above this pressure it is above critical point, so it is not correct to compare, for system behavior is not the same; secondly, it cannot be assumed that mixture composition is the same in the three trials, for the amount of THF added to reach steady state was a calculated average, but still there is a big gap between pressures (and densities), which makes the composition slightly different from trial to trial. Figure 2 Mole fraction of β carotene in supercritical mixture of CO 2 /THF as a function of pressure. The best relationship established in this study is shown in Figure 3 and relates the solubility of β carotene with the mole fraction of THF. The presence of an increasing amount of a co solvent (THF) enhances solubility of β carotene, for the affinity of the solute for supercritical mixture rises exponentially, as can be seen in Figure 3. Another important conclusion taken from the analysis of this figure is the independency of pressure at constant temperature. It s easy to see in Figure 3 that the solubility point at 100 bar correlates with the solubility data at 130 bar. Thus, the difference between the solubility at these pressures in Figure 2 results from the difference in the mixture composition, meaning that THF fraction is much more relevant than pressure, at constant temperature. The effect of co solvent was also observed in other studies [33]. For this system, the presence of 5% of THF means an increase in β carotene solubility of about two orders of magnitude, relatively to pure supercritical CO 2 [32]. Supercritical Micronization of β Carotene 5

6 Figure 3 Mole fraction of β carotene in supercritical mixture of CO 2 /THF as a function of molar fraction of THF Micronization results Micronization of synthetic β carotene was conducted, in order to evaluate particle size and morphology under different operating pressures. Micronization took place all around in the precipitation vessel, starting from its walls, except when the pressure was 75 bar, below mixture critical point, where crystals formed and accumulated at the bottom of the vessel. These observations are shown in Figure 4. Macroscopically, crystals appear to be like plain leaves colored dark red with some silver reflection, which was not verified in the initial product. (a) (b) Figure 4 Precipitation vessel after micronization: above critical point (a); below critical point (b) Product purity Product purity has been analyzed by HPLC and spectrophotometry and micronized and soluble product have been compared with raw β carotene. Two different wavelengths have been used to detect β carotene isomers (454 nm) and degradation oxides (260 nm), according to Randolph et al. [13]. Supercritical Micronization of β Carotene 6

7 Through HPLC, it has been detected no degradation or isomerization, as no peak was observed at 260 nm and one only peak was detected at 454 nm, which corresponds to all trans β carotene, by comparison with raw β carotene. Additionally, no significant difference between micronized, soluble and raw β carotene has been observed in their spectra. These results prove that isomerization and product oxidation don t happen in this process, as was also verified by Miguel et al. [16]. This can be explained by two reasons. First, CO 2 is inert. Second, even if there is a small amount of oxygen mixed with CO 2, its contact with β carotene only happens in the short period of time before micronization takes place. After that, oxidation reaction should proceed as an heterogeneous reaction, which is much slower than the homogeneous reaction Effect of operating pressure on particle size and morphology Micronization of β carotene has been successful for pressures above 75 bar, at 40 C. A trial has been made at 65 bar, but no powder has been obtained and the initial β carotene was recovered in the liquid solution in the expansion vessel. Thus, three different samples were analyzed, which were processed at 75, 100 and 130 bar, the former being formed below critical point. The initial product and processed powders were analyzed by scanning electron microscopy (SEM) and their size, size distribution and morphology were compared. Observing Figure 5, one can easily confirm the tridimensional aspect of raw β carotene crystals and their narrow particle size distribution. On the other hand, processed β carotene looks completely different in shape and size uniformity. (a) (b) Figure 5 Raw β carotene (SEM images, zoom 1000 (a) and 3000 (b)). Figure 6 shows the aspect of processed β carotene when the operating pressure is 75 bar (below critical point). In this figure, it s particularly evident the almost bidimensional structure of crystals, as well as its irregular leaf like shape, which can prove the aggregation of small particles in further stages of crystal growth, as proposed by Bristow et al. [34]. Particles formed below critical pressure aggregate more easily than those formed at higher pressures, which can be explained by their precipitation that occurs inside THF rich droplets, where submicrometric particles start to nucleate, coalesce and fuse during the growth stage that proceeds the nucleation step. Additionally to particle shape change relatively to raw β carotene, it has been observed that particles formed at 75 bar show a porous structure, which, Supercritical Micronization of β Carotene 7

8 Sofia Antunes September 2007 despite the increase in particle size, increases the surface area and can, thus, give way to interesting applications. (a) (b) Figure 6 β carotene processed at 75 bar (SEM images, zoom 100 (a) and 500 (b)). For the two trials above 75 bar, the mixture where precipitation occurred was already in a supercritical state, completely miscible and there exists only one phase inside the precipitation vessel. The precipitation mechanism is now gas phase nucleation, but one can easily assume that crystal growth has also occurred, because of the particle size obtained. At 100 bar, particle size distribution is relatively uniform, as can be observed in Figure 7, and particle size and morphology are quite different from those obtained at 75 bar. Particles formed at 100 bar show irregular rectangular or triangular shape, smooth surfaces and they are thin but still tridimensional. Cocero & Ferrero [14] have obtained similar particles in their study of β carotene precipitation. (a) (b) Figure 7 β carotene processed at 100 bar (SEM images, zoom 500 (a) and 5000 (b)). At 130 bar, the mixture inside the precipitation vessel is also in a supercritical state, but its density is higher than at 100 bar. Particles obtained in this trial show morphology similar to those obtained at 75 bar: they re thin and leaf like, particle size distribution is relatively large, but their surface is smooth like those obtained at 100 bar. Some particles also reveal possible aggregation during the process, due to their shape and size. Supercritical Micronization of β Carotene 8

9 (a) (b) Figure 8 β carotene processed at 130 bar (SEM images, zoom 200 (a) and 500 (b)). Particle size distributions obtained in different trials are shown in Figure 9. It s easy to verify again that particles obtained at 75 and 130 bar have similar particle size distributions. Smaller particles were obtained at 100 bar, but particle size distribution was less narrow. Figure 9 Particle size distributions obtained at different pressures. Above the critical point, there is a clear tendency for the increase of particle size with the increase of pressure (14 to 78 μm when pressure rises from 100 to 130 bar). Similar tendencies have been verified by other authors [16, 25, 27, 28, 29, 34]. The reason why this happens is the increase in density that comes from the increase of pressure. At higher densities, solubility of β carotene is also higher and supersaturation is lower. As particle size increases when supersaturation decreases, particle size increases with pressure. Through Peng Robinson equation of state, it is possible to estimate densities of the mixture where precipitation happens, at different pressures. Representation of average particle size as a function of density is in Figure 10, which shows a strong relationship between particle size and density of the mixture. Additionally, this relationship is extended to all the experimental points, including that one below critical point. Supercritical Micronization of β Carotene 9

10 Figure 10 Average particle size as a function of density of the mixture where precipitation occurs. Former affirmations about the similarity between particles obtained at 75 and 130 bar can now be confirmed by the comparable densities of mixtures where micronization takes place. Higher densities provoke not only lower supersaturation but also more aggregation, leading to bigger particles. The influence of pressure on particle size below critical point has not been studied. In this case, there are two unmixable phases and micronization occurs in the liquid THF rich phase by a mechanism of volumetric expansion, when CO 2 dissolves into THF. The higher the pressure, the larger the volumetric expansion of THF rich droplets and this fact leads to higher supersaturation and formation of smaller particles. Thus, if the micronization of β carotene at 65 bar had been successful, particles obtained would be even bigger than those obtained at 75 bar. This thesis is also supported by Figure 10, because, below critical point, higher pressure means lower density of droplets, and it was confirmed by other authors [4, 8, 19] Precipitation of a natural extract of β carotene In order to evaluate micronization as a purification process, a trial has been run using a natural extract of β carotene. The resulting micronized product was analyzed by HPLC and compared to the liquid. The micronized crystals of the extract look like the micronized synthetic β carotene, but their color is orange, instead of red. Due to the nature of the extract produced by microorganisms one can conclude with some conviction that impurities present in the extract are mostly low molecular weight compounds, typically products of microbial metabolism, that do not absorb at the same wavelength β carotene does. Otherwise, it was not possible to quantify β carotene present in this extract, because its real composition is unknown and there may also be some carotenoids or other impurities absorbing at β carotene wavelength. Thus, Lambert Beer law determined for synthetic β carotene is not appropriate. HPLC analyses (not shown) proved that liquid and micronized products obtained in this trial are quite similar, meaning that purification didn t occur. Spectra analyses don t allow to conclude anything. Supercritical Micronization of β Carotene 10

11 4. CONCLUSION Supercritical antisolvent micronization of synthetic and natural β carotene has been successful in this work. It has been shown that it is possible to micronize this carotenoid under experimental conditions below or above the mixture critical point and the minimal pressure for the precipitation to occur equals 75 bar. Depending on the pressure, it was possible to obtain microparticles ranging from 14 to 100 μm. Morphology and crystal aggregation also depends on pressure. It has also been established a linear relationship between average particle size and the density of the mixture where precipitation occurs, meaning that it s possible to control particle size by manipulating experimental conditions. A solubility study has also given way to some interesting data about this system s thermodynamics. Two main results show the exponential decrease of β carotene solubility in the supercritical mixture CO 2 /THF with the temperature increase, as a consequence of the decrease of density, and the exponential increase of solubility with the increase of THF molar fraction, independently of pressure, due to β carotene affinity for this organic solvent. Additionally, the presence of a co solvent (THF) enhances β carotene solubility by a factor of about two orders of magnitude, relatively to pure CO 2. This micronization process also allowed the precipitation of a biological extract of β carotene, but no purification was detected. This fact shows that this process can be applied in food and drug industries, for processing biological products, because carbon dioxide is inert, non toxic and low cost and the final product is free of organic solvents. However, experimental conditions must be optimized, in order to obtain some purification. REFERENCES 1 Reverchon, E., Journal of Supercritical Fluids, 15, Rogers, T. L., Johnston, K. P., Williams, R. O., Drug Development and Industrial Pharmacy, 27 (10), Reverchon, E., Caputo, G., De Marco, I., Ind. Eng. Chem. Res., 42, Costa, M. S., Duarte, A. R. C., Cardoso, M. M., Duarte, C. M. M., International Journal of Pharmaceutics, 328, De Marco, I., Reverchon, E., Eighth Conference on Supercritical Fluids and Their Applications. 6 Reverchon, E., De Marco, I., Caputo, G., Della Porta, G., Journal of Supercritical Fluids, 26, Reverchon, E., Della Porta, G., De Rosa, I., Subra, P., Letourneur, D., Journal of Supercritical Fluids 18, Reverchon, E., Della Porta, G., Powder Technology, 106, Reverchon, E., Journal of Supercritical Fluids, 15, Supercritical Micronization of β Carotene 11

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