A Comparative Study of Several Techniques to Obtain Fatty Acid Nanoparticles: Palmitic Acid R. Benages, L. Baye´s, R. Cordobilla, E. Moreno, T. Calvet, and Miquel A. Cuevas-Diarte* Departament de Cristal·lografia, Mineralogia i Dipo`sits Minerals, Facultat de Geologia, UniVersitat de Barcelona, Martı´ i Franque`s s/n, E-08028 Barcelona, Spain
CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 4 1762–1766
ReceiVed July 25, 2008; ReVised Manuscript ReceiVed NoVember 11, 2008
ABSTRACT: The aim of this study is to obtain fatty acid nanoparticles, especially of palmitic acid. Some synthetic paths were carried out: reprecipitation method, hot homogenization technique, and microemulsions. A qualitative comparison of particle size obtained with these and traditional methods such as dissolution/evaporation, melting/quenching, and sublimation were performed. The hot homogenization technique with addition of small amounts of aluminum chloride has been found to be suitable to obtain a narrow distribution of nanoparticles. Electron diffraction was attempted to characterize the crystal form obtained from the hot homogenization technique; nevertheless, the obtained phase cannot be identified due to overlapping reflections of the different fatty acid phases. Introduction The physical and chemical properties of nanoparticles are between those known to be associated with crystals and isolated particles.1 It is assumed that they will have interesting properties in terms of energy storage, catalysis,2 electronics, optics,3,4 and biomedical science, among others.1 Numerous works have been undertaken in the past 10 years by our team dealing with the use of long chain compounds (n-alkanes,5 n-alkanols,6 and n-carboxylic acids7) as molecular alloys phase change materials (MAPCM) for energy storage and thermal protection applications.8 In particular, some applications require the incorporation of small amounts of phase change materials (PCM) in complex matrices (i.e., porous bricks, concrete) in which the control and reduction of the particle size is decisive. In addition, we think that the thermal properties could be enhanced by increasing the heat transfer area using nanoparticles, and this should allow us to expand our range of applications. The general methods used to obtain nanoparticles include gasphase techniques (evaporation/condensation in inert gas), thermal decomposition, laser pyrolysis, colloidal, and ultrasonic approaches.2 However, although these procedures can be applied to inorganic substances they may not be appropriate for thermically unstable organic compounds.9 Different techniques have thus been developed to obtain organic nanoparticles. Reprecipitation Method.3,4,9-11 This technique is used to synthesize nanocrystals of polydiacetylene10,11 and perylene,3 among others. It consists in dissolving the reagent in alcohol or acetone at millimolar concentrations, and injecting afterward a few microliters of this into a solvent in which the reagent is poorly soluble (water is commonly used); this mixture is then shaken vigorously. Water and the organic solvent must be miscible.9 Hot Homogenization Technique. It is used to obtain nanoparticles of oleic acid with stearic acid (in the form of nanostructured lipid carriers12 (NLC)) and monostearin with clobetasol propionate (in the form of solid lipid nanoparticles13 (SLN)). The method consists of dissolving a small amount of * To whom correspondence should be addressed. Phone: (0048) 93 402 13 50. Fax: (0048) 93 402 13 40. E-mail:
[email protected]. Web: http:// www.ub.es.
reagent (around 400 mg) in a mixture of organic solvents (ethanol and acetone). This is then dispersed rapidly in 120 mL of distilled water with magnetic stirring at 70 °C (above the melting point of the reagent from which we wish to obtain the nanoparticles). The dispersion product is cooled to room temperature and the pH is adjusted to 1.20 with 0.1 M hydrochloric acid. The product is then centrifuged and the precipitate is redispersed in distilled water and lyophilized. Micro- and Nanoemulsions. This method is used to obtain cholesterol and retinol,14 stearic acid, behenic acid, and Acidan N12,15 hydrocortisone and progesterone complexes with β-cyclodextrin or 2-hydroxypropyl-β-cyclodextrin17 nanoparticles. Different surfactants are employed to obtain nanoparticles. The first step consists of melting of the reagent, and then the surfactant, the cosurfactant and hot water are added successively, shaking vigorously throughout. The product is a transparent microemulsion at a temperature close to the reagent’s melting point.15 Nanoparticles of oleic acid (and some of its esters) have also been obtained with the help of ultrasound.16 The preparation consists in mixing the water and oil in a given ratio. This mixture is first shaken for 1 min in a vortex mixer and then subjected to ultrasound for 8 min. Different techniques using compressed fluids, usually CO2, have been developed to obtain micro- and nanostructured materials. There are four main techniques in which CO2 acts as (1) solvent: rapid expansion of supercritical solutions (RESS);18 (2) antisolvent: the gas antisolvent process (GAS)19 and the aerosol solvent extraction system (ASES);20 and (3) cosolvent: depressurization of an expanded liquid organic solution (DELOS).21 These techniques are based on the decompression of a compressed fluid, which causes a drastic fall in temperature and, consequently, homogeneous nucleation throughout the reactor, limiting crystal growth. This procedure normally yields particles with a narrow size distribution, homogeneous morphology, and high polymorphic purity. A wide variety of drugs has been obtained in the size range of nanometers to a few microns.22 Nanoparticles of stearic acid using DELOS process are obtained with a very high polymorphic purity (Em phase).23 The aim of the present study is to obtain pure nanoparticles of fatty acids, particularly of palmitic acid. We also sought to establish the crystallization phase when the grain size is reduced and conduct a qualitative comparison of the particle size
10.1021/cg8008128 CCC: $40.75 2009 American Chemical Society Published on Web 02/05/2009
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Figure 1. Images of FE-SEM (a) dissolution of palmitic acid in ethanol and evaporation at 23 °C (D+E); (b) melting of C16OOH and quenching in liquid nitrogen (M+Q); and (c) sublimated sample.
and requires the combination of various techniques. A detailed study of the polymorphism of palmitic acid has already been conducted by our group.24 In some cases we have performed a similar work in stearic acid (C18H35O2H) with identical results. We think that the behavior could be extended to all the family. Experimental Procedures
Figure 2. FE-SEM micrograph for a reprecipitation method sample.
obtained via what we have called traditional methods and specific methods. We have considered as traditional methods those crystallization techniques commonly used in pharmaceutical investigation, polymorphism studies, and in general those methods used to obtain powders of crystalline material with no aim to have nanosized particles. Dissolution/evaporation, melting/quenching, and sublimation were chosen as traditional methods. We have considered as specific methods those crystallization techniques in which the main purpose is to obtain particles of reduced size, as described above. Reprecipitation, microemulsions, and hot homogenization technique were chosen as specific methods. Palmitic acid (C16OOH) is a normal saturated organic acid with 16 carbon atoms and no side groups. Its melting temperature is 334.3 ( 0.5 K.24 In the solid state seven different phases were identified and characterized: the triclinic A2 (P1j) and Asuper (P1j, Z ) 6); the monoclinic Em (P21/c, Z ) 4) and Bm (P21/c, Z ) 4); the orthorhombic Eo (Pbca, Z ) 8) and Bo (Pbca, Z ) 8); and the monoclinic C (P21/c, Z ) 4) phase (the most stable at high temperatures). The occurrence of each phase depends on many factors such as temperature and rate of crystallization, the type of solvent, and the purity of the acid itself. The structural characterization of the different phases is complicated
Palmitic acid (Fluka) was of 99% purity or higher, according to gas chromatography. Aluminum chloride hexahydrate (AlCl3 · 6H2O) was purchased by Fluka; its purity was g99%. Analytical grade ethanol and acetone and distilled water were also used. The following proceduress were tried to obtain palmitic acid nanoparticles: reprecipitation method, microemulsion, and hot homogenizaton technique. In the reprecipitation method, 150 µL (15 syringes of 10 µL) of a 2 mM solution of palimitic acid in ethanol is injected into 10 mL of vigorously shaken water. Microemulsions were performed using sodium dodecylsulphate (SDS) as surfactant. The experimental procedure involved hightemperature homogenization (in contrast to the micro/nanoemulsions described in the Introduction) with the addition of different concentrations of SDS. In the hot homogenization technique, a given amount of palmitic acid (around 190 mg) is dissolved in a mixture of acetone (6 mL) and ethanol (6 mL). This solution is then poured into 110 mL of distilled water at 70 °C and the product is shaken for 5 min; this produces a whitish suspension. Finally, the pH is adjusted to 1.2 with hydrochloric acid in order to enable the formation of clusters (which are separated by density). Detailed description of the parameters varied to optimize the precipitation conditions for palmitic acid are given in Results and Discussion. The morphology and size of the formed particles were analyzed using a Hitachi S-4100 field-emission scanning electron microscope (FESEM) at room temperature. Samples were mounted on a slide (12 mm diameter) with a conductive adhesive (Agar Scientific) and covered with a fine carbon layer (ca. 20 nm). Transmission electron microscopes (TEM: Hitachi H-800-MT at 100 and 200 kV; and Philips CM-30 at 300 kV) were used to obtain electron diffraction patterns at various chamber lengths. Prior to TEM observa-
Figure 3. H-800-MT TEM micrographs from microemulsion samples with a SDS concentration of (a) 0.5%; (b) 1%; (c) 5%; (d) 10%. Samples were diluted prior to the observation. Bars indicate 2 µm.
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Figure 6. Micrographs obtained by FE-SEM (a) lyophilized sample; (b) vacuum-dried sample at room temperature.
Figure 4. DSC analysis of synthesis with surfactant (SDS). From bottom to top: commercial sodium dodecylsulphate; commercial palmitic acid; 0.5% SDS; 1% SDS; 5% SDS; and 10% SDS. The different samples were vacuum-dried at room temperature. tions the sample was diluted and a drop was evaporated at room temperatures over a holey carbon grid. Differential scanning calorimetry (DSC) was performed at atmospheric pressure using a Perkin-Elmer DSC-7 calorimeter. Between 4.7 and 4.9 mg of the sample was placed in sealed aluminum capsules, and this was heated at 2 °C/min until melting.
Results and Discussion The experimental section is divided into two main parts: a qualitative estimation of particle size using the traditional methods or crystallization methods commonly used to obtain powder (dissolution/evaporation: D+E; melting/quenching: M+Q and sublimation) and a qualitative estimation of particle size using specific methods directed to obtain particles of nanometer size, principally of palmitic acid. Traditional Methods. Grain size was compared using scanning electron microscope (FE-SEM) images. In all three samples, a wide size distribution was observed. The general trends are that particles obtained with D+E methods are larger than M+Q, and these are larger than those obtained by the sublimation method. Clusters of nanoparticles deposited on the grains were observed with the melting/quenching procedure. Figure 1 shows a photograph taken with the scanning electron microscope (FE-SEM) for each test method. It can be seen in these images that the particle size, in all three samples, was in the micrometer range. Specific Methods. Experiments were conducted with three methods. The reprecipitation method yielded clusters with a rounded morphology displayed in Figure 2.
Compared with conventional methods a greater proportion of small particles (not reaching nanometric size) were observed. Because of the small amount of sample obtained we opted to search for other methods. In Microemulsions the addition of the following concentrations of SDS: 0.5%, 1%, 5%, and 10% (g of SDS/mL of H2O) give different morphologies. The experimental observations in the transmission electron microscope (Figure 3) show us that the proportion of nanoparticles decreases as the surfactant concentration increases. In contrast, it can be seen that an increasingly extensive continuous layer appears. In order to obtain significant micelle formation the concentration of the surfactant must be greater than the critical micelle concentration (CMC), which for SDS is 25 mM.25 The most diluted test, that is, 0.5%, was below the CMC and therefore no micelles were formed. The endothermic peak in the DSC analysis (Figure 4) confirmed the presence of palmitic acid. As the concentration of surfactant increases the melting peak of the acid becomes less energetic, thus indicating that the amount of C16OOH decreases. The presence of SDS is also confirmed by the low endothermic energy signal around 17 °C. Since the objective is to obtain pure nanoparticles, in other words, without any foreign element that might interfere with the determination of the properties of palmitic acid, it was decided to test the hot homogenization technique. In the hot homogenization technique, several parameters are taken into account: effect of pH, effect of the drying method, effect of the organic solution addition, and the influence of the aluminum chloride (AlCl3) addition. To evaluate the effect of pH, several samples were compared, adjusting the pH to 1.20 and not adjusting it. It was found that tests under an acid pH yield samples that in terms of grain size and morphology are more homogeneous (Figure 5a) than those in which the pH was not adjusted (Figure 5b). Furthermore, it was observed that when the pH was adjusted prior to adding the organic solvent the product contained a greater proportion
Figure 5. Micrographs obtained by FE-SEM (a) adjusting pH to 1.2; (b) without adjusting the pH; (c) adjusting the pH to 1.0 prior to adding the organic solution.
Techniques to Obtain Fatty Acid Nanoparticles
Crystal Growth & Design, Vol. 9, No. 4, 2009 1765 Table 1
Figure 7. Micrographs obtained by FE-SEM (a) addition all at once; (b) dropwise addition.
Figure 8. Micrograph obtained by CM-30 TEM (300 kV). Bar indicates 1 µm.
Figure 9. Micrographs and electron diffraction obtained with H-800MT microscopy at 200 kV of a sample 10% dispersed in water. The circle corresponds to the selected area electron diffraction (SAED).
spot no.
d-spacing (nm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.438 0.433 0.437 0.216 0.217 0.217 0.219 0.219 0.218 0.125 0.125 0.124 0.125 0.125 0.124
of small-grain particles (Figure 5c). In all samples rounded morphology was achieved. The three samples were vacuumdried at room, temperature. Different drying procedures were tested (lyophilization, vacuum-drying at room temperature and at 10 °C, drying at room temperature and in the refrigerator at 4 °C) in order to observe their effect on grain size. Here we only compare two procedures: lyophilization and drying at room temperature. As can be observed in Figure 6 the lyophilized samples showed fewer large-grain particles (Figure 6a) than those dried at room temperature (Figure 6b) did. As to the organic solution addition two experiments have to be pointed out: first, dropwise addition of the organic solvent, and second, adding it all at once. The former yielded a higher proportion of micrometric particles (smaller than 10 µm). This was also observed by Kasai1 in the reprecipitation method. The latter produced larger crystals, although there were also particles smaller than one micron. Figure 7 shows the comparison of two samples in which the synthetic path followed was the addition of the organic solvent (all at once, Figure 7a; and dropwise, Figure 7b) to 110 mL of acidified distilled water (pH ∼ 1.20) at 70 °C, the product then being quenched in liquid nitrogen from this temperature and lyophilized. As these three parameters were studied in the hot homogenization technique, no clear variation in particle size was observed. Aluminum chloride addition. We selected aluminum chloride as an additional reagent because we observed corrosion in
Figure 10. (left) HR-TEM image of the nanocrystals of palmitic acid (bar 10 nm) and (right) amplification showing the interplanar arrangement.
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aluminum films (which was used to cover the samples) of the acidified samples. TEM observations of one of those samples demonstrate homogeneous particle size. Thermal data and energy dispersive X-ray analysis (EDX) analysis gave evidence that some aluminum chloride was formed. A series of experiments were carried out by addition of small amounts of aluminum chloride (in ppm’s order). We conclude that the best performance was achieved by the addition of 500 ppm of AlCl3 in 110 mL of water of the hot homogenization technique and addition of the solution of palmitic acid dropwise. The role played by aluminum chloride could be explained by means of avoiding coagulation of the formed particles due to its adsorption on the hydrophobic part of the chains and its high charge density. Figure 8 shows a photograph of this experiment, in which a narrow distribution of nanoparticles is displayed. The sample was diluted in water (1%) after the observation. Structural Characterization. In order to determine the crystal form obtained from the hot homogenization technique we use the electron diffraction data obtained by transmission electron microscopes (Hitachi H-800-MT and Philips CM-30). The observation grid was mounted from the emulsion obtained in the synthesis after dilution between 1 and 10% in water, as appropriate. The diffraction patterns were acquired with a CCD camera and processed using the Digital Micrograph software in order to obtain a list of spacings and compare them with the reflections of phases C, A2, Bm and Em of palmitic acid. Figure 9 shows a micrograph of a crystal, as well as the selected area electron diffraction (SAED), and Table 1 gives some spots data. Unfortunately, with this kind of diffraction analysis, we are not able to establish unequivocally the present phase as the different structures of C16OOH are very similar. Some experiments changing the sample orientation have been run out by us, but no nearer spots suitable to identify the crystal phase have been detected. Routine electron diffraction experiments refer to an equivalent X-ray region in the range from 2θ ) 15° onward, where the reflections of these four phases overlap. Distinguishing the phase which crystallizes requires the observation, in the electron diffraction, of spots closer to the center. This would be equivalent to the long spacing region, where these four phases do not have overlap reflections. We have performed high resolution transmission electron microscopy (HR-TEM) studies in order to identify the crystal phase present in the sample. We found that the shorter interplanar distances are in the 0.3 nm region, which is not enough information to establish the present phase. Amplification of Figure 10 shows that the arrangement is quite homogeneous. Moreover, we cannot use epitaxial crystallization described by Dorset26 in similar compounds because it may lose its crystalline form in the process. Acknowledgment. We are very grateful to Quim Portillo of Serveis Cientificote`cnics of the University of Barcelona (SCT-
Benages et al.
UB) for his support and helpful explanations in the transmission electron microscopy field. We also thank the Ministerio de Ciencia y Tecnologia for the financial support through the CICYT (Grant No. BES-20021855, Project No. MAT20013352), the Generalitat de Catalunya through the Grup Consolidat (1996SGR0039), and Xarxa de Refere`ncia R+D+I en Materials Avanc¸ats per l’Energia (XARMAE).
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CG8008128
