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Effect of Compressed CO2 on the Properties of Lecithin Reverse Micelles Yueju Zhao, Jianling Zhang, Buxing Han,* Chaoxing Zhang, Wei Li, Xiaoying Feng, Minqiang Hou, and Guanying Yang Center for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China ReceiVed May 8, 2008. ReVised Manuscript ReceiVed June 17, 2008 Lecithin is a very useful biosurfactant. In this work, the effects of compressed CO2 on the critical micelle concentration (cmc) of lecithin in cyclohexane and solubilization of water, lysozyme, and PdCl2 in the lecithin reverse micelles were studied. The micropolarity and pH value of the polar cores of the reverse micelles with and without CO2 were also investigated. It was found that CO2 could reduce the cmc of the micellar solution and enhance the capacity of the reverse micelles to solubilize water, the biomolecule, and the inorganic salt significantly. Moreover, the water pools could not be formed in the reverse micelles in the absence of CO2 because of the limited amount of water solubilized. However, the water pools could be formed in the presence of CO2 because large amounts of water could be solubilized. All of these provide more opportunity for effective utilization of this green surfactant. The possible mechanism for tuning the properties of the reverse micelles by CO2 is discussed.
1. Introduction Surfactants in apolar solvents can form reverse micelles spontaneously. Reverse micelles have been used in many fields, including extraction and fractionation of proteins,1 enzyme catalysis,2 chemical reaction,3–5 and preparation of nanoparticles.6–10 Lecithin is a major component of the cell membrane as well as a very useful biosurfactant (Figure 1). The aggregation and interfacial properties of lecithin systems are of fundamental interest in biochemistry for understanding their biological function and different industrial applications.11–14 The structural and dynamic properties of lecithin reverse micelles have been extensively studied15–18 since Scartazzini and Luisi first reported their gel-like, viscoelastic lecithin/organic solvent/water reverse * Corresponding author. Fax: 6-10-62559373; tel: 86-10-62562821; e-mail:
[email protected]. (1) Freeman, K. S.; Tang, T. T.; Shah, R. D. E.; Kiserow, D. J.; McGown, L. B. J. Phys. Chem. B 2000, 104, 9312. (2) Mitra, R. N.; Dasgupta, A.; Das, D.; Roy, S.; Debnath, S.; Das, P. K. Langmuir 2005, 21, 12115. (3) Cuccovia, I. M.; Dias, L. G.; Maximiano, F. A.; Chaimovich, H. Langmuir 2001, 17, 1060. (4) Kane, M. A.; Baker, G. A.; Pandey, S.; Bright, F. V. Langmuir 2000, 16, 4901. (5) Jacobson, G. B.; Lee, C. T.; Johnston, K. P. J. Org. Chem. 1999, 64, 1201. (6) Vestal, C. R.; Zhang, Z. J. Chem. Mater. 2002, 14, 3817. (7) Ji, M.; Chen, X. Y.; Wai, C. M.; Fulton, J. L. J. Am. Chem. Soc. 1999, 121, 2631. (8) Kitchens, C. L.; Roberts, C. B. Ind. Eng. Chem. Res. 2004, 43, 4070. (9) Liu, J. C.; Raveendran, P.; Shervani, Z.; Ikushima, Y.; Hakuta, Y. Chem. Eur. J. 2005, 11, 1854. (10) Naoe, K.; Petit, C.; Pileni, M. P. Langmuir 2008, 24, 2792. (11) Mureseanu, M.; Galarneau, A.; Renard, G.; Fajula, F. Langmuir 2005, 21, 4648. (12) Avramiotis, S.; Papadimitriou, V.; Hatzara, E.; Bekiari, V.; Lianos, P.; Xenakis, A. Langmuir 2007, 23, 4438. (13) Madamwar, D.; Thakar, A. Appl. Biochem. Biotechnol. 2004, 118, 361. (14) Van Horn, W. D.; Simorellis, A. K.; Flynn, P. F. J. Am. Chem. Soc. 2005, 127, 13553. (15) Schurtenberger, P.; Scartazzini, R.; Magid, L. J.; Leser, M. E.; Luisi, P. L. J. Phys. Chem. 1990, 94, 3695. (16) Capitani, D.; Rossi, E.; Seger, A. L. Langmuir 1993, 9, 685. (17) Schurtenberger, P.; Cavaco, C. Langmuir 1994, 10, 100. (18) Tung, S. H.; Huang, Y. E.; Raghavan, S. R. J. Am. Chem. Soc. 2006, 128, 5751.
micellar solutions.19 The lecithin reverse micelles have been used in enzymatic interesterification,20,21 lipase-catalyzed hydrolysis,22 fatty acid esterification,23 the food industry,24 and nanomaterials synthesis.25,26 The lecithin reverse micelles have many advantages, but their ability to solubilize water is poor, which limits its application to a great extent. Scartazzini and Luisi determined the maximum values of water-to-lecithin molar ratio (W0max) in different organic solvents, and all the values were smaller than 12.19 Different short-chain alcohols (1-propanol, 1-butanol, 1-pentanol, 1-hexanol, and 1-octanol) have been employed as cosolvents to enhance water solubilization,27,28 which suffers from economic and environmental costs, as well as contamination or modification of products by these additives. Development of effective, controllable, and environmentally benign methods to enhance the solubilization of polar substances in lecithin reverse micelles is desirable and challenging. CO2 is plentiful, inexpensive, and nontoxic. Supercritical or compressed CO2 has been used in different fields, such as selective extraction of phosphatidylcholine (PC) from deoiled soybean lecithin,29 preparation of encapsulated proteins,30,31 extraction of protein from organisms,32 enzymatic catalysis with supercritical (19) Scartazzini, R.; Luisi, P. L. J. Phys. Chem. 1988, 92, 829. (20) Marangoni, A. G.; McCurdy, R. D.; Brown, E. D. J. Am. Oil. Chem. Soc. 1993, 70, 737. (21) Komatsu, T.; Nagayama, K.; Imai, M. Colloids Surf., B 2004, 38, 175. (22) Nagayama, K.; Matsu-ura, S.; Doi, T.; Imai, M. J. Mol. Catal. B: Enzym. 1998, 4, 25. (23) Nagayama, K.; Yamasaki, N.; Imai, M. Biochem. Eng. J. 2002, 12, 231. (24) Patel, N.; Schmid, U.; Lawrence, M. J. J. Agric. Food Chem. 2006, 54, 7817. (25) Sahiner, N.; Singh, M. Polymer 2007, 48, 2827. (26) Simmons, B. A.; Li, S. C.; John, V. T.; McPherson, G. L.; Bose, A.; Zhou, W. L.; He, J. B. Nano Lett. 2002, 2, 263. (27) Yamazaki, K.; Imai, M.; Suzuki, I. Colloids Surf., A 2007, 293, 241. (28) Avramiotis, S.; Bekiari, V.; Lianos, P.; Xenakis, A. J. Colloid Interface Sci. 1997, 194, 326. (29) Teberikler, L.; Koseoglu, S.; Akgerman, A. J. Am. Oil. Chem. Soc. 2001, 78, 115. (30) Sellers, S. P.; Clark, G. S.; Sievers, R. E.; Carpenter, J. F. J. Pharm. Sci. 2001, 90, 785. (31) Chaitanya, V. S. V.; Senapati, S. J. Am. Chem. Soc. 2008, 130, 1866. (32) Dunford, N. T.; Temelli, F.; LeBlanc, E. J. Food Sci. 1997, 62, 2899.
10.1021/la801427b CCC: $40.75 2008 American Chemical Society Published on Web 07/23/2008
Properties of Lecithin ReVerse Micelles
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Figure 1. The chemical structure of lecithin.
CO2 as the solvent,33,34 enhancing the solubilization of water in sodium bis-2-ethylhexylsulfosuccinate (AOT) and Triton X-100 reverse micellar systems,35,36 induction of nanoemulsions,37 and as an antisolvent for different applications.38 In this work, we studied the effect of compressed CO2 on the solubilization of water, lysozyme, and PdCl2 in lecithin reverse micelles. The effects of CO2 on the micropolarity and pH value of the polar domains of the reverse micelles were also studied. It was found that CO2 could enhance the ability of the reverse micelles to solubilize water, the biomolecule, and the inorganic salt significantly. Another interesting point was that water pools could be formed in the reverse micelles in the presence of CO2, while water pools cannot be formed in the CO2-free system because of the limited amount of water solubilized. All of these provide more opportunity for effective utilization of this green surfactant.
2. Materials and Methods Materials. Lecithin (biological purity) purchased from Beijing Chemical Reagent Company was kept in a desiccator and used without any further purification. Lysozyme (biological purity) was purchased from Sinopharm Chemical Reagent Co. Ltd. Cyclohexane, ethanol, and methyl orange (MO) (all are analytical grade) were supplied by Beijing Chemical Plant. CO2 (>99.995%) was purchased from Beijing Analytical Instrument Factory. Palladium chloride (PdCl2) (analytical grade) was purchased from Beijing Chemical Reagent Company. Bromophenol blue (BPB) was supplied by Beijing Xudong Chemical Reagent Company. Citric acid (analytical grade, Chengdu Chemical Plant) and trisodium citrate dihydrate (analytical grade, Beijing Yili Fine Chemical Plant) were used to prepare the buffer solutions of different pH values. p-tert-Butylphenol (t-BP) was purchased from Shanghai Chemical Reagent Co. Ltd. HCl (analytical grade) was purchased from Beijing Chemical Reagent Company. The double-distilled water purchased from Beijing Kebanzhengye Pure Water Corporation was used throughout the experiments. Phase Behavior of the Lecithin/Cyclohexane Solution in the Presence of CO2. The apparatus and procedures to determine the phase behavior of the lecithin/cyclohexane solution were similar to those used previously.39,40 The apparatus consisted mainly of a highpressure view cell, a constant-temperature water bath, a high-pressure syringe pump (DB-80), a magnetic stirrer, a gas cylinder, and a pressure gauge which was accurate to (0.025 MPa in the pressure range of 0-20 MPa. The YKKY A2 digital controller was used to control the temperature of the water bath, of which the accuracy was (0.1 K. In the experiment, a suitable amount of the lecithin/ cyclohexane solution ([lecithin] ) 40 mM) was added into the view cell after the air in the cell was replaced by CO2. Then the temperature (33) Holmes, J. D.; Steytler, D. C.; Rees, G. D.; Robinson, B. H. Langmuir 1998, 14, 6371. (34) Novak, Z.; Habulin, M.; Krmelj, V.; Knez, Z. J. Supercrit. Fluids 2003, 27, 169. (35) Shen, D.; Zhang, R.; Han, B. X.; Dong, Y.; Wu, W. Z.; Zhang, J. L. Chem. Eur. J. 2004, 10, 5123. (36) Shen, D.; Han, B. X.; Dong, Y.; Wu, W. Z.; Chen, J. W.; Zhang, J. L. Chem. Eur. J. 2005, 11, 1228. (37) Zhang, J. L.; Han, B. X.; Zhang, C. X.; Li, W.; Feng, X. Y. Angew. Chem., Int. Ed. 2008, 47, 3012. (38) Jessop, P. G.; Subramaniam, B. Chem. ReV. 2007, 107, 2666. (39) Zhang, H. F.; Lu, J.; Han, B. X. J. Supercrit. Fluids 2001, 20, 65. (40) Zhang, R.; Liu, J.; He, J.; Han, B. X. Macromolecules 2002, 35, 7869.
of the system was controlled at 303.2 K. After the thermal equilibrium had been reached, CO2 was charged into the cell until a suitable pressure was reached. The magnetic stirrer was used to enhance the mixing of CO2 and the solution. The pressure and the volume at equilibrium condition were recorded. More CO2 was added, and the volume of the liquid phase at another pressure was determined. The volume-expansion coefficient was obtained based on the liquid volumes before and after the dissolution of CO2. The solution became cloudy as the pressure was high enough because of the antisolvent effect of CO2. We defined this pressure as the cloud-point pressure. Water Solubilization of the Lecithin Reverse Micelles with and without CO2. The apparatus and procedures to study the water solubilization in the lecithin reverse micelles were also similar to those for the study of the phase behavior described above.39,40 In a typical experiment, the air in the view cell was replaced by CO2. Then the desired amount of double-distilled water was loaded into the high-pressure view cell with the lecithin/cyclohexane solution ([lecithin] ) 40 mM). The cell was placed into the constanttemperature water bath of 303.2 K, and the stirrer was started. CO2 was charged into the cell slowly until the hazy and milky liquid solution became transparent and completely clear, which was an indication of the solubilization of all the water.40–42 More CO2 was added until the lecithin/cyclohexane/water solution became cloudy again. Both transparent point and cloud point were recorded, so that the phase diagram could be obtained. Micropolarity of the Lecithin Reverse Micelles by UV-vis Study. The apparatus and procedures to study the micropolarity of the lecithin reverse micelles at different CO2 pressures were the same as those used previously.39 The apparatus consisted mainly of a gas cylinder, a high-pressure pump, a pressure gauge, a temperature controller, and a high-pressure UV sample cell. The UV-vis spectrometer was produced by the Beijing Instrument Company (TU-1901). In a typical experiment, the desired amount of methyl orange (MO)/alcohol solution was loaded into the sample cell and the alcohol was removed by blowing nitrogen slowly. The lecithin/ cyclohexane/H2O solution was added into the sample cell, which was maintained at 303.2 K. Then compressed CO2 was added until the desired pressure was reached. The stirrer was started to promote equilibration. The concentration of the probe MO was 4.0 × 10-5 M. The absorbance spectrum measurement was started, and the spectrum was recorded. pH of the Lecithin Reverse Micelles. The UV-vis spectra of BPB in citric acid buffers of different pH values were first determined and used as working curve. The high-pressure UV sample cell and the experimental procedures used to determine the pH values of the polar cores of the reverse micelles in the presence of CO2 were similar to those used to study the micropolarity described above. In the experiment, known amount of BPB aqueous solution ([BPB] ) 4 × 10-6 M) was added into the reverse micellar solution. Then the suitable amount of water was injected into the solution to reach the desired W0. The UV-vis spectra of BPB were recorded at different CO2 pressures. The pH values of water cores in lecithin reverse micelles at different CO2 pressures were obtained after comparing with the working curve of the buffers. Critical Micelle Concentration (cmc) of the Lecithin/Cyclohexane Solution. The apparatus and procedures to determine the cmc of lecithin/cyclohexane solution were similar to those used for the micropolarity measurement, as described above. In a typical (41) Alexandridis, P.; Andersson, K. J. Colloid Interface Sci. 1997, 194, 166. (42) Alexandridis, P.; Andersson, K. J. Phys. Chem. A 1997, 101, 8103.
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Figure 2. The dependence of volume expansion coefficient ∆V of lecithin/cyclohexane solution ([lecithin] ) 40 mM) on CO2 pressure at 303.2 K.
experiment, the desired amount of the t-BP/cyclohexane and lecithin/ cyclohexane solutions were loaded into the sample cell, and then the cell was charged with CO2. The concentration of the probe was 5 × 10-5 M after the solution was expanded by CO2. The differential signals of ∆A/∆C (where A and C stand for the value of UV absorbance and concentration of lecithin, respectively) were plotted against the lecithin concentrations to get the cmc values. Solubilization of Lysozyme in CO2/Reverse Micellar Solutions. The apparatus and procedures were similar to those used for determining the water solubilization described above. In a typical experiment, 4 mL of reverse micellar solution containing excessive lysozyme was loaded into the high-pressure view cell, which was immersed in the water bath. After the thermal equilibrium was reached, CO2 was pumped slowly into the cell until the solution became absolutely transparent and clear, indicating that lysozyme was completely solubilized. As the CO2 pressure reached a sufficiently high value, the solution became turbid again, which was indicative of the precipitation of lysozyme. Solubilization of PdCl2 in CO2/Reverse Micellar Solutions. The apparatus and procedures to study the solubilization of PdCl2 were also similar to those used for determining solubilization of water in the reverse micellar solution described above. In the experiment, the desired amount of PdCl2 aqueous solution of known concentration was added into the view cell that was filled with lecithin/ cyclohexane solution. The view cell was placed into the constanttemperature water bath of 303.2 K, and the stirrer was started. CO2 was charged into the cell slowly until the hazy liquid solution became transparent and completely clear, which was an indication of complete solubilization of the PdCl2. As the CO2 pressure reached a sufficiently high value, the solution became turbid again, which was indicative of the precipitation of the PdCl2.
3. Results and Discussion Phase Behavior of the Lecithin/Cyclohexane Solution in the Presence of CO2. CO2 is a nonpolar molecule and can dissolve in apolar organic solvents and expand the solvents. The volumeexpansion coefficient ∆V (∆V ) (V - V0)/V0, where V and V0 are the volumes of the CO2-saturated and CO2-free solution) of lecithin/cyclohexane solution expanded by CO2 was determined, which is shown in Figure 2. As we can see, the ∆V value increases with increasing pressure. In the low pressure range, the ∆V changes slowly and increases sharply at higher pressure. As the pressure of CO2 is high enough, the lecithin/cyclohexane solution became cloudy due to the surfactant beginning to precipitate. The cloud-point pressure determined in this work for the reverse micellar solution is 5.50 MPa. In this work, all the experiments were conducted below 5.50 MPa to avoid the precipitation of the surfactant. Effect of CO2 on Water Solubilization in Reverse Micelles. Figure 3 shows the effect of CO2 on the maximum water-tosurfactant molar ratio (W0max) in the lecithin reverse micelles at
Zhao et al.
Figure 3. The dependence of maximum water-to-surfactant molar ratio (W0max) in the lecithin reverse micellar solution ([lecithin] ) 40 mM) on CO2 pressure at 303.2 K.
303.2 K and different pressures. In the absence of compressed CO2, the W0max of the lecithin micelles is as small as 9. However, the W0max is much larger in the presence of CO2 in suitable pressure range. For example, the W0max can reach 45 at 4.95 MPa. A similar solubilization phenomenon was observed previously for the Triton X-100/cyclohexane and AOT/n-alkane reverse micellar systems.35,36 As the pressure of CO2 was too high, the solubilized water precipitated because of the antisolvent effect of CO2. The pressure at which the water begins to precipitate is defined as the precipitation pressure. At a fixed W0 value, both the clear-point pressure and the precipitation pressure were determined, and the results are shown in Figure 3. The system is the single-phase region between the two curves, and all the water can be solubilized in the reverse micelles in this region. The system is separated into two phases outside this region. The precipitation pressure decreases with increasing amount of water, while the clear-point pressure increases with increasing amount of water. The single-phase becomes narrow with increasing amount of water. Micropolarity in Reverse Micelles at Different CO2 Pressures. Methyl orange (MO) is a commonly used solvatochromic probe to study the micropolarity of microemulsions because its absorption maximum λmax is sensitive to the polarity of its environment.43,44 The λmax of MO shifts to longer wavelengths (red shift) with increasing polarity. In this work, we used MO as the probe to detect the micropolarity of the dispersed droplets in the lecithin/cyclohexane/water microemulsions. As example, Figure 4a shows some UV-vis spectra of MO in the reverse micelles at different W0 values and at CO2 pressure of 5.00 MPa. Figure 4b presents the λmax of MO as a function of W0 with and without CO2. In the absence of CO2 the W0 is limited to 9, and the λmax of MO in the lecithin/cyclohexane/ water reverse micelles increases from 405.5 to 414.5 nm. This indicates that the micropolarity of the reverse micelles is increased with addition of water, which is similar to those reported in literature.35,36 In the presence of CO2, the W0 can be much higher. The λmax of MO increases with W0, as W0 is less than 15. However, λmax is independent of amount of water at larger W0. A reasonable explanation for this phenomenon is that MO mainly exists in the polar head region of the surfactants. In the low W0 region, water molecules are mainly bound to the polar heads of the surfactants. The polarity of the polar region increases with the increase of W0 because more water molecules exist near the polar heads of the surfactants. After W0 exceeds 15, water pools are formed and further increase of the amount of water only enlarges the size of the water pools. Therefore, the polarity in the polar head (43) Zhu, D. M.; Schelly, Z. A. Langmuir 1992, 8, 48. (44) Clarke, M. J.; Harrison, K. L.; Johnson, K. P.; Howdle, S. M. J. Am. Chem. Soc. 1997, 119, 6399.
Properties of Lecithin ReVerse Micelles
Figure 4. (a) UV-vis spectra of MO in the lecithin/cyclohexane/H2O reverse micelle solutions ([lecithin] ) 40 mM) with different W0 values at CO2 pressure of 5.00 MPa; (b) the λmax of MO as a function of the W0 in the lecithin/cyclohexane/H2O reverse micelle solutions ([lecithin] ) 40 mM) with (∆) and without (9) CO2.
region keeps unchanged. Therefore, we can deduce that water pools cannot be formed in the system in the absence of CO2, while they can be formed in the presence of CO2. In many cases, the water pools are crucial for effective applications of microemulsions. Therefore, CO2 has great potential to widen the applications of the biosurfactant. Furthermore, the λmax of MO without CO2 is larger than that in the presence of CO2 at the same W0 value, as can be known from Figure 4b. The main reason for this phenomenon may be that some CO2 molecules exist in the polar head region, which reduces the polarity because CO2 is a nonpolar compound. The MO also exists mainly in this region, and the λmax of MO is smaller in the presence of CO2. pH of the Polar Cores of the Reverse Micelles at Different CO2 Pressures. Dissolution of CO2 in water can produce ions (e.g., H+, HCO3-) that alter the pH in the water cores.45 Herein the pH values of the water cores inside the lecithin reverse micelles at different CO2 pressures were measured using UV-vis spectra. An acid-sensitive indicator (BPB) was used as the probe.45–47 The UV spectra of BPB in citric acid buffers with different pH values were first determined, two absorption peaks appear at 430 and 590 nm, and the spectrum is related to the proton dissociation of BPB.46,47 At lower pH, BPB exists as a neutral molecule, which has an electronic transition at 430 nm, while at higher pH, it exists as an ionized species and an electronic transition occurs at 590 nm. With the decreasing pH, the absorbance at 430 nm increases, while the absorbance at 590 nm decreases. The absorbance intensity ratio I430/I590 vs pH curve of the solutions with known pH values can be used as a working curve to obtain the pH values of other solutions.47 pH value measurement of the water cores inside the reverse micelles were carried out at different CO2 pressures in the W0 (45) Liu, D. X.; Zhang, J. L.; Han, B. X.; Fan, J. F.; Mu, T. C.; Liu, Z. M.; Wu, W. Z.; Chen, J. J. Chem. Phys. 2003, 119, 4873. (46) Fattah, A. A. A.; Kelany, M. E.; Rehim, F. A.; Miligy, A. A. E. J. Photochem. Photobiol. 1997, 110, 291. (47) Wei, Y. J.; Li, K. A.; Tong, S. Y. Talanta 1996, 43, 1.
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Figure 5. (a) UV-vis spectra of BPB (4 × 10-6 M) in the water/ lecithin/cyclohexane reverse micelles ([lecithin] ) 40 mM, W0 ) 9) at different CO2 pressures; (b) the dependence of pH of the water cores on pressure at different W0 values.
range from 9 and 20. As examples, Figure 5a demonstrates some spectra of the BPB in the water cores of the reverse micelles at W0 ) 9 and CO2 pressures. It can be seen that the absorbance around 430 nm increases with the increasing CO2 pressure, while the absorbance around 590 nm is decreased. The pH value of the water cores of the lecithin reverse micelles under different CO2 pressures were obtained from the working curve of citric acid buffers. Figure 5b shows the pH values as a function of CO2 pressure at W0 of 9 and 20, respectively. We can see that at both W0 values, the pH of the water cores decreases with the increasing CO2 pressure. This indicates that CO2 in the reverse micelles can be ionized. According to Henry’s law, an increase in pressure should enhance the solubility of CO2 in the water cores. Therefore, more CO2 is ionized and the acidity of the water cores increases. In addition, Figure 5b shows clearly that at the same CO2 pressure, the pH at W0 ) 9 is larger than that at W0 ) 20. This results mainly from the fact that there is no water pool in the reverse micelles at W0 ) 9, while water pools exist at W0 ) 20, as discussed above. In other words, the degree of ionization of CO2 in the water pools is larger than that in the bound water domains. cmc at Different CO2 Pressures. UV-vis spectroscopy is one of the useful techniques to determine the cmc of surfactant solutions,48–52 and tert-butylphenol (t-BP) is often used as the probe. The absorbance of t-BP at 276 nm increases with increasing concentration of lecithin in the premicellar concentration region. When the lecithin concentration reaches the cmc value, the absorbance has a reduction trend. In this work, the cmc values of lecithin in cyclohexane with or without compressed CO2 were (48) Murali, M. K.; Jayakumar, R.; Rakshit, S. K. Langmuir 1996, 12, 4068. (49) Chen, J.; Zhang, J. L.; Han, B. X.; Feng, X. Y.; Hou, M. Q.; Li, W. J.; Zhang, Z. F. Chem. Eur. J. 2006, 12, 8067. (50) Toews, K. L.; Robert, M. S.; Wai, C. M.; Smart, N. G. Anal. Chem. 1995, 67, 4040. (51) Jayakumar, R.; Mandal, A. B.; Manoharan, P. T. J. Chem. Soc. Chem. Commun. 1993, 853. (52) Jayakumar, R.; Jeevan, R. G.; Mandal, A. B.; Manoharan, P. T. J. Chem. Soc. Faraday Trans. 1994, 90, 2725.
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Figure 8. Dependence of maximum lysozyme concentration ([lysozyme]max) in the lecithin/cyclohexane reverse micellar solution ([lecithin] ) 40 mM) on the pressure of CO2 at 303.2 K and W0 ) 20.
Figure 6. (a) UV-vis spectra of t-BP (5 × 10-5 M) in lecithin/ cyclohexane solution at various lecithin concentrations in absence of CO2; (b) the plot of the differential signals of UV absorbance (∆A/∆C) of t-BP.
Figure 7. Dependence of cmc value of the lecithin/cyclohexane reverse micelles on the pressure of CO2 at 303.2 K.
determined by UV-vis method using t-BP as probe. As examples, Figure 6a shows some absorption spectra of t-BP in lecithin/ cyclohexane solutions with different lecithin concentrations at 303.2 K in the absence of CO2. The dependence of the differential signals of UV absorbance (∆A/∆C, where A and C are the UV absorbance and the concentration of lecithin, respectively) of t-BP in lecithin/cyclohexane solution on the lecithin concentration at 303.2 K is shown in Figure 6b. The shape is similar to those of other surfactant solutions.48,49 The absorbance of t-BP shows a discontinuous and abrupt change at the cmc value, where ∆A/ ∆C is zero. From Figure 6b, the cmc of lecithin in cyclohexane was found to be 10.3 mM, which agrees with the value measured by other method.53 The cmc at different CO2 pressures were determined, and the results are given in Figure 7. The cmc of lecithin in cyclohexane decreases with increasing pressure of CO2 in a low-pressure range. This indicates that addition of CO2 is favorable to the formation of the reverse micelles. In the high-pressure range, however, an increase in pressure results in increase in the cmc. Therefore, the ability of the surfactant to form reverse micelles can be tuned by CO2 pressure. It has been reported that cmc is (53) Koike, H. Biochem. Eng. J. 2007, 36, 38.
often related with the solubilization capacity, and the solubilization capacity is increased with the decrease of cmc.54,55 The decrease of cmc with increasing CO2 pressure in the low pressure range can partly explain the enhancement of water solubilization by the addition of CO2. Effect of CO2 on the Solubilization of Lysozyme in Reverse Micelles. Reverse micelles or water-in-oil microemulsions have many applications in biotechnology because of their ability to solubilize biomolecules in the water cores. For example, they can be used in purification of proteins,56 enzymatic reactions,57 and drug delivery.58 Enhancement of the biomolecules solubilization in microemulsions is crucial to the applications. Our previous work showed that the solubilization of BSA could be enhanced by compressed CO2 in the AOT/isooctane reverse micelles. 59 The effect of compressed CO2 on the solubilization of lysozyme in the lecithin reverse micelles of W0 ) 20, and the results are shown in Figure 8. Apparently, with the adding of CO2, the maximum concentration is increased significantly in the low-pressure range and then decreases with increasing CO2 pressure after passing through the maximum. At 5.1 MPa, the micellar solution loses the ability to solubilize lysozyme. This result is very interesting for application. For example, in protein extraction and recovery, we can enhance the extraction efficiency at lower CO2 pressure and then precipitate the protein completely from reverse micelles at higher pressure. In addition, through the easy control of CO2 pressure, the precipitation of surfactant can be avoided because the pressure at which the surfactant begins to precipitate is 5.50 MPa, as discussed above. Thus, pure protein can be obtained and postprocess is much easier than the traditional demulsifying method.60,61 Effect of CO2 on the Solubilization of PdCl2 in Reverse Micelles. We also investigated the effect of CO2 on the solubilization of inorganic salt PdCl2 in the reverse micelles, and the results are shown in Figure 9. At W0 ) 20, with the continuous adding of CO2, maximum PdCl2 concentration is increased with CO2 pressure. At CO2 pressure of about 4.90 MPa, the PdCl2 solubility reached a maximum value of 0.165 mM. As the pressure is higher than 4.91 MPa, the solubility of PdCl2 in the reverse micellar solutions decreases with the pressure increasing, indicating that higher pressure is not favorable to the solubilization (54) Zhou, W. J.; Zhu, L. Z. Colloids Surf. A 2005, 255, 145. (55) Negm, N. A. J. Surfactants Deterg. 2007, 10, 71. (56) Pires, M. J.; Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Prog. 1996, 12, 290. (57) Klyachko, N. L.; Levashov, A. V. Curr. Opin. Colloid Interface Sci. 2003, 8, 179. (58) Lawrence, M. J.; Rees, G. D. AdV. Drug. DeliVery ReV. 2000, 45, 89. (59) Feng, X. Y.; Zhang, J. L.; Chen, J.; Han, B. X.; Shen, D. Chem. Eur. J. 2006, 12, 2087. (60) Lye, G. J.; Asenjo, J. A.; Pyle, D. L. Biotechnol. Bioeng. 1995, 47, 509. (61) Shin, Y. O.; Weber, M. E.; Vera, J. H. Biotechnol. Prog. 2003, 19, 928.
Properties of Lecithin ReVerse Micelles
Figure 9. Dependence of PdCl2 concentration in the lecithin reverse micelles ([lecithin] ) 40 mM, W0 ) 20) on the pressure of CO2 at 303.2 K.
Figure 10. The effect of pH of HCl aqueous solution ([lecithin] ) 40 mM) on the W0max of lecithin/cyclohexane reverse micelles at 303.2 K.
of PdCl2 in reverse micelles. At 5.08 MPa, the PdCl2 can be completely precipitated from the lecithin reverse micelles. The method to tune the solubilization of inorganic salts in the reverse micelles has potential applications in different fields, such as preparation of nanoparticles and catalytic reactions. We also studied the solubilization of lysozyme and PdCl2 in the revise micelles at W0 ) 9 without CO2, and it was demonstrated that the solubilization amount was negligibly small. The main reason may be that in the absence of CO2, the water has no bulk nature because of bonding to the head groups of the surfactants. In the presence of CO2, the W0 is much larger and water pools exist in the reverse micelles. Therefore the solubilization amount can be enhanced significantly. Possible Mechanism. The effect of CO2 on the properties of the reverse micelles is very complex because it distributes among organic continuous phase, interfacial film of the reverse micelles, and the polar cores of the reverse micelles. It is very difficult to give a convinced explanation for the effect of CO2 on the stability of the reverse micelles. However, we can deduce that CO2 influences the stability of the reverse micelles mainly in three ways: (1) CO2 is a small molecule, and it can insert itself between the surfactant tails,35,36,62 which leads to a more rigid interfacial film and consequently stabilizes the micelles; (2) the CO2 in the organic phase reduces the solvent strength of the solvent, 37 which should reduce the ability to solubilize water; (3) CO2 changes the pH in the water cores of the reverse micelles. In order to obtain some information about how pH affects the stability of the reverse micelles, we studied the effect of pH on the solubilization of water in the absence of CO2 by using HCl solution with known pH values. The W0max at different pH values is demonstrated in Figure 10. It can be seen that the W0max is almost (62) Senapati, S.; Berkowitz, M. L. J. Phys. Chem. B 2003, 107, 12906.
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independent of pH within the pH range of 3-6. Therefore, it can be deduced that the changes in pH originated from the dissolution of CO2 is not the main reason for the change of the stability of the reverse micelles. In other words, the effects of CO2 on the properties of the interfacial film and organic continuous phase are the dominant factors. In the low pressure region the first factor plays a main role, and the reverse micelles are more stable and more water, lysozyme and PdCl2 can be solubilized. Therefore, the W0max increases with the CO2 pressure increasing. In the high pressure region, the concentration of CO2 in the organic phase becomes very high, as can be known from Figure 2. Therefore, the second factor above becomes dominant, and further addition of CO2 reduces the stability of the reverse micelles.
4. Conclusion The effects of compressed CO2 on the cmc of the lecithin in cyclohexane, the solubilization capacity of the lecithin reverse micelles for different substances, and micropolarity and pH of the polar cores of the reverse micelles have been studied at 303.2 K. The main conclusions are as follow. The W0max of the lecithin reverse micelles is 9 in the absence of CO2 and increases with increasing CO2 pressure in the lowpressure range and can reach 45 at 4.95 MPa. There are no obvious water pools in the reverse micelles at W0 of less than 15, and water pools can be formed as W0 exceeds 15. Therefore, the water pools cannot be formed in the system in the absence of CO2, while they can be formed in the presence of CO2. CO2 has great potential to widen the application of the biosurfactant because in many cases the water pools are crucial for effective applications of microemulsions. In the low-pressure range, CO2 can enhance the solubilization of lysozyme and PdCl2 significantly, and the solubilized biomolecule and inorganic salt can be precipitated completely at higher pressure, while the surfactant remains in the solution. The method to tune the solubilization of substances in the reverse micelles of this green surfactant may be used in different fields, such as extraction and fractionation of biomolecules, catalytic reactions, and preparation of nanoparticles. The cmc of lecithin in cyclohexane decreases with the increasing CO2 pressure in a low-pressure region, while increase in pressure results in increase in the cmc in high-pressure region. In other words, the stability of the reverse micelles can be controlled by CO2 pressure, which is consistent with the fact that the solubilization of the substances can be tuned by CO2 pressure. The pH value of the polar domains inside the reverse micelles decreases with the increasing CO2 pressure because of partial ionization of CO2 in the reverse micelles. At the same CO2 pressure, the pH at W0 ) 9 is larger than that at W0 ) 20 because there is no water pool in the reverse micelles at W0 ) 9, while water pools exists at W0 ) 20. This indicates that the degree of ionization of CO2 in the water pools is larger than that in the water domains bound to the head groups of the surfactants. The change in pH originated from the dissolution of CO2 in the polar domains of the reverse micelles is not the main reason for the change of the stability of the reverse micelles. The insertion of CO2 into the interfacial film of the reverse micelles to enhance its rigidity and the reduction of the solvent power of the organic continuous phase by dissolution of CO2 may be the main reasons. Acknowledgment. The authors are grateful to the National Natural Science Foundation of China (20633080) for financial support. LA801427B
