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Purification process of octacosanol extracts from rice bran wax by molecular distillation
Journal of Food Engineering 79 (2007) 63–68 www.elsevier.com/locate/jfoodeng
Purification process of octacosanol extracts from rice bran wax by molecular distillation Fang Chen, Zhengfu Wang, Guanghua Zhao, Xiaojun Liao, Tongyi Cai, Linyu Guo, Xiaosong Hu * College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China Received 24 September 2005; accepted 15 January 2006 Available online 6 March 2006
Abstract Molecular distillation (MD) was used to increase the purity of octacosanol extracts from transesterified rice bran wax (RBW), and the effects of MD parameters on the purification process and efficiency were studied. The octacosanol content, triacontanol content and the ratio of octacosanol content (Co) to triacontanol content (Ct) in distillates and residues, the yields of distillates and residues, and split ratio (yield of distillates to the yield of residues) were investigated in the circumstance of temperature and pressure changes. Results indicated that distilling temperature and distilling pressure are very important factors in purification process. The increase of distilling temperature or the decrease of distilling pressure could increase the mean free path (MFP) of molecules; therefore, the components with larger molecular weight could be separated and concentrated onto the condenser, and the yield of distillates increased and the split ratio was higher, but the content of octacosanol in distillates did not increase accordingly. Purified by MD at 150 C and 0.5 Torr, the octacosanol content in distillates can be up to 37.6%, increasing by 48.0% over that in the raw octacosanol extracts. The purification processes of octacosanol extracts with MD were further explained by comparing the components of distillates with residues at 0.5 Torr, and the components of distillates at 0.5 Torr with those at 2.0 Torr as well. 2006 Elsevier Ltd. All rights reserved. Keywords: Octacosanol; Molecular distillation (MD); Purification; Rice bran wax (RBW)
1. Introduction Rice bran wax (RBW) is a main unsaponifiable component in rice bran oil extracts. It is composed of esters with 46 up to 60 carbon atoms (Tolloch, 1976). RBW has been reported to be one of the best sources containing octacosanol, and its benefits to health, such as blood lipid or cholesterol lowering and athletic performance improving, have been studied and proven to be completely without side-effects (Kato et al., 1995; Rapport, 2000; Shimura,
*
Corresponding author. Address. Letter Box 303, College of Food Science and Nutritional Engineering, China Agricultural University, No. 17, Qinghuadonglu, Haidian District, Beijing 100083, PR China. Tel./fax: +86 10 62737434. E-mail address: [email protected] (X. Hu). 0260-8774/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2006.01.030
Hasegawa, Takano, & Suzuki, 1987). The high stability and encouragingly biological activities of octacosanol make it become a potential candidate as a supplement in foods, medicine and cosmetics. Molecular distillation (MD) is performed in a high-vacuum environment where materials just stay a little while. Therefore, the distillation of high molecular weight species such as higher carbon fatty alcohols can be performed without thermally degrading the products. MD is especially suitable for the separation or purification of materials with high boiling point (Batistella & Maciel, 1996; Ridway, 1956; Terry et al., 2004). In contrast to conventional methods, such as column chromatography and recrystallization, MD can avoid using of any organic solvents, resulting in much smaller waste and higher safety of products during the purification of octacosanol extracts (Shunqing & Feng, 2004).
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F. Chen et al. / Journal of Food Engineering 79 (2007) 63–68
In the present study, the effects of the distilling temperature and distilling pressure on the purification process of octacosanol extracts by MD were investigated. Comparison of compositions between distillates and residues at the same condition and comparison of compositions in distillates between different conditions were done, in order to explain the purification processes of octacosanol extracts, which were influenced by different MD conditions. 2. Materials and methods 2.1. Materials Octacosanol extracts were obtained through MD at 176.1 C and 1.29 Torr from the crude octacosanol extracts, which were extracted from transesterified RBW. In the octacosanol extracts, the contents of octacosanol and triacontanol were 25.4% and 51.5%, respectively (Chen et al., 2005). All solvents and chemicals used were of analytical grade or GC grade. Cyclohexane, hexadecanol, octacosanol, triacontanol, lacceroic alcohol, tetratriacontanol and 1,3,5-triphenylbenzene (TPB) were purchased from Sigma Chemical Co. in Beijing, PR China. The MD apparatus with the capacity of 2000 mL (Pope Scientific Co, USA) was used for purification and its schematic diagram is shown in Fig. 1.
2.2. Purification of octacosanol extracts by MD MD was used to further purify the octacosanol extracts. The flow rate of feed, temperature of condenser, and rotate rate of scraper were set at 3 mL/min, 90 C and 50 rpm, respectively. The feed was put into the feed flask after being heated and melted. Then, the vacuum pump was turned on to remove the gas from the feed. When the vacuum was steady and the distillation temperature was fixed, the feeding valve was turned on, and then the degassed feed liquid immediately flowed down and the scraper quickly spun it into a very thin film spreading over onto the evaporating surface. Heated walls and high-vacuum made more volatile components, which consist of light cuts, concentrate onto the closely positioned internal condensing surface and became the distillate, while the less volatile components, which consist of heavy components, flowed down along the cylinder and became the residue. Thus, the resulting fractions were separated and discharged through the individual outlets, respectively. The yield was calculated and the octacosanol content could be determined after the distillates were collected. 2.3. Analysis of compositions of octacosanol extracts According to the methods of Kazuko, Kumlko, and Yohel (1991) Gonzalez, Magraner, and Acosta (1996),
Fig. 1. Schematic diagram of wiped-film molecular evaporator.
F. Chen et al. / Journal of Food Engineering 79 (2007) 63–68
the main components of purified products, such as tetracosanol, hexadecanol, octacosanol, triacontanol, and lacceroic alcohol, were determined. TPB (0.200 g) was dissolved into 100.00 mL of cyclohexane; therefore, the TPB with a concentration of 2.000 mg/mL was prepared, and calibration curves were obtained by injecting mixed standard solutions with concentrations ranging from 100 lg/mL to 900 lg/mL. Purified product (0.025 g) prepared with MD was dissolved in 3.0 mL of cyclohexane under the help of ultrasonic wave at 40 C, and then 1.5 mL of TPB solution with a concentration of 2.000 mg/mL, as an internal standard solution, was added to the above solution. Before being subjected to GLC analysis, the resulting solution was added with cyclohexane until the volume was up to 5.0 mL. GLC was performed on a Hewlett Packard 6890A and a HP-5 column of 30 · 320 · 0.25 lm. The gas flow rates of N2, H2 and air were 45, 40, and 450 mL/min, respectively. The operating temperatures were set and changed as follows: injector, 320 C; detector, 330 C; initial oven temperature, 230 C, rested for 6 min, and then increased with a rate of 10 C min 1 to 280 C, again rested for 20 min, and then increased with a rate of 20 C min 1– 300 C.
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and triacontanol in distillates and residues were determined to indicate the purification efficiency by MD. 3.1. Influence of distilling temperature on the purification of octacosanol extracts during MD The purification of octacosanol extracts was conducted at 1.0 Torr. The influence of distilling temperature ranging from 140 C to 180 C on the contents of octacosanol and triacontanol in distillates and residues is shown in Fig. 2. With the increase of the temperature, the octacosanol content decreased and the triacontanol content increased in distillates. It is known that when the distilling temperature increases, the vapor pressure rapidly increases; at the same time, the mean free path (MFP) of the molecule becomes long, causing more frequent collisions of molecules in the gap between the evaporator and the condenser (Lutisˇan & Cvengrosˇ, 1995; Lutisˇan, Cvengrosˇ, & Micov, 2002; Ridway, 1956). Therefore, heavier fractions with larger molecular weight, such as triacontanol, can reach the condenser with increase of temperature. Because the MFP of octacosanol molecule was closed to the one of triacontanol molecule under the given condition, and the triacontanol was the most dominant component in extracts, as a result, there were more molecules of concentrated triacontanol in
2.4. Statistical analysis 70
The mean value of triplicate determinations was analyzed. ORIGIN7.5 software was used to make the graphs.
60
Octacosanol in distillates Octacosanol in residues Triacontanol in distillates Triacontanol in residues
3. Results and discussion 50
Content (%)
The distilling temperature and distilling pressure are two major factors affecting the purification of octacosanol extracts. The optimum conditions of first-stage MD have been obtained in our preliminary study (Chen et al., 2005), and the compositions of purified products are given in Table 1. In order to increase the purity of octacosanol extracts, the purification process by second-stage MD was studied. In the octacosanol extracts obtained by firststage MD, octacosanol is a medium molecular weight composition, and triacontanol content is in the majority. Therefore, the goal of the present study is to remove heavier fractions with larger molecular weight than octacosanol as much as possible. And the contents of octacosanol
40
30
20
140
150
160
170
180
Distilling temperature (°C)
Fig. 2. Effect of distilling temperature on the contents of octacosanol and triacontanol in distillates and residues obtained by MD (distilling pressure = 1.0 Torr).
Table 1 Effect of different purification conditions on the components of products obtained by MD Components Tetracosanol Hexadecanol Octacosanol Triacontanol Lacceroic alcohol a
Raw octacosanol extracts a
3.4 ± 0.4 9.2 ± 0.3 25.4 ± 0.5 51.5 ± 0.7 9.6 ± 0.6
Mean values of triplicate determinations.
Distillates (150 C, 0.5 Torr)
Distillates (150 C, 2.0 Torr)
Residues (150 C, 0.5 Torr)
3.6 ± 0.4 10.8 ± 0.3 37.6 ± 0.6 40.2 ± 0.5 6.0 ± 0.2
17.7 ± 0.9 23.2 ± 0.4 29.7 ± 0.4 22.0 ± 1.0 3.6 ± 0.2
1.4 ± 0.3 3.3 ± 0.6 16.9 ± 0.8 72.6 ± 1.1 5.0 ± 0.3
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comparison with the octacosanol on the condenser. So the triacontanol content increased significantly in distillates. On the other hand, 150 C became a turning point for the changes in octacosanol content and triacontanol content in residues. At temperature less than 150 C, the octacosanol content in residues decreased slightly while the triacontanol content in residues increased. This suggested that octacosanol could only just evaporate to the condenser, but the fractions with larger molecular weight in comparison with octacosanol could not reach the condenser and were collected in the residues. With increase of temperature, a few of the molecules in heavier fractions began to get enough energy to escape from the evaporator and to be condensed into the distillates, but a few of the molecules in lighter fractions moved more randomly in the gap, neither in distillates nor in residues. When the temperature was above 180 C, it was almost impossible to collect the residues and the separation efficiency was not satisfactory. Based on the above observations, the increase in yield of distillates, decrease in the yield of residues, and the increase in split ratio were illustrated with the increase of temperature (Fig. 3). The split ratio is a ratio of the yield of distillates to the yield of residues (Terry et al., 2004), which represents the evaporation degree. At the higher temperature (180 C), the split ratio was 4.8 times that at the lower temperature (140 C), because heat transfer to molecules occurred more efficiently and the molecules power could increase to reach the condenser. Although the increased mass in the distillates would be expected during MD, this higher split ratio decreased the purity of octacosanol extracts. When distillates with a certain octacosanol content were obtained, the ratio of octacosanol content (Co) to triacontanol content (Ct) reflected the difference in behavior of both kinds of molecules during MD, and the closer the
ratio of Co/Ct in raw material is, the lower the separation efficiency is. The ratio of Co/Ct was also followed over the temperature range studied (Fig. 4). With the increase of temperature, the ratio of Co/Ct in distillates decreased but that in the residues increased; interestingly, a cross point (0.49) was observed at 178 C, and that is exactly the ratio of Co/Ct in raw octacosanol extracts. This suggested that MD conditions have nearly identical influence on the evaporation of octacosanol molecules and triacontanol molecules; therefore, removal of triacontanol from distillates was impossible at temperature more than 178 C. 3.2. Influence of distilling pressure on the purification of octacosanol extracts during MD The purification of octacosanol extracts was conducted at 150 C. Under distilling pressure ranging from 0.2 Torr to 2.0 Torr, different changes had taken place in octacosanol content and triacontanol content in distillates and residues which are shown in Fig. 5. The octacosanol content in distillates increased as the distilling pressure increased from 0.2 Torr to 0.5 Torr, and then decreased from 0.5 Torr to 2.0 Torr; whereas the triacontanol content in distillates increased continuously as the distilling pressure increased. Because the MFP of molecules of octacosanol and triacontanol decreased sharply as the distilling pressure increased, as a result, more and more molecules could not reach the condenser; the contents of octacosanol and triacontanol became less and less in the distillates. When the distilling pressure increased to 0.5 Torr, most of the heavier fractions with larger molecular weight in comparison with octacosanol could not reach the condenser. So octacosanol became the main component in the distillates. And when the pressure increased over 0.5 Torr, it became difficult for a portion of octacosanol molecules to reach the condenser, but some components, of which the
3.2 100
Yield (%)
2.4
Residues
80
2.8
60 40 Distillates
Split ratio
20
2.0 0 140
150
160
170
180
Distilling temperature (°C)
1.6
1.2
0.8
0.4 140
150
160
170
180
Distilling temperature (°C)
Fig. 3. Effect of distilling temperature on the yields of distillates and residues, and split ratio during MD (distilling pressure = 1.0 Torr).
F. Chen et al. / Journal of Food Engineering 79 (2007) 63–68 1.8
2.4
100
2.2
1.6
Yield(%)
1.4
1.8 1.2
residues
80
2.0
60 40
distillates
1.6
Split ratio
Ratio of Co/Ct
67
distillates
1.0
cross-point
0.8
20
1.4 0 0.4
1.2
0.8
1.2
1.6
2.0
Distilling pressure (Torr)
1.0
residues
0.8
0.6
0.6 0.4
0.4 0.2
0.2 0.2
0.4
0.6
0.0
1.0
1.2
1.4
1.6
1.8
2.0
Distilling pressure (Torr) 140
150
160
170
180
Distilling temperature (°C)
Fig. 4. Effect of distilling temperature on the ratio of octacosanol content to triacontanol content in distillates and residues obtained by MD (distilling pressure = 1.0 Torr).
70
60
Octacosanol in distillates Octacosanol in residues Triacontanol in distillates Triacontanol in residues
50
Content (%)
0.8
40
30
20
Fig. 6. Effect of distilling pressure on the yields of distillates and residues, and split ratio during MD (distilling temperature = 150 C).
As shown in Fig. 7, the ratio of Co/Ct in distillates increased as the distilling pressure increased, and the ratio of Co/Ct increased faster under 0.2 Torr to 0.5 Torr than those under 0.5 Torr to 2.0 Torr. The lowest ratio of Co/Ct in residues was observed under 0.5 Torr, at which the octacosanol content was the highest. This suggested that better purification efficiency of octacosanol extracts during MD should be obtained under 0.5 Torr. Eventually, by integrating the octacosanol content in distillates and the yield of distillates, the purification of octacosanol extracts was conducted by MD at 150 C and 0.5 Torr. In these conditions, the purification yield can be up to 53.8% and the octacosanol content in distillates can be up to 37.6%, which increased by 48.0% over that in the raw octacosanol extracts.
10
0.0
0.2
0.4
0.6
0.8
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1.2
1.4
1.6
1.8
2.0
2.2
Distilling pressure (Torr)
1.4
Fig. 5. Effect of distilling pressure on the contents of octacosanol and triacontanol in distillates and residues obtained by MD (distilling temperature = 150 C).
1.2
Distillates
molecular weight is less than that of octacosanol, began to be condensed. Besides, when the distilling pressure increased, octacosanol content in residues increased continuously, but a turning point between the increase and decrease of triacontanol content in residues was observed under 1.0 Torr. For the heavier fractions than triacontanol molecules, it became impossible to reach the condenser under pressure more than 1.0 Torr, and the larger the molecular weight was, the sharper the decline of molecule quantity was. Therefore, the yield of distillates declined and the yield of residues increased, and the split ratio decreased with increase of distilling pressure (Fig. 6). In such circumstance, sacrifice of the yield of distillates did not bring an increase in the purity of distillates.
Ratio of Co/Ct
1.0
0.8
0.6
Residues 0.4
0.2
0.0 0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Distilling pressure (Torr)
Fig. 7. Effect of distilling temperature on the ratio of octacosanol content to triacontanol content in distillates and residues obtained by MD (distilling temperature = 150 C).
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In order to investigate the influence of MD parameters on the purification process of octacosanol extracts, the main components of distillates obtained by MD at 150 C and under the pressure of 0.5 Torr and 2.0 Torr were compared. It is shown in Table 1 that the contents of tetracosanol and hexadecanol (lighter fraction) in distillates under 0.5 Torr were more than that under 2.0 Torr, and the contents of octacosanol, triacontanol and lacceroic alcohol (heavier fraction) in distillates under 0.5 Torr were less than those under 2.0 Torr, respectively, which indicated that for the components with larger molecular weight it is more difficult to reach the condenser under 2.0 Torr. Therefore, these components were in the minority in distillates; by comparison, the components with lighter molecular weight were in the majority in distillates. Comparison of components between distillates and residues obtained by MD at 150 C and 0.5 Torr indicated that the contents of tetracosanol, hexadecanol and octacosanol in distillates were more than those in the residues, and the contents of triacontanol and lacceroic alcohol in residues were more than those in the distillates, respectively. Because the components with larger molecular weights had a smaller MFP of the molecule, making them more difficult to reach the condenser, these components consisted of a larger proportion in residues than in distillates. In contrast, the components with a lesser molecular weight had a larger MFP of the molecule which made them easier to reach the condenser, and accordingly, their proportion in distillates was larger than that in the residues. Similarly, the differences in compositions between raw octacosanol extracts and distillates obtained by MD at 150 C and 0.5 Torr could be elucidated according to the above purification process as well. 4. Conclusion The present work investigated the contents of octacosanol and triacontanol in distillates and residues, the yields of distillates and residues, and split ratio (ratio of yield of distillates to the yield of residues), ratio of Co/Ct in distillates and residues during purification process by MD. The results indicated that MD was effective for purification of the octacosanol extracts. Distilling temperature and distilling pressure have significant effect on the purification of octacosanol extracts by MD, so the choice of these parameters is very important in purification process. When distilling temperature decreased and distilling pressure increased, the MFP of molecules decreased sharply; therefore, more and more molecules could not reach the condenser and get into the residues. So the yield of distillates and split ratio decreased. However, octacosanol content in distillates was determined not only by the absolute amount of octacosanol molecules, but also by the relative amount of other components that could reach the condenser, which is help-
ful for choosing the parameters suitable for the purification of natural products through MD. With respect to the octacosanol content in distillates and the yield of distillates, the better temperature and pressure in the purification of octacosanol extracts through MD should be 150 C and 0.5 Torr; under such circumstance, the octacosanol content in distillates can reach as high as 37.6%, increasing by 48.0% over the raw octacosanol extracts, and the yield of distillates was 53.8%. Acknowledgements The research is supported by fund from China HighTech (863) Project (No: 2002AA248011), fund from Beijing Natural Science Fund Committee (No: 6041003) and Research Starting Fund from China Agricultural University (No: 2004005). Accordingly, we gratefully acknowledge the financial supports. References Batistella, C.B., Maciel, M. R. W. (1996). Comparison between falling film and centrifugal molecular distillators for separation of fine chemicals, 3er Congreso Interamericano de Computacion Aplicada a la Industria de Argentina (pp.133–136). Chen, F., Cai, T., Zhao, G., Liao, X., Guo, L., & Hu, X. (2005). Optimizing conditions for the purification of crude octacosanol extract from rice bran wax by molecular distillation analyzed using response surface methodology. Journal of Food Engineering, 70, 47–53. Gonzalez, B. L. J., Magraner, H. Z. L., & Acosta, G. P. C. (1996). Analytical procedure for the determination of 1-octacosanol in plasma by solvent extraction and capillary gas chromatography. Journal of Chromatography B, 682, 359–363. Kato, S., Karino, K., Hasegawa, J., Nagasaki, A., Eguchi, M., & Ichinose, T. (1995). Octacosanol affects lipid metabolism in rats fed on a high fat diet. British Journal of Nutrition, 73, 433–442. Kazuko, K., Kumlko, A., & Yohel, H. (1991). Free primary alcohols in oils and waxes from germs, kernels and other components of nuts, seeds, fruits and cereals. Journal of the American Oil Chemists’ Society, 68(11), 869–872. Lutisˇan, J., & Cvengrosˇ, J. (1995). Mean free path of molecules on molecular distillation. The Chemical Engineering Journal and the Biochemical Engineering Journal, 56(2), 39–50. Lutisˇan, J., Cvengrosˇ, J., & Micov, M. (2002). Heat and mass transfer in the evaporating film of a molecular evaporator. Chemical Engineering Journal, 85, 225–234. Rapport, L. J. (2000). Nutraceuticals (3): octacosanol. Pharmaceutical Journal, 265, 170–171. Ridway, W. (1956). Molecular distillation. London: Chapman and Hall Press (pp. 1–63). Shimura, S., Hasegawa, T., Takano, S., & Suzuki, T. (1987). Studies on the effect of octacosanol on motor endurance in mice. Nutritional Reproduction International, 36, 1029–1038. Shunqing, Z., & Feng, T. (2004). Application of molecular distillation in separation of natural products. Fine Chemistry, 21, 49–51 (in Chinese). Isbell, T. A., & Cermak, S. C. (2004). Purification of meadowfoam monoestolide from polyestolide. Industrial Crops and Products, 19, 113–118. Tolloch, A. P. (1976). Chemistry and biochemistry of natural waxes. Elsevier: Amsterdam (pp. 50–86).
Purification process of octacosanol extracts from rice bran wax by molecular distillation Fang Chen, Zhengfu Wang, Guanghua Zhao, Xiaojun Liao, Tongyi Cai, Linyu Guo, Xiaosong Hu * College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China Received 24 September 2005; accepted 15 January 2006 Available online 6 March 2006
Abstract Molecular distillation (MD) was used to increase the purity of octacosanol extracts from transesterified rice bran wax (RBW), and the effects of MD parameters on the purification process and efficiency were studied. The octacosanol content, triacontanol content and the ratio of octacosanol content (Co) to triacontanol content (Ct) in distillates and residues, the yields of distillates and residues, and split ratio (yield of distillates to the yield of residues) were investigated in the circumstance of temperature and pressure changes. Results indicated that distilling temperature and distilling pressure are very important factors in purification process. The increase of distilling temperature or the decrease of distilling pressure could increase the mean free path (MFP) of molecules; therefore, the components with larger molecular weight could be separated and concentrated onto the condenser, and the yield of distillates increased and the split ratio was higher, but the content of octacosanol in distillates did not increase accordingly. Purified by MD at 150 C and 0.5 Torr, the octacosanol content in distillates can be up to 37.6%, increasing by 48.0% over that in the raw octacosanol extracts. The purification processes of octacosanol extracts with MD were further explained by comparing the components of distillates with residues at 0.5 Torr, and the components of distillates at 0.5 Torr with those at 2.0 Torr as well. 2006 Elsevier Ltd. All rights reserved. Keywords: Octacosanol; Molecular distillation (MD); Purification; Rice bran wax (RBW)
1. Introduction Rice bran wax (RBW) is a main unsaponifiable component in rice bran oil extracts. It is composed of esters with 46 up to 60 carbon atoms (Tolloch, 1976). RBW has been reported to be one of the best sources containing octacosanol, and its benefits to health, such as blood lipid or cholesterol lowering and athletic performance improving, have been studied and proven to be completely without side-effects (Kato et al., 1995; Rapport, 2000; Shimura,
*
Corresponding author. Address. Letter Box 303, College of Food Science and Nutritional Engineering, China Agricultural University, No. 17, Qinghuadonglu, Haidian District, Beijing 100083, PR China. Tel./fax: +86 10 62737434. E-mail address: [email protected] (X. Hu). 0260-8774/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2006.01.030
Hasegawa, Takano, & Suzuki, 1987). The high stability and encouragingly biological activities of octacosanol make it become a potential candidate as a supplement in foods, medicine and cosmetics. Molecular distillation (MD) is performed in a high-vacuum environment where materials just stay a little while. Therefore, the distillation of high molecular weight species such as higher carbon fatty alcohols can be performed without thermally degrading the products. MD is especially suitable for the separation or purification of materials with high boiling point (Batistella & Maciel, 1996; Ridway, 1956; Terry et al., 2004). In contrast to conventional methods, such as column chromatography and recrystallization, MD can avoid using of any organic solvents, resulting in much smaller waste and higher safety of products during the purification of octacosanol extracts (Shunqing & Feng, 2004).
64
F. Chen et al. / Journal of Food Engineering 79 (2007) 63–68
In the present study, the effects of the distilling temperature and distilling pressure on the purification process of octacosanol extracts by MD were investigated. Comparison of compositions between distillates and residues at the same condition and comparison of compositions in distillates between different conditions were done, in order to explain the purification processes of octacosanol extracts, which were influenced by different MD conditions. 2. Materials and methods 2.1. Materials Octacosanol extracts were obtained through MD at 176.1 C and 1.29 Torr from the crude octacosanol extracts, which were extracted from transesterified RBW. In the octacosanol extracts, the contents of octacosanol and triacontanol were 25.4% and 51.5%, respectively (Chen et al., 2005). All solvents and chemicals used were of analytical grade or GC grade. Cyclohexane, hexadecanol, octacosanol, triacontanol, lacceroic alcohol, tetratriacontanol and 1,3,5-triphenylbenzene (TPB) were purchased from Sigma Chemical Co. in Beijing, PR China. The MD apparatus with the capacity of 2000 mL (Pope Scientific Co, USA) was used for purification and its schematic diagram is shown in Fig. 1.
2.2. Purification of octacosanol extracts by MD MD was used to further purify the octacosanol extracts. The flow rate of feed, temperature of condenser, and rotate rate of scraper were set at 3 mL/min, 90 C and 50 rpm, respectively. The feed was put into the feed flask after being heated and melted. Then, the vacuum pump was turned on to remove the gas from the feed. When the vacuum was steady and the distillation temperature was fixed, the feeding valve was turned on, and then the degassed feed liquid immediately flowed down and the scraper quickly spun it into a very thin film spreading over onto the evaporating surface. Heated walls and high-vacuum made more volatile components, which consist of light cuts, concentrate onto the closely positioned internal condensing surface and became the distillate, while the less volatile components, which consist of heavy components, flowed down along the cylinder and became the residue. Thus, the resulting fractions were separated and discharged through the individual outlets, respectively. The yield was calculated and the octacosanol content could be determined after the distillates were collected. 2.3. Analysis of compositions of octacosanol extracts According to the methods of Kazuko, Kumlko, and Yohel (1991) Gonzalez, Magraner, and Acosta (1996),
Fig. 1. Schematic diagram of wiped-film molecular evaporator.
F. Chen et al. / Journal of Food Engineering 79 (2007) 63–68
the main components of purified products, such as tetracosanol, hexadecanol, octacosanol, triacontanol, and lacceroic alcohol, were determined. TPB (0.200 g) was dissolved into 100.00 mL of cyclohexane; therefore, the TPB with a concentration of 2.000 mg/mL was prepared, and calibration curves were obtained by injecting mixed standard solutions with concentrations ranging from 100 lg/mL to 900 lg/mL. Purified product (0.025 g) prepared with MD was dissolved in 3.0 mL of cyclohexane under the help of ultrasonic wave at 40 C, and then 1.5 mL of TPB solution with a concentration of 2.000 mg/mL, as an internal standard solution, was added to the above solution. Before being subjected to GLC analysis, the resulting solution was added with cyclohexane until the volume was up to 5.0 mL. GLC was performed on a Hewlett Packard 6890A and a HP-5 column of 30 · 320 · 0.25 lm. The gas flow rates of N2, H2 and air were 45, 40, and 450 mL/min, respectively. The operating temperatures were set and changed as follows: injector, 320 C; detector, 330 C; initial oven temperature, 230 C, rested for 6 min, and then increased with a rate of 10 C min 1 to 280 C, again rested for 20 min, and then increased with a rate of 20 C min 1– 300 C.
65
and triacontanol in distillates and residues were determined to indicate the purification efficiency by MD. 3.1. Influence of distilling temperature on the purification of octacosanol extracts during MD The purification of octacosanol extracts was conducted at 1.0 Torr. The influence of distilling temperature ranging from 140 C to 180 C on the contents of octacosanol and triacontanol in distillates and residues is shown in Fig. 2. With the increase of the temperature, the octacosanol content decreased and the triacontanol content increased in distillates. It is known that when the distilling temperature increases, the vapor pressure rapidly increases; at the same time, the mean free path (MFP) of the molecule becomes long, causing more frequent collisions of molecules in the gap between the evaporator and the condenser (Lutisˇan & Cvengrosˇ, 1995; Lutisˇan, Cvengrosˇ, & Micov, 2002; Ridway, 1956). Therefore, heavier fractions with larger molecular weight, such as triacontanol, can reach the condenser with increase of temperature. Because the MFP of octacosanol molecule was closed to the one of triacontanol molecule under the given condition, and the triacontanol was the most dominant component in extracts, as a result, there were more molecules of concentrated triacontanol in
2.4. Statistical analysis 70
The mean value of triplicate determinations was analyzed. ORIGIN7.5 software was used to make the graphs.
60
Octacosanol in distillates Octacosanol in residues Triacontanol in distillates Triacontanol in residues
3. Results and discussion 50
Content (%)
The distilling temperature and distilling pressure are two major factors affecting the purification of octacosanol extracts. The optimum conditions of first-stage MD have been obtained in our preliminary study (Chen et al., 2005), and the compositions of purified products are given in Table 1. In order to increase the purity of octacosanol extracts, the purification process by second-stage MD was studied. In the octacosanol extracts obtained by firststage MD, octacosanol is a medium molecular weight composition, and triacontanol content is in the majority. Therefore, the goal of the present study is to remove heavier fractions with larger molecular weight than octacosanol as much as possible. And the contents of octacosanol
40
30
20
140
150
160
170
180
Distilling temperature (°C)
Fig. 2. Effect of distilling temperature on the contents of octacosanol and triacontanol in distillates and residues obtained by MD (distilling pressure = 1.0 Torr).
Table 1 Effect of different purification conditions on the components of products obtained by MD Components Tetracosanol Hexadecanol Octacosanol Triacontanol Lacceroic alcohol a
Raw octacosanol extracts a
3.4 ± 0.4 9.2 ± 0.3 25.4 ± 0.5 51.5 ± 0.7 9.6 ± 0.6
Mean values of triplicate determinations.
Distillates (150 C, 0.5 Torr)
Distillates (150 C, 2.0 Torr)
Residues (150 C, 0.5 Torr)
3.6 ± 0.4 10.8 ± 0.3 37.6 ± 0.6 40.2 ± 0.5 6.0 ± 0.2
17.7 ± 0.9 23.2 ± 0.4 29.7 ± 0.4 22.0 ± 1.0 3.6 ± 0.2
1.4 ± 0.3 3.3 ± 0.6 16.9 ± 0.8 72.6 ± 1.1 5.0 ± 0.3
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comparison with the octacosanol on the condenser. So the triacontanol content increased significantly in distillates. On the other hand, 150 C became a turning point for the changes in octacosanol content and triacontanol content in residues. At temperature less than 150 C, the octacosanol content in residues decreased slightly while the triacontanol content in residues increased. This suggested that octacosanol could only just evaporate to the condenser, but the fractions with larger molecular weight in comparison with octacosanol could not reach the condenser and were collected in the residues. With increase of temperature, a few of the molecules in heavier fractions began to get enough energy to escape from the evaporator and to be condensed into the distillates, but a few of the molecules in lighter fractions moved more randomly in the gap, neither in distillates nor in residues. When the temperature was above 180 C, it was almost impossible to collect the residues and the separation efficiency was not satisfactory. Based on the above observations, the increase in yield of distillates, decrease in the yield of residues, and the increase in split ratio were illustrated with the increase of temperature (Fig. 3). The split ratio is a ratio of the yield of distillates to the yield of residues (Terry et al., 2004), which represents the evaporation degree. At the higher temperature (180 C), the split ratio was 4.8 times that at the lower temperature (140 C), because heat transfer to molecules occurred more efficiently and the molecules power could increase to reach the condenser. Although the increased mass in the distillates would be expected during MD, this higher split ratio decreased the purity of octacosanol extracts. When distillates with a certain octacosanol content were obtained, the ratio of octacosanol content (Co) to triacontanol content (Ct) reflected the difference in behavior of both kinds of molecules during MD, and the closer the
ratio of Co/Ct in raw material is, the lower the separation efficiency is. The ratio of Co/Ct was also followed over the temperature range studied (Fig. 4). With the increase of temperature, the ratio of Co/Ct in distillates decreased but that in the residues increased; interestingly, a cross point (0.49) was observed at 178 C, and that is exactly the ratio of Co/Ct in raw octacosanol extracts. This suggested that MD conditions have nearly identical influence on the evaporation of octacosanol molecules and triacontanol molecules; therefore, removal of triacontanol from distillates was impossible at temperature more than 178 C. 3.2. Influence of distilling pressure on the purification of octacosanol extracts during MD The purification of octacosanol extracts was conducted at 150 C. Under distilling pressure ranging from 0.2 Torr to 2.0 Torr, different changes had taken place in octacosanol content and triacontanol content in distillates and residues which are shown in Fig. 5. The octacosanol content in distillates increased as the distilling pressure increased from 0.2 Torr to 0.5 Torr, and then decreased from 0.5 Torr to 2.0 Torr; whereas the triacontanol content in distillates increased continuously as the distilling pressure increased. Because the MFP of molecules of octacosanol and triacontanol decreased sharply as the distilling pressure increased, as a result, more and more molecules could not reach the condenser; the contents of octacosanol and triacontanol became less and less in the distillates. When the distilling pressure increased to 0.5 Torr, most of the heavier fractions with larger molecular weight in comparison with octacosanol could not reach the condenser. So octacosanol became the main component in the distillates. And when the pressure increased over 0.5 Torr, it became difficult for a portion of octacosanol molecules to reach the condenser, but some components, of which the
3.2 100
Yield (%)
2.4
Residues
80
2.8
60 40 Distillates
Split ratio
20
2.0 0 140
150
160
170
180
Distilling temperature (°C)
1.6
1.2
0.8
0.4 140
150
160
170
180
Distilling temperature (°C)
Fig. 3. Effect of distilling temperature on the yields of distillates and residues, and split ratio during MD (distilling pressure = 1.0 Torr).
F. Chen et al. / Journal of Food Engineering 79 (2007) 63–68 1.8
2.4
100
2.2
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Yield(%)
1.4
1.8 1.2
residues
80
2.0
60 40
distillates
1.6
Split ratio
Ratio of Co/Ct
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distillates
1.0
cross-point
0.8
20
1.4 0 0.4
1.2
0.8
1.2
1.6
2.0
Distilling pressure (Torr)
1.0
residues
0.8
0.6
0.6 0.4
0.4 0.2
0.2 0.2
0.4
0.6
0.0
1.0
1.2
1.4
1.6
1.8
2.0
Distilling pressure (Torr) 140
150
160
170
180
Distilling temperature (°C)
Fig. 4. Effect of distilling temperature on the ratio of octacosanol content to triacontanol content in distillates and residues obtained by MD (distilling pressure = 1.0 Torr).
70
60
Octacosanol in distillates Octacosanol in residues Triacontanol in distillates Triacontanol in residues
50
Content (%)
0.8
40
30
20
Fig. 6. Effect of distilling pressure on the yields of distillates and residues, and split ratio during MD (distilling temperature = 150 C).
As shown in Fig. 7, the ratio of Co/Ct in distillates increased as the distilling pressure increased, and the ratio of Co/Ct increased faster under 0.2 Torr to 0.5 Torr than those under 0.5 Torr to 2.0 Torr. The lowest ratio of Co/Ct in residues was observed under 0.5 Torr, at which the octacosanol content was the highest. This suggested that better purification efficiency of octacosanol extracts during MD should be obtained under 0.5 Torr. Eventually, by integrating the octacosanol content in distillates and the yield of distillates, the purification of octacosanol extracts was conducted by MD at 150 C and 0.5 Torr. In these conditions, the purification yield can be up to 53.8% and the octacosanol content in distillates can be up to 37.6%, which increased by 48.0% over that in the raw octacosanol extracts.
10
0.0
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0.8
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1.2
1.4
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2.2
Distilling pressure (Torr)
1.4
Fig. 5. Effect of distilling pressure on the contents of octacosanol and triacontanol in distillates and residues obtained by MD (distilling temperature = 150 C).
1.2
Distillates
molecular weight is less than that of octacosanol, began to be condensed. Besides, when the distilling pressure increased, octacosanol content in residues increased continuously, but a turning point between the increase and decrease of triacontanol content in residues was observed under 1.0 Torr. For the heavier fractions than triacontanol molecules, it became impossible to reach the condenser under pressure more than 1.0 Torr, and the larger the molecular weight was, the sharper the decline of molecule quantity was. Therefore, the yield of distillates declined and the yield of residues increased, and the split ratio decreased with increase of distilling pressure (Fig. 6). In such circumstance, sacrifice of the yield of distillates did not bring an increase in the purity of distillates.
Ratio of Co/Ct
1.0
0.8
0.6
Residues 0.4
0.2
0.0 0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Distilling pressure (Torr)
Fig. 7. Effect of distilling temperature on the ratio of octacosanol content to triacontanol content in distillates and residues obtained by MD (distilling temperature = 150 C).
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In order to investigate the influence of MD parameters on the purification process of octacosanol extracts, the main components of distillates obtained by MD at 150 C and under the pressure of 0.5 Torr and 2.0 Torr were compared. It is shown in Table 1 that the contents of tetracosanol and hexadecanol (lighter fraction) in distillates under 0.5 Torr were more than that under 2.0 Torr, and the contents of octacosanol, triacontanol and lacceroic alcohol (heavier fraction) in distillates under 0.5 Torr were less than those under 2.0 Torr, respectively, which indicated that for the components with larger molecular weight it is more difficult to reach the condenser under 2.0 Torr. Therefore, these components were in the minority in distillates; by comparison, the components with lighter molecular weight were in the majority in distillates. Comparison of components between distillates and residues obtained by MD at 150 C and 0.5 Torr indicated that the contents of tetracosanol, hexadecanol and octacosanol in distillates were more than those in the residues, and the contents of triacontanol and lacceroic alcohol in residues were more than those in the distillates, respectively. Because the components with larger molecular weights had a smaller MFP of the molecule, making them more difficult to reach the condenser, these components consisted of a larger proportion in residues than in distillates. In contrast, the components with a lesser molecular weight had a larger MFP of the molecule which made them easier to reach the condenser, and accordingly, their proportion in distillates was larger than that in the residues. Similarly, the differences in compositions between raw octacosanol extracts and distillates obtained by MD at 150 C and 0.5 Torr could be elucidated according to the above purification process as well. 4. Conclusion The present work investigated the contents of octacosanol and triacontanol in distillates and residues, the yields of distillates and residues, and split ratio (ratio of yield of distillates to the yield of residues), ratio of Co/Ct in distillates and residues during purification process by MD. The results indicated that MD was effective for purification of the octacosanol extracts. Distilling temperature and distilling pressure have significant effect on the purification of octacosanol extracts by MD, so the choice of these parameters is very important in purification process. When distilling temperature decreased and distilling pressure increased, the MFP of molecules decreased sharply; therefore, more and more molecules could not reach the condenser and get into the residues. So the yield of distillates and split ratio decreased. However, octacosanol content in distillates was determined not only by the absolute amount of octacosanol molecules, but also by the relative amount of other components that could reach the condenser, which is help-
ful for choosing the parameters suitable for the purification of natural products through MD. With respect to the octacosanol content in distillates and the yield of distillates, the better temperature and pressure in the purification of octacosanol extracts through MD should be 150 C and 0.5 Torr; under such circumstance, the octacosanol content in distillates can reach as high as 37.6%, increasing by 48.0% over the raw octacosanol extracts, and the yield of distillates was 53.8%. Acknowledgements The research is supported by fund from China HighTech (863) Project (No: 2002AA248011), fund from Beijing Natural Science Fund Committee (No: 6041003) and Research Starting Fund from China Agricultural University (No: 2004005). Accordingly, we gratefully acknowledge the financial supports. References Batistella, C.B., Maciel, M. R. W. (1996). Comparison between falling film and centrifugal molecular distillators for separation of fine chemicals, 3er Congreso Interamericano de Computacion Aplicada a la Industria de Argentina (pp.133–136). Chen, F., Cai, T., Zhao, G., Liao, X., Guo, L., & Hu, X. (2005). Optimizing conditions for the purification of crude octacosanol extract from rice bran wax by molecular distillation analyzed using response surface methodology. Journal of Food Engineering, 70, 47–53. Gonzalez, B. L. J., Magraner, H. Z. L., & Acosta, G. P. C. (1996). Analytical procedure for the determination of 1-octacosanol in plasma by solvent extraction and capillary gas chromatography. Journal of Chromatography B, 682, 359–363. Kato, S., Karino, K., Hasegawa, J., Nagasaki, A., Eguchi, M., & Ichinose, T. (1995). Octacosanol affects lipid metabolism in rats fed on a high fat diet. British Journal of Nutrition, 73, 433–442. Kazuko, K., Kumlko, A., & Yohel, H. (1991). Free primary alcohols in oils and waxes from germs, kernels and other components of nuts, seeds, fruits and cereals. Journal of the American Oil Chemists’ Society, 68(11), 869–872. Lutisˇan, J., & Cvengrosˇ, J. (1995). Mean free path of molecules on molecular distillation. The Chemical Engineering Journal and the Biochemical Engineering Journal, 56(2), 39–50. Lutisˇan, J., Cvengrosˇ, J., & Micov, M. (2002). Heat and mass transfer in the evaporating film of a molecular evaporator. Chemical Engineering Journal, 85, 225–234. Rapport, L. J. (2000). Nutraceuticals (3): octacosanol. Pharmaceutical Journal, 265, 170–171. Ridway, W. (1956). Molecular distillation. London: Chapman and Hall Press (pp. 1–63). Shimura, S., Hasegawa, T., Takano, S., & Suzuki, T. (1987). Studies on the effect of octacosanol on motor endurance in mice. Nutritional Reproduction International, 36, 1029–1038. Shunqing, Z., & Feng, T. (2004). Application of molecular distillation in separation of natural products. Fine Chemistry, 21, 49–51 (in Chinese). Isbell, T. A., & Cermak, S. C. (2004). Purification of meadowfoam monoestolide from polyestolide. Industrial Crops and Products, 19, 113–118. Tolloch, A. P. (1976). Chemistry and biochemistry of natural waxes. Elsevier: Amsterdam (pp. 50–86).