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Supercritical extraction of long chain n-alcohols from sugar cane crude wax
J. of Supercritical Fluids 41 (2007) 267–271
Supercritical extraction of long chain n-alcohols from sugar cane crude wax Antonio de Lucas a , Alberto Garc´ıa b , Amaury Alvarez b , Ignacio Gracia a,∗ a
Departamento de Ingenier´ıa Qu´ımica, Universidad de Castilla-La Mancha, Facultad de Ciencias Qu´ımicas, Avda. Camilo Jos´e Cela n◦ 10, 13004 Ciudad Real, Spain b ICIDCA, Institute of Sugar Cane Byproducts, Via Blanca 804 San Miguel del Padr´ on, 11 000 Havana, Cuba Received 21 April 2006; received in revised form 27 September 2006; accepted 29 September 2006
Abstract We have studied the supercritical CO2 extraction of long chain alcohols from sugar cane crude wax, a by product of sugar cane production. The aim of this work has been to determine the effect on extraction yield of some operational variables such as pressure (300–350 bar), temperature (50–100 ◦ C) and the ratio of KOH in the saponification stage (1–20%, w/w). To assess the n-alcohol composition of the supercritical extracts the fractions were analyzed by gas cromatography (GC). Response Surface Methodology was used to obtain mathematical expressions relating the yield as a function of the operational variables studied. These correlations have permitted us to determine the best extraction conditions of the experimental range analyzed (P = 350 bar, T = 100 ◦ C, 20% KOH/crude wax). These results were compared to those obtained by the existing industrial organic solvent multiple stage refining process. The quality of the supercritical extract was considerably superior, because of its higher n-alcohols purity, 78.24% (w/w), compared to the organic solvent extraction yield, 22.00% (w/w). © 2006 Elsevier B.V. All rights reserved. Keywords: Long chain n-alcohols; Response Surface Methodology; Sugar cane wax; Supercritical extraction
1. Introduction Several researchers have reported the biological effects of active compounds extracted from sugar cane crude wax (CW) such as long chain n-alcohols, fatty acids or ethanolic extracts, which have application to atherosclerotic vascular, coronary heart diseases [1,2] and dermatologic diseases [3,4]. CW is obtained by heptane extraction from the sugar cane filter mud, a residue resulting from the sugar cane production containing 75% water, a large variety of fats, waxy esters, free alcohols, sterols and a resinous fraction mainly composed of calcium salts of heavy polyesters [5]. The industrial process for the separation of these alcohols shown in Fig. 1, consists of different successive and multiple steps, permitting a first fractionation of resin compounds, and the separation in a second step, of fats and a refined wax. Further solvent processing of the refined wax is focused on the isolation of a natural mixture of high molecular weight aliphatic alcohols of the series C24 –C34 , which once purified, have medical application (like a medicant called
∗
Corresponding author. Tel.: +34 926295300; fax: +34 926295318. E-mail address: [email protected] (I. Gracia).
0896-8446/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2006.09.013
PolicosanolTM ). This remedy is used for the treatment of low density lipoprotein and cholesterol reduction [2,3]. This long current process has several problems related to both, complexity and the use of organic solvents (toxic, expensive, generation of residues, low selectivity) requiring solvent recovery, as well as being energy-intensive operation. The use of CO2 under supercritical conditions has been expanded to the isolation of bioactive compounds from natural materials like lanolin, jojoba esters and popolis [6]. Due to CO2 ’s properties, selectivity of triglycerides and waxes has also been achieved for substances containing a high level of lipid material [7]. This procedure has been used for the separation of natural waxes [8,9] and aliphatic primary alcohols from the sugar cane wax [10,11] and rice bran [12]. All these results indicate that supercritical CO2 technology is a promising alternative to the actual organic solvent extraction of the long chain alcohols from CW. In the present study, we present supercritical fluid extraction (SFE) as a technically viable alternative process for the extraction of high molecular weight n-alcohols from the CW. We have analyzed the effect of pressure (300–350 bar), temperature (50–100 ◦ C) and the amount of KOH used in the previous saponification (1–20%, w/w) on extraction yield of long chain
268
A. de Lucas et al. / J. of Supercritical Fluids 41 (2007) 267–271
Fig. 1. Industrial process for n-alcohol isolation from sugar cane crude wax. Table 1 Sugar cane crude wax properties and composition Melting point (◦ C)
Color
Fats (%, w/w)
Waxes (%, w/w)
LCAa (%, w/w)
Resins (%, w/w)
56–61
Dark green
33.8
55.2
2–3
15.1
a
LCA: long chain n-alcohols (included in the waxy fraction).
alcohols. A Response Surface Methodology (RSM) based on the statistical analysis of the experimental data was used to obtain mathematical expressions relating the operational variables and extraction yield. 2. Experimental procedures 2.1. Materials Sugar cane crude wax flakes were provided by the Guiteras Sugar Mill (Puerto Padre, Delicias, Provincia las Tunas, Cuba). Table 1 shows the main physical properties and composition of CW used in the study. Liquid CO2 (purity 99.5%) was supplied by Gases Industriales S.A. (La Habana, Cuba). 2.2. Supercritical CO2 extraction 2.2.1. Sample preparation Previous to CO2 extraction, CW must be saponified as described in Fig. 2: 1000 g of CW were melted to 85 ◦ C and mixed with solid KOH and 10% (w/w) ethanol, being stirred
Fig. 2. Supercritical CO2 processing of crude wax.
A. de Lucas et al. / J. of Supercritical Fluids 41 (2007) 267–271
during 2 h at 85 ◦ C. The liquid waxy material was cooled to 25 ◦ C in a flat mold, in order to obtain flakes of 2 mm thickness. 2.2.2. Apparatus and extraction procedure The flow diagram of the extraction equipment has been described in detail in reference [13]. Liquid carbon dioxide was provided from a steel cylinder. After cooling and filtering, the CO2 was compressed by a positive displacement HPLC pump. The pressure was regulated by a back-pressureregulator and checked by a manometer. The compressed fluid at 300–350 bar was passed through a vertically mounted extractor from the bottom. The extractor was a 75 ml stainless steel cylinder (17.48 mm i.d. × 304.8 mm). To keep the extractor temperature at the desired value a digital controller regulated the electric current through a resistor that surrounded the extractor cylinder. The temperature in the extractor was displayed in a temperature indicator. In the first extraction stage the wax-laden gas from the extractor was passed through a heated metering valve where the supercritical CO2 was depressurized, and the extract was collected in two successive cooled separators at 0 ◦ C. The gas flow through the extractor was measured by a turbine flow meter and totalled by a digital flow computer. In all experiments, the extraction was run with samples of 100 g of saponified CW flakes, placed in the extractor between two layers of glass wool to prevent losses of small particles. The experiments were accomplished in 2.0 h due to that longer extraction times did not significantly increased n-alcohols yield. The extract was collected in two consecutive separators. A white powder rich in alcohols was collected in a first separator and it was determined gravimetrically and analyzed according to the procedure described below. A second yellow fraction of highly soluble material was recovered in the wax separator and it was discarded as a byproduct owing that there is not alcohol occurrence in that fraction [10]. 2.3. Organic solvent extraction from crude wax In order to compare supercritical extracts to those obtained in the industrial process, 1000 g of crude wax flakes were extracted in a Soxhlet apparatus with analytical grade ethanol at 70 ◦ C (Panreac, Montplet & Esteban, S.A., Barcelona, Spain) for 2.5 h in order to separate in a first stage a soluble fraction formed by waxes and fats from the insoluble resins. After solvent recovery the soluble material was determined gravimetrically and fractionated in a second cold-extraction stage. The sample was melted at 60 ◦ C and stirred at 400 rpm in a 5000 ml beaker with 3500 ml ethanol at 20 ◦ C during 10 h. The mixture was filtered with filtering paper Whatman No. 3 in a glass funnel. The filter cake residue, called refined wax, free of fats, was dried during 4 h at 105 ◦ C to constant weight. In order to isolate the alcohol rich-fraction from the refined wax, 250 g refined wax flakes were saponified by melting the wax to 85 ◦ C and mixed with solid KOH pearls with 10% (w/w) ethanol, being stirred during 2 h at 85 ◦ C. The liquid waxy material was poured and cooled to 25 ◦ C in a flat mold, in order to obtain flakes of 2 mm thickness. The saponified refined wax was then extracted in a Soxhlet apparatus with analytical grade acetone (Panreac, Montplet
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& Esteban, S.A.) for 4.0 h. After extraction the solvent was evaporated and the acetone-soluble material determined gravimetrically. 2.4. GC analysis of long chain alcohols The aliphatic primary alcohols determination in the supercritical extracts and in the organic solvents extract was carried out according to European Food Legislation [14]. The fatty substance, with 1-eicosanol added as internal standard, is saponified with ethanolic potassium hydroxide and then the unsaponifiable matter extracted with ethyl ether. The alcoholic fraction is separated from the unsaponifiable matter by chromatography on a basic silica gel plate; the alcohols recovered from the silica gel are transformed into trimethylsilyl ethers and analyzed by capillary gas chromatography. It was used a PU 4600 Phillips gas chromatograph, equipped with a 2.1 mm × 3 m capillary column coated with 10% OV-101 on 100–120 Chromosorb W (Supelco/Sigma–Aldrich Inc., Madrid, Spain). Conditions were: injection and detection temperature 320 ◦ C; oven temperature programmed from 130 to 145 ◦ C at 10 ◦ C/min, and the carrier gas, Ar at 30 mL/min. The injection volume was 3 mL using chloroform as solvent. The alcohol derivatives were prepared with the silanizing agent N-methylN-(trimethylsilyl)trifluoroacetamide (Sigma–Aldrich Inc.). 3. Results and discussion 3.1. Supercritical extraction from CW Response Surface Methodology is commonly used in the study of empirical relationships between measured responses and independent variables, minimizing experimentation and leading to correlations which can be used for optimization purposes [15]. The response studied in this work was long chain alcohols yield (Y), defined as the weight percentage of alcohol fraction extracted at supercritical conditions from the starting sugar cane crude wax material. The operation variables selected were: pressure, temperature and the ratio of KOH/CW used in the saponification stage. The levels of each factor are indicated in Table 2, and were determined by taking into account previous experiments [16]. Saponification ratios varied from 20% (g KOH/100 g CW), the value used in the industrial process, and a lower percentage (1%) to reduce operations costs. Pressure ranges used were 300 and 350 bar, since extraction below 300 bar led to low extraction rates, whereas over 350 bar n-alcohols yield increased slightly. Temperature levels (50 and 100 ◦ C) were selected over the critical temperature of the solvent (31.1 ◦ C) but under and above the Table 2 Levels of factors Factor
Lower level (−1)
Higher level (+1)
KOH (%, w/w) Temperature (◦ C) Pressure (bar)
1 50 300
20 100 350
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A. de Lucas et al. / J. of Supercritical Fluids 41 (2007) 267–271
Table 3 Experimental matrix and results for the full 23 factorial design
Table 4 Main effects and interactions from the 23 factorial design
Experiment
KOH
T
P
KOH (%)
T (◦ C)
P (bar)
Y (g/100 g CW)
1 2 3 4 5 6 7 8 9 10 11 12
−1 +1 +1 −1 +1 −1 +1 −1 0 0 0 0
+1 +1 −1 −1 +1 −1 −1 +1 0 0 0 0
−1 +1 −1 −1 −1 +1 +1 +1 0 0 0 0
1 20 20 1 20 1 20 1 10.5 10.5 10.5 10.5
100 100 50 50 100 50 50 100 75 75 75 75
300 350 300 300 300 350 350 350 325 325 325 325
0.44 2.43 0.60 0.36 0.70 0.50 1.90 1.70 0.82 0.84 0.87 0.86
melting point of the octacosanol (82 ◦ C), respectively, the most represented aliphatic alcohol in the sugar CW. The solvent flow range was set in all experiments at 1 SL/min (l/min at standard conditions), taking into account that lower CO2 flows resulted in very low extract yields, but higher flow results resulted in entrainment of the substrate [9]. In order to allow a direct comparison of each variable effect, the independent variables were normalized in the range −1 to +1 according to: 2(Xi − Xmin ) (Xmax − Xmin ) − 1
Effect % alcohols
p value
KOH T P KOH–T KOH–P T–P Curvature
−0.4363 −0.1752 −0.0202 6.0 × 10−4 1.7 × 10−3 6.2 × 10−4 0.6351
0.0706 0.0562 0.3154 0.2834 0.0297 0.0352 0.1760
See Tables 1 and 2 for abbreviations.
Conditions—time of extraction: 2.0 h; weight of sugar cane crude wax: 100 g; CO2 flow: 1 SL/min.
xi =
Factor of interaction
(1)
where xi is the normalized value of variable X at condition i, Xi the dimensional value, and Xmin and Xmax represent the inferior and superior limits, respectively. Codified levels are indicated in Table 2. The RSM analysis consisted on a full factorial 23 experimental design to which four central points were added to evaluate the curvature effect. Experiments were run at random. The experimentation matrix is shown in Table 3. Columns 2–4 give the variable levels coded −1 to +1 in the dimensionless coordinate, while 5–7 give the dimensional variable levels. Experiments were run randomly. Table 3 also shows the experimental values for the yield of alcohols (Y). A statistical analysis was performed for these results using commercial software Statgraphics 5.1 Plus (Manugistics, Inc. Rockville, MD, USA). The analysis of the main effects and interactions for the chosen responses, together with the curvature check of the response surface results are shown in Table 4. The test of statistical significance, p value, was determined accord-
ing to the total error criteria considering a confidence level of 95%. The influence of a factor will be significant if the value of critical level (p) is lower than 0.05; discarding the meaningless parameters for p values over 0.05 [15]. Table 4 shows the main effects for the variables and the binary interactions considered. As it can be seen, the effect of the variables KOH, P and T cannot be separately discussed, because the KOH–P and T–P interactions are significant. This fact is reasonable due to that an increase in temperature produces opposite effects on these variables, i.e., increasing the vapor pressure of n alcohols, but also decreasing the CO2 density and its solvent power. The KOH–P interaction can be understood taking into account that the chemical composition of the crude wax is modified with KOH value, since approximately 50% of the n-alcohols are in form of esters [5]. Also, the amount of CO2 soluble compounds changes according of the amount of KOH in the saponification reaction. As shown in Table 4, the curvature effect was no significant, indicating the validity of pure fractional design in the experimental range analyzed, so that second-order models were not necessary. Thus, the equation that relates alcohols yield and variables can be expressed in the followings terms, where independent variables are codified: Y (g/100 g CW) = 2.9394 + 8.6 × 10−4 KOH P + 3.1 × 10−4 PT
(2)
From Eq. (2) the best operation conditions in the experimental range analyzed are: 20% KOH, 350 bar and 100 ◦ C. The extraction is favored for the higher quantity of saponified material, and P and T values where solubilization of n-alcohols in CO2 prevails over resins and soaps [7]. Table 5 shows the corresponding alcohol composition determined by GC together with the certified analysis of a commercial sample of PolicosanolTM (Smart City SA, Luxemburg). As observed the supercritical extract is mainly formed
Table 5 CG analysis of long chain aliphatic alcohol composition in the CO2 extract and comparison with a commercial sample of PolicosanolTM Alcohol Cn samplea
CO2 (mg/100 g) CO2 samplea (%, w/w) PolicosanolTM (%, w/w)b a b
Sample from Experiment 2. Commercial sample.
C24
C26
C27
C28
C29
C30
C32
C34
Total
0.08 0.10 0.13
4.25 5.42 10.24
0.63 0.80 0.53
53.19 67.85 61.83
0.20 0.25 3.70
12.35 15.75 20.87
6.26 7.99 –
1.43 1.82 –
78.32 100.00 99.41
A. de Lucas et al. / J. of Supercritical Fluids 41 (2007) 267–271
271
For a nutraceutical application, these poor quality characteristics require a further extract purification. To compare extraction yield and purity combined, row 5 in Table 6 shows the total amount (g) of n-alcohols extracted from 100 g of original crude wax. This result indicates that, in spite of the lower yields, the high purity of supercritical extracts, 70.42%, compared to a 22.00% organic solvent extract, results in a more efficient recovery of the n-alcohols from the CW (1.92 g/100 g CW versus 1.70 g/100 g CW). References
Fig. 3. Gas chromatogram corresponding to n-alcohol composition from Experiment 2.
by octacosanol C28 , containing 68% (w/w) of the total alcohols fraction, triacontanol C30 , dotriacontanol C32 and hexacosanol C26 ; whereas tetratiacontanol C34 , nonacosanol C29 and heptacosanol C27 are minor components. Fig. 3 shows the gas chromatogram corresponding to the quantitative analysis of the results in Table 5. It is noticeable that the octacosanol content of the extract (67.85%, w/w) is similar to that corresponding to a commercial sample of PolicosanolTM (60–75%, w/w) [16]. This fact, in addition to the similar n-alcohol distribution of the samples, indicates the value of the supercritical extract. 3.2. Comparison with industrial organic solvent extraction Table 6 shows the extraction yield and some physical characteristics of the alcohols obtained by the two methods studied, supercritical CO2 extraction and the industrial organic solvent (OS) process. With regard to physical characteristics, the green color and lower melting point of the organic extract indicate a low quality extract. The SC extract characteristics were rather similar to those corresponding to the pure compounds: melting point 80–82 ◦ C, white color for 98% C24 –C34 Sigma–Aldrich reagent. This shows that OS extract, in spite of higher extraction yield, contains a lot of impurities as confirmed by the row 4. Table 6 Comparison of organic solvent refining (OS) and supercritical CO2 (SC-CO2 ) extraction from sugar cane crude wax
(◦ C)
Melting point Color Extraction yield (%, w/w) n-Alcohols concentration in extract (%, w/w) n-Alcohols recovered (g/100 g CW)
OS
SC-CO2
64–68 Green 7.80 22.00 1.70
81–82 White 2.43 78.32 1.92
[1] R. Mas, Drugs of the future: policosanol, a review, Am. Heart J. 25 (2000) 569–586. [2] I. Gouni-Berthold, Policosanol: clinical pharmacology and therapeutic significance of a new lipid-lowering, Am. Heart J. 143 (2002) 356–654. [3] J.L. Hargrove, P. Greenspan, D.K. Hatle, Nutricional significance and metabolism of very long chain alcohols and acids from dietary waxes, Exp. Biol. Med. 229 (2004) 215–226. [4] N. Led´on, A. Casaco, V. Rodr´ıguez, J. Cruz, Anti-inflammatory and analgesic effect of a mixture of fatty acids isolated and purified from sugar cane wax oil, Planta Med. 69 (2003) 367–369. [5] A. Garc´ıa, Cera y grasas de la cachaza de ca˜na de az´ucar, in: Subproductos y Derivados de la Industria, Azucarera, GEPLACEA/PNUD (Eds.), M´exico, 1988, p. 315. [6] A. Lagunas, R. Magraner, Obtenci´on de mezclas de alcoholes alif´aticos de cera de ca˜na de az´ucar, Patente Cubana 229 (29) (1992). [7] E. Stahl, K.W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer-Verlag, Berl´ın, 1988, p. 127. [8] E. Stahl, K.W. Quirin, Process for the extractive production of valuable natural waxes, US Patent 4,548,755 (1985). [9] L. Fragernas, Extraction of waxes from peat with supercritical fluids, Acta Chem. Scand. 40 (1986) 538–543. [10] A. Garc´ıa, A. Alvarez, R. Ca˜nete, H. Sovov´a, Supercrital fluid extraction of sugar cane crude wax, in: G. Brunner, M. Perrut (Eds.), Proceedings of the 3rd International Symposium on Supercritical Fluids, Tome 3, 1994, p. 234. [11] K. Furukawa, K. Honda, I. Soschiciro, T. Matsui, Process for recovery primary normal aliphatic higher alcohols; extraction of sugar cane with supercritical fluid, US Patent 4,714,791 (1987). [12] A. Lucas, A. Garc´ıa, J. Rinc´on, A. Alvarez, I. Gracia, M.A. Garc´ıa, Supercritical carbon dioxide extraction of fatty and waxy material from rice bran, J. Am. Oil Chem. Soc. 73 (1997) 1127–1131. [13] A. de Lucas, E. Mart´ınez de la Ossa, J. Rinc´on, M.A. Blanco, I. Gracia, Supercritical fluid extraction of tocopherol concentrates from olive tree leaves, J. Supercrit. Fluids 22 (2002) 221–228. [14] Commission Regulation (EC) No. 796/2002, Annex XIX, Determination of aliphatic alcohols content by capillary gas chromatography, Off. J. Eur. Community 128 (2002) 23–28. [15] J.E.P. Box, W.G. Hunter, J.S. Hunter, Statistics for Experimenters: Design, Discovery and Innovation, second ed., John Wiley & Sons, New Jersey, 2005. [16] A. Garc´ıa, A. Lagunas, I. Gracia, A. Alvarez, Extracci´on de n-alcoholes de cadena larga a partir de cera cruda de ca˜na de az´ucar mediante CO2 supercr´ıtico, in: Diversificaci´on 2004, VII Congreso Internacional de Az´ucar y Derivados de la ca˜na, La Habana, 2004, p. 87.
Supercritical extraction of long chain n-alcohols from sugar cane crude wax Antonio de Lucas a , Alberto Garc´ıa b , Amaury Alvarez b , Ignacio Gracia a,∗ a
Departamento de Ingenier´ıa Qu´ımica, Universidad de Castilla-La Mancha, Facultad de Ciencias Qu´ımicas, Avda. Camilo Jos´e Cela n◦ 10, 13004 Ciudad Real, Spain b ICIDCA, Institute of Sugar Cane Byproducts, Via Blanca 804 San Miguel del Padr´ on, 11 000 Havana, Cuba Received 21 April 2006; received in revised form 27 September 2006; accepted 29 September 2006
Abstract We have studied the supercritical CO2 extraction of long chain alcohols from sugar cane crude wax, a by product of sugar cane production. The aim of this work has been to determine the effect on extraction yield of some operational variables such as pressure (300–350 bar), temperature (50–100 ◦ C) and the ratio of KOH in the saponification stage (1–20%, w/w). To assess the n-alcohol composition of the supercritical extracts the fractions were analyzed by gas cromatography (GC). Response Surface Methodology was used to obtain mathematical expressions relating the yield as a function of the operational variables studied. These correlations have permitted us to determine the best extraction conditions of the experimental range analyzed (P = 350 bar, T = 100 ◦ C, 20% KOH/crude wax). These results were compared to those obtained by the existing industrial organic solvent multiple stage refining process. The quality of the supercritical extract was considerably superior, because of its higher n-alcohols purity, 78.24% (w/w), compared to the organic solvent extraction yield, 22.00% (w/w). © 2006 Elsevier B.V. All rights reserved. Keywords: Long chain n-alcohols; Response Surface Methodology; Sugar cane wax; Supercritical extraction
1. Introduction Several researchers have reported the biological effects of active compounds extracted from sugar cane crude wax (CW) such as long chain n-alcohols, fatty acids or ethanolic extracts, which have application to atherosclerotic vascular, coronary heart diseases [1,2] and dermatologic diseases [3,4]. CW is obtained by heptane extraction from the sugar cane filter mud, a residue resulting from the sugar cane production containing 75% water, a large variety of fats, waxy esters, free alcohols, sterols and a resinous fraction mainly composed of calcium salts of heavy polyesters [5]. The industrial process for the separation of these alcohols shown in Fig. 1, consists of different successive and multiple steps, permitting a first fractionation of resin compounds, and the separation in a second step, of fats and a refined wax. Further solvent processing of the refined wax is focused on the isolation of a natural mixture of high molecular weight aliphatic alcohols of the series C24 –C34 , which once purified, have medical application (like a medicant called
∗
Corresponding author. Tel.: +34 926295300; fax: +34 926295318. E-mail address: [email protected] (I. Gracia).
0896-8446/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2006.09.013
PolicosanolTM ). This remedy is used for the treatment of low density lipoprotein and cholesterol reduction [2,3]. This long current process has several problems related to both, complexity and the use of organic solvents (toxic, expensive, generation of residues, low selectivity) requiring solvent recovery, as well as being energy-intensive operation. The use of CO2 under supercritical conditions has been expanded to the isolation of bioactive compounds from natural materials like lanolin, jojoba esters and popolis [6]. Due to CO2 ’s properties, selectivity of triglycerides and waxes has also been achieved for substances containing a high level of lipid material [7]. This procedure has been used for the separation of natural waxes [8,9] and aliphatic primary alcohols from the sugar cane wax [10,11] and rice bran [12]. All these results indicate that supercritical CO2 technology is a promising alternative to the actual organic solvent extraction of the long chain alcohols from CW. In the present study, we present supercritical fluid extraction (SFE) as a technically viable alternative process for the extraction of high molecular weight n-alcohols from the CW. We have analyzed the effect of pressure (300–350 bar), temperature (50–100 ◦ C) and the amount of KOH used in the previous saponification (1–20%, w/w) on extraction yield of long chain
268
A. de Lucas et al. / J. of Supercritical Fluids 41 (2007) 267–271
Fig. 1. Industrial process for n-alcohol isolation from sugar cane crude wax. Table 1 Sugar cane crude wax properties and composition Melting point (◦ C)
Color
Fats (%, w/w)
Waxes (%, w/w)
LCAa (%, w/w)
Resins (%, w/w)
56–61
Dark green
33.8
55.2
2–3
15.1
a
LCA: long chain n-alcohols (included in the waxy fraction).
alcohols. A Response Surface Methodology (RSM) based on the statistical analysis of the experimental data was used to obtain mathematical expressions relating the operational variables and extraction yield. 2. Experimental procedures 2.1. Materials Sugar cane crude wax flakes were provided by the Guiteras Sugar Mill (Puerto Padre, Delicias, Provincia las Tunas, Cuba). Table 1 shows the main physical properties and composition of CW used in the study. Liquid CO2 (purity 99.5%) was supplied by Gases Industriales S.A. (La Habana, Cuba). 2.2. Supercritical CO2 extraction 2.2.1. Sample preparation Previous to CO2 extraction, CW must be saponified as described in Fig. 2: 1000 g of CW were melted to 85 ◦ C and mixed with solid KOH and 10% (w/w) ethanol, being stirred
Fig. 2. Supercritical CO2 processing of crude wax.
A. de Lucas et al. / J. of Supercritical Fluids 41 (2007) 267–271
during 2 h at 85 ◦ C. The liquid waxy material was cooled to 25 ◦ C in a flat mold, in order to obtain flakes of 2 mm thickness. 2.2.2. Apparatus and extraction procedure The flow diagram of the extraction equipment has been described in detail in reference [13]. Liquid carbon dioxide was provided from a steel cylinder. After cooling and filtering, the CO2 was compressed by a positive displacement HPLC pump. The pressure was regulated by a back-pressureregulator and checked by a manometer. The compressed fluid at 300–350 bar was passed through a vertically mounted extractor from the bottom. The extractor was a 75 ml stainless steel cylinder (17.48 mm i.d. × 304.8 mm). To keep the extractor temperature at the desired value a digital controller regulated the electric current through a resistor that surrounded the extractor cylinder. The temperature in the extractor was displayed in a temperature indicator. In the first extraction stage the wax-laden gas from the extractor was passed through a heated metering valve where the supercritical CO2 was depressurized, and the extract was collected in two successive cooled separators at 0 ◦ C. The gas flow through the extractor was measured by a turbine flow meter and totalled by a digital flow computer. In all experiments, the extraction was run with samples of 100 g of saponified CW flakes, placed in the extractor between two layers of glass wool to prevent losses of small particles. The experiments were accomplished in 2.0 h due to that longer extraction times did not significantly increased n-alcohols yield. The extract was collected in two consecutive separators. A white powder rich in alcohols was collected in a first separator and it was determined gravimetrically and analyzed according to the procedure described below. A second yellow fraction of highly soluble material was recovered in the wax separator and it was discarded as a byproduct owing that there is not alcohol occurrence in that fraction [10]. 2.3. Organic solvent extraction from crude wax In order to compare supercritical extracts to those obtained in the industrial process, 1000 g of crude wax flakes were extracted in a Soxhlet apparatus with analytical grade ethanol at 70 ◦ C (Panreac, Montplet & Esteban, S.A., Barcelona, Spain) for 2.5 h in order to separate in a first stage a soluble fraction formed by waxes and fats from the insoluble resins. After solvent recovery the soluble material was determined gravimetrically and fractionated in a second cold-extraction stage. The sample was melted at 60 ◦ C and stirred at 400 rpm in a 5000 ml beaker with 3500 ml ethanol at 20 ◦ C during 10 h. The mixture was filtered with filtering paper Whatman No. 3 in a glass funnel. The filter cake residue, called refined wax, free of fats, was dried during 4 h at 105 ◦ C to constant weight. In order to isolate the alcohol rich-fraction from the refined wax, 250 g refined wax flakes were saponified by melting the wax to 85 ◦ C and mixed with solid KOH pearls with 10% (w/w) ethanol, being stirred during 2 h at 85 ◦ C. The liquid waxy material was poured and cooled to 25 ◦ C in a flat mold, in order to obtain flakes of 2 mm thickness. The saponified refined wax was then extracted in a Soxhlet apparatus with analytical grade acetone (Panreac, Montplet
269
& Esteban, S.A.) for 4.0 h. After extraction the solvent was evaporated and the acetone-soluble material determined gravimetrically. 2.4. GC analysis of long chain alcohols The aliphatic primary alcohols determination in the supercritical extracts and in the organic solvents extract was carried out according to European Food Legislation [14]. The fatty substance, with 1-eicosanol added as internal standard, is saponified with ethanolic potassium hydroxide and then the unsaponifiable matter extracted with ethyl ether. The alcoholic fraction is separated from the unsaponifiable matter by chromatography on a basic silica gel plate; the alcohols recovered from the silica gel are transformed into trimethylsilyl ethers and analyzed by capillary gas chromatography. It was used a PU 4600 Phillips gas chromatograph, equipped with a 2.1 mm × 3 m capillary column coated with 10% OV-101 on 100–120 Chromosorb W (Supelco/Sigma–Aldrich Inc., Madrid, Spain). Conditions were: injection and detection temperature 320 ◦ C; oven temperature programmed from 130 to 145 ◦ C at 10 ◦ C/min, and the carrier gas, Ar at 30 mL/min. The injection volume was 3 mL using chloroform as solvent. The alcohol derivatives were prepared with the silanizing agent N-methylN-(trimethylsilyl)trifluoroacetamide (Sigma–Aldrich Inc.). 3. Results and discussion 3.1. Supercritical extraction from CW Response Surface Methodology is commonly used in the study of empirical relationships between measured responses and independent variables, minimizing experimentation and leading to correlations which can be used for optimization purposes [15]. The response studied in this work was long chain alcohols yield (Y), defined as the weight percentage of alcohol fraction extracted at supercritical conditions from the starting sugar cane crude wax material. The operation variables selected were: pressure, temperature and the ratio of KOH/CW used in the saponification stage. The levels of each factor are indicated in Table 2, and were determined by taking into account previous experiments [16]. Saponification ratios varied from 20% (g KOH/100 g CW), the value used in the industrial process, and a lower percentage (1%) to reduce operations costs. Pressure ranges used were 300 and 350 bar, since extraction below 300 bar led to low extraction rates, whereas over 350 bar n-alcohols yield increased slightly. Temperature levels (50 and 100 ◦ C) were selected over the critical temperature of the solvent (31.1 ◦ C) but under and above the Table 2 Levels of factors Factor
Lower level (−1)
Higher level (+1)
KOH (%, w/w) Temperature (◦ C) Pressure (bar)
1 50 300
20 100 350
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Table 3 Experimental matrix and results for the full 23 factorial design
Table 4 Main effects and interactions from the 23 factorial design
Experiment
KOH
T
P
KOH (%)
T (◦ C)
P (bar)
Y (g/100 g CW)
1 2 3 4 5 6 7 8 9 10 11 12
−1 +1 +1 −1 +1 −1 +1 −1 0 0 0 0
+1 +1 −1 −1 +1 −1 −1 +1 0 0 0 0
−1 +1 −1 −1 −1 +1 +1 +1 0 0 0 0
1 20 20 1 20 1 20 1 10.5 10.5 10.5 10.5
100 100 50 50 100 50 50 100 75 75 75 75
300 350 300 300 300 350 350 350 325 325 325 325
0.44 2.43 0.60 0.36 0.70 0.50 1.90 1.70 0.82 0.84 0.87 0.86
melting point of the octacosanol (82 ◦ C), respectively, the most represented aliphatic alcohol in the sugar CW. The solvent flow range was set in all experiments at 1 SL/min (l/min at standard conditions), taking into account that lower CO2 flows resulted in very low extract yields, but higher flow results resulted in entrainment of the substrate [9]. In order to allow a direct comparison of each variable effect, the independent variables were normalized in the range −1 to +1 according to: 2(Xi − Xmin ) (Xmax − Xmin ) − 1
Effect % alcohols
p value
KOH T P KOH–T KOH–P T–P Curvature
−0.4363 −0.1752 −0.0202 6.0 × 10−4 1.7 × 10−3 6.2 × 10−4 0.6351
0.0706 0.0562 0.3154 0.2834 0.0297 0.0352 0.1760
See Tables 1 and 2 for abbreviations.
Conditions—time of extraction: 2.0 h; weight of sugar cane crude wax: 100 g; CO2 flow: 1 SL/min.
xi =
Factor of interaction
(1)
where xi is the normalized value of variable X at condition i, Xi the dimensional value, and Xmin and Xmax represent the inferior and superior limits, respectively. Codified levels are indicated in Table 2. The RSM analysis consisted on a full factorial 23 experimental design to which four central points were added to evaluate the curvature effect. Experiments were run at random. The experimentation matrix is shown in Table 3. Columns 2–4 give the variable levels coded −1 to +1 in the dimensionless coordinate, while 5–7 give the dimensional variable levels. Experiments were run randomly. Table 3 also shows the experimental values for the yield of alcohols (Y). A statistical analysis was performed for these results using commercial software Statgraphics 5.1 Plus (Manugistics, Inc. Rockville, MD, USA). The analysis of the main effects and interactions for the chosen responses, together with the curvature check of the response surface results are shown in Table 4. The test of statistical significance, p value, was determined accord-
ing to the total error criteria considering a confidence level of 95%. The influence of a factor will be significant if the value of critical level (p) is lower than 0.05; discarding the meaningless parameters for p values over 0.05 [15]. Table 4 shows the main effects for the variables and the binary interactions considered. As it can be seen, the effect of the variables KOH, P and T cannot be separately discussed, because the KOH–P and T–P interactions are significant. This fact is reasonable due to that an increase in temperature produces opposite effects on these variables, i.e., increasing the vapor pressure of n alcohols, but also decreasing the CO2 density and its solvent power. The KOH–P interaction can be understood taking into account that the chemical composition of the crude wax is modified with KOH value, since approximately 50% of the n-alcohols are in form of esters [5]. Also, the amount of CO2 soluble compounds changes according of the amount of KOH in the saponification reaction. As shown in Table 4, the curvature effect was no significant, indicating the validity of pure fractional design in the experimental range analyzed, so that second-order models were not necessary. Thus, the equation that relates alcohols yield and variables can be expressed in the followings terms, where independent variables are codified: Y (g/100 g CW) = 2.9394 + 8.6 × 10−4 KOH P + 3.1 × 10−4 PT
(2)
From Eq. (2) the best operation conditions in the experimental range analyzed are: 20% KOH, 350 bar and 100 ◦ C. The extraction is favored for the higher quantity of saponified material, and P and T values where solubilization of n-alcohols in CO2 prevails over resins and soaps [7]. Table 5 shows the corresponding alcohol composition determined by GC together with the certified analysis of a commercial sample of PolicosanolTM (Smart City SA, Luxemburg). As observed the supercritical extract is mainly formed
Table 5 CG analysis of long chain aliphatic alcohol composition in the CO2 extract and comparison with a commercial sample of PolicosanolTM Alcohol Cn samplea
CO2 (mg/100 g) CO2 samplea (%, w/w) PolicosanolTM (%, w/w)b a b
Sample from Experiment 2. Commercial sample.
C24
C26
C27
C28
C29
C30
C32
C34
Total
0.08 0.10 0.13
4.25 5.42 10.24
0.63 0.80 0.53
53.19 67.85 61.83
0.20 0.25 3.70
12.35 15.75 20.87
6.26 7.99 –
1.43 1.82 –
78.32 100.00 99.41
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For a nutraceutical application, these poor quality characteristics require a further extract purification. To compare extraction yield and purity combined, row 5 in Table 6 shows the total amount (g) of n-alcohols extracted from 100 g of original crude wax. This result indicates that, in spite of the lower yields, the high purity of supercritical extracts, 70.42%, compared to a 22.00% organic solvent extract, results in a more efficient recovery of the n-alcohols from the CW (1.92 g/100 g CW versus 1.70 g/100 g CW). References
Fig. 3. Gas chromatogram corresponding to n-alcohol composition from Experiment 2.
by octacosanol C28 , containing 68% (w/w) of the total alcohols fraction, triacontanol C30 , dotriacontanol C32 and hexacosanol C26 ; whereas tetratiacontanol C34 , nonacosanol C29 and heptacosanol C27 are minor components. Fig. 3 shows the gas chromatogram corresponding to the quantitative analysis of the results in Table 5. It is noticeable that the octacosanol content of the extract (67.85%, w/w) is similar to that corresponding to a commercial sample of PolicosanolTM (60–75%, w/w) [16]. This fact, in addition to the similar n-alcohol distribution of the samples, indicates the value of the supercritical extract. 3.2. Comparison with industrial organic solvent extraction Table 6 shows the extraction yield and some physical characteristics of the alcohols obtained by the two methods studied, supercritical CO2 extraction and the industrial organic solvent (OS) process. With regard to physical characteristics, the green color and lower melting point of the organic extract indicate a low quality extract. The SC extract characteristics were rather similar to those corresponding to the pure compounds: melting point 80–82 ◦ C, white color for 98% C24 –C34 Sigma–Aldrich reagent. This shows that OS extract, in spite of higher extraction yield, contains a lot of impurities as confirmed by the row 4. Table 6 Comparison of organic solvent refining (OS) and supercritical CO2 (SC-CO2 ) extraction from sugar cane crude wax
(◦ C)
Melting point Color Extraction yield (%, w/w) n-Alcohols concentration in extract (%, w/w) n-Alcohols recovered (g/100 g CW)
OS
SC-CO2
64–68 Green 7.80 22.00 1.70
81–82 White 2.43 78.32 1.92
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