Hydrolysis optimization and characterization study of preparing fatty acids from Jatropha curcasseed oil
© (Salimon) et al 2011
Received: 1 August 2011
Accepted: 1 November 2011
Published: 1 November 2011
Fatty acids (FAs) are important as raw materials for the biotechnology industry. Existing methods of FAs production are based on chemical methods. In this study potassium hydroxide (KOH)-catalyzed reactions were utilized to hydrolysis Jatropha curcas seed oil.
The parameters effect of ethanolic KOH concentration, reaction temperature, and reaction time to free fatty acid (FFA%) were investigated using D-Optimal Design. Characterization of the product has been studied using Fourier transforms infrared spectroscopy (FTIR), gas chromatography (GC) and high performance liquid chromatography (HPLC). The optimum conditions for maximum FFA% were achieved at 1.75M of ethanolic KOH concentration, 65°C of reaction temperature and 2.0 h of reaction time.
This study showed that ethanolic KOH concentration was significant variable for J. curcas seed oil hydrolysis. In a 18-point experimental design, FFA% of hydrolyzed J. curcas seed oil can be raised from 1.89% to 102.2%, which proved by FTIR and HPLC.
Hydrolysis of oils and fats is the applied term to the operation in which ethanolic KOH reacts with oil to form glycerol and fatty acids (FAs). Production of FAs and glycerol from oils are important especially in oleochemical industries. FAs and glycerol are widely used as raw materials in food, cosmetics, pharmaceutical industries [1, 2], soap production, synthetic detergents, greases, cosmetics, and several other products .
Lipid hydrolysis is usually carried out in the laboratory by refluxing oils and fats with different catalysts . The reaction can be catalyzed by acid, base, or lipase, but it also occurs as an un-catalyzed reaction between fats and water dissolved in the fat phase at suitable temperatures and pressures .
Researchers have been used several methods to prepare FAs and glycerol such as enzymatic hydrolysis using lipases from Aspergillus niger, Rhizopus javanicus and Penicillium solitum , C. rugosa , and subcritical water . Nowadays, researchers have used potassium hydroxide catalyzed hydrolysis of esters is sometimes known as saponification because of its relationship with soap making. There are two big advantages of doing this. The reactions are one-way rather than reversible, and the products are easier to separate as shown in . On a laboratory scale, alkaline hydrolysis is carried out with only a slight excess of alkali, typically potassium or sodium hydroxide in ethanol, refluxing for 1 h, and the FAs recovered after acidification of the reaction mixture. This is a sufficiently mild procedure that most FAs, including polyunsaturates, epoxides, and cyclopropenes, are unaltered .
Today ethanol is given emphasis over methanol in the world. Methanol was preferred in the seventies and eighties but the interest for methanol ended and instead ethanol programs were initialized. The grain-ethanol production, which today dominates the Europe alternative fuel market, may decide the technological path for decades to come. Today some European countries have commercial plants and pilot plant on ethanol production but no plant on methanol production .
Several reports have appeared on the hydrolysis of oils and fats using enzymes. Fats and oils can hydrolyze in the presence of natural enzymes. Enzyme reactions require milder conditions, less solvent, and give cleaner products attributes of green chemistry. There is increasing interest in the use of lipase enzymes for large-scale reactions. Reaction generally occurs under milder conditions of temperature and pH and there is reduced danger of undesirable side-reactions .
Though several studies which have appeared on the use of enzymes for hydrolysis of fats and oils. These studies have used to hydrolysis of FAs depends on the types of catalysts, types of vegetable oils and also depends on the different variables such as temperature and time to achieve 100% hydrolysis of vegetable oils. Hydrolysis of J. curcas seed oil by using sodium hydroxide NaOH  and potassium hydroxide KOH  have been studied. The literatures [11, 12] showed that with using methanol and NaOH, the experiment was conducted with optimum molar ratio (6:1) keeping the catalyst concentration (1% NaOH), reaction temperature (65°C) and reaction time (1 h). But by using KOH, the experiment was conducted with optimum molar ratio (8:1) keeping the catalyst concentration (1% KOH), reaction temperature (70°C) and reaction time (31/2 h).
This study is executed for the factors that affect the process of hydrolysis of J. curcas seed oil. D-optimal design was used to evaluate the effect of three factors, such as concentrations of ethanolic KOH concentration, temperature and time reaction were studied for the optimum hydrolysis.
Results and discussion
Effect of Process Parameters and Statistical Analysis
D-optimal design optimization of J. curcas seed oil hydrolysis and response for FFA%.
Coded independent variable levels
FFA, % (responses)
Ethanolic KOH (M, X 1 )
Temperature (°C, X 2 )
Time (h, X 3 )
The results show the hydrolysis performance of the ethanolic KOH effects on the hydrolysis reactions when submitted to different experimental conditions. Hydrolysis reactions were carried out at various ethanolic KOH concentrations ranging from 1.00 to 2.00 M. Table 1 demonstrates the effect of ethanolic KOH concentration on the FFA%. The FFA% at 1.00 M was low, however, it increased with increasing ethanolic KOH concentration, it can be clearly seen that the maximum FFA% obtained at 1.75 M was about 102.2%. A different observation was reported by other researchers for hydrolysis of various vegetable using C. rugosa lipase [13–15]. Increase in enzyme concentration did not give any significant changes in the reaction rate .
The effect of the reaction temperature on the FFA% is shown in Table 1. The FFA% increased when the temperature was increased from 50 to 70°C. At a reaction temperature of approximately 65°C, a percentage of FFA was achieved at 102.2%. This result indicates that the temperature is important factor in the hydrolysis of J. curcas seed oil. This theory was reported by  using enzyme C. rugosa lipase.
Table 1 indicates the FFA% using different times (1.5, 2.0 and 2.5h) with different variables, such as concentration of KOH and reaction temperatures. The FFA% increased with an increase in the reaction time, as shown in Table 1; 2.0 h was chosen to obtain the highest percentage of FFA (102.2%).
Regression coefficients of the predicted quadratic polynomial model for response variables Y (FFA%).
Coefficients (ß) % FFA (Y)
Analysis of variance (ANOVA) of the response Y (FFA%) of the D-optimal design
Sum of squares
GC-FID Analysis of Fatty Acids Composition
Fatty acid composition before and after J. curcas seed oil hydrolysis
FA% before hydrolysisa
FA% after hydrolysis 1.00Mb
FA% after hydrolysis 1.50Mc
FA% after hydrolysis 1.75Md
Table 4 shows a comparison of the FAs composition before the hydrolysis (a) and after the hydrolysis at different ethanolic KOH concentration (b and c, respectively), as determined directly by GC-FID, through FAMEs analysis. Intermediate products formed in the hydrolysis, as well as the methyl esters by FAMEs . The comparative data indicate that the hydrolysis does not cause the decomposition of the FAs.
FTIR Analysis of Fatty Acids
The main wavelengths in the FTIR functional groups of J. curcas seed oil hydrolysis
Wavelength of oila
Wavelength of 1.00Mb
Wavelength of 1.75Mc
C = C bending vibration (aliphatic)
C-H stretching vibration (aliphatic)
C = O stretching vibration (ester)
C = O stretching vibration (carboxylic acid)
C-H scissoring and bending for methylene
C-O stretching asymmetric (carboxylic acid)
C-O bending vibration (ester)
O-H bending vibration (carboxylic acid)
C-H group vibration (aliphatic)
For carboxylic acid carbonyl functional groups (C = O), FTIR spectrum showed absorption bands of hydrolysis oil (b and c) at 1711 cm-1 for stretching vibration, 1283-1285 cm-1 for stretching asymmetric while at 1413 and 918-937 cm-1 for bending vibration of carboxylic acid . The hydrolysis of J. curcas seed oil at 1.75M show disappeared completely of ester groups at 1746 and 1163 cm-1.
Figure 6 show the main change of hydrolysis J. curcas seed oil (b and c), which (b) 1.00M ethanolic KOH solution show low hydrolysis with C = O (ester carbonyl) at 1739, 1180 cm-1 while (c) at 1.75M ethanolic KOH solution shows high hydrolysis with strong absorption. Peaks at 2925- 2854 cm-1 indicated the CH2 and CH3 scissoring of both J. curcas seed oil and hydrolysis oil which showed on Figure 6(a), (b) and 6(c). FTIR spectrum also showed absorption bands at 722 cm-1 for (C-H) group vibration.
HPLC Analysis of Fatty Acids
Figure 7 illustrates a typical profile of triacylglycerol obtained of non hydrolyzed J. curcas seed oil. Figures 8, 9 and 10 illustrate the variation of the chromatographic profile of hydrolyzed J. curcas seed oil as a function of the ethanolic KOH concentration effects at 1.00, 1.50 and 1.75M, respectively.
HPLC chromatogram results showed, that with increasing ethanolic KOH concentration an increase in the FFA% and decreases of the concentration of the triacylglycerol is observed, fact that supports the hydrolysis model under investigation. A different observation was reported by other researchers for hydrolysis of various vegetable using C. rugosa lipase [13–15]. Increase in enzyme concentration did not give any significant changes in the reaction rate . Therefore, further increase in ethanolic KOH solution concentration show improvement in the conversion.
Procedure of J. curcasSeed Oil Hydrolysis
Independent variables and their levels for D-optimal design of the hydrolysis reaction
Determination of the FFA%
Gas Chromatography Method Analysis of Fatty Acids Composition
Gas chromatography method (GC) analysis was performed on Shimadzu equipped with flame ionization detector and capillary column (30 m × 0.25 mm × 0.25 mm film). The parameters of GC have been carried out according to .
Fourier Transforms Infrared Spectroscopy analysis of Fatty Acids
Fourier transforms infrared spectroscopy (FTIR) has been carried out according to . FTIR of the products was recorded on a Perkin Elmer Spectrum GX spectrophotometer in the range 400-4000 cm-1. FTIR was used to measure functional groups of FA. A very thin film of FA was covered on NaCl cells (25 mmi.d × 4 mm thickness) and was used for analysis.
High Performance Liquid Chromatography Method Analysis of fatty Acids
High performance liquid chromatography (HPLC) was performed on waters model 1515 equipped with refractive index detector and Spherisorb C18 column (250 mm × 4.8 mm × 3 mm) was used for analysis the TAG, DAG, MAG and FFA. The parameters of HPLC have been carried out according to . The samples were dissolved in 10 mL of the mixture acetone: acetonitrile before 20 mL of the sample inject into HPLC.
Experimental Design (D-Optimal) and Statistical Analysis
Where ß 0 ; ß i ; ß ii and ß ij are constant, linear, square and interaction regression coefficient terms, respectively, and xi and xj are independent variables. The Minitab software version 14 (Minitab Inc., USA) was used for multiple regression analysis, analysis of variance (ANOVA), and analysis of ridge maximum of data in the response surface regression (RSREG) procedure. The goodness of fit of the model was evaluated by the coefficient of determination R 2 and the analysis of variance (ANOVA) .
J. curcas seed oil hydrolysis, under optimum conditions, a highest hydrolysis was achieved. Hydrolysis occurs rapidly at 1.75M of ethanolic KOH, yielding 102.2% of the FFA. The analyses made by GC-FID had a positive identification of FAs composition and there is no significant difference under the optimum conditions p < 0.05. FTIR analyses showed strong absorption of carboxylic acid peaks at 1711 and 1285 cm-1. The results by using HPLC showed with increasing in ethanolic KOH concentration shows improvement in the J. curcas seed oil hydrolysis.
We would like to thank UKM and the Ministry of Science and Technology for research grant UKM-GUP-NBT-08-27-113 and UKM-OUP-NBT-29-150/2011.
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