Green Synthesis of Isopropyl Ricinoleate

Transcription

1 Journal of Oleo Science Copyright 2013 by Japan Oil Chemists Society Green Synthesis of Isopropyl Ricinoleate Nishat R. Khan and Amit P. Pratap * Department of Oils, Oleochemicals and Surfactants Technology, Institute of Chemical Technology (University under Section 3 of UGC Act 1956; Formerly UDCT/ UICT), Nathalal Parekh Road, Matunga (East), Mumbai INDIA Abstract: Increased environmental awareness is slowly driving the industry to develop alternatives to chemical routes for synthesis. Lipase catalysed synthesis is one such alternative route that is environmentally more acceptable. In this study, immobilized Candida antarctica lipase (Lipozyme 435) was used for the esterification of ricinoleic acid and isopropyl alcohol. Molecular sieves were used to remove the water formed during esterification to drive the reaction in forward direction. The optimal conditions observed were 40 temperature, 4% enzyme concentration, 1:1 acid: alcohol ratio and 4 hours time interval. Under the described conditions, the reusability of lipase was tested and it was found that above 80% esterification was observed for over three cycles. Key words: Candida antarctica lipase (Lipozyme 435), enzymatic esterification, ricinoleic acid, isopropyl alcohol, isopropyl ricinoleate 1 Introduction Fatty esters are non-greasy and non-ionic derivatives of oils. They find applications in a large number of cosmetic formulations as surfactants, emollients, emulsifiers, thickening agents, etc. The current synthesis of these fatty esters having an emollient effect on skin is done by reacting a fatty acid and an alcohol at high temperature using a metal catalyst. These energy intensive conditions lead to degradation of esters and formation of undesired side products 1. Enzyme catalyzed reactions have gained a considerable importance in synthetic organic chemistry, pharmaceuticals and food industry 2. The chemical process for the esterification of long-chain fatty acids and fatty alcohols requires temperatures as high as F, which can generate raw products that are dark-coloured and do not meet the required quality criteria for cosmetic products in terms of purity, colour and odour. For this reason, the products undergo a host of reprocessing steps in which they are steamed, bleached, and filtered to remove the undesired colour and odour caused by the impurities. The enzymatic methods have the advantages of low energy requirements and minimal thermal degradation. The biocatalytic process runs at F under nearly physiological reaction conditions and supplies highly selective ultra-pure, colourless products that obviate the need for expensive time-consuming reprocessing and cleaning 3. The use of immobilized enzyme for the production of fatty acid esters improves the quality of the final product but also reduces the cost of the enzyme and is friendly to the environment 4. The formation of water poses a problem in the esterification reaction due to equilibria 5. Activated molecular sieves or salt hydrates can be added to the system to remove water produced by esterification 6. The kinetics of palmitic acid esterification catalyzed catalyst concentration wt by immobilized lipase Candida antarctica Novozyme 435 showed 70 reaction in 120 min at Similarly results were obtained for synthesis of 2-ethylhexyl palmitate and lauryl palmitate 8, 9. Present study deals with the synthesis of a novel ester isopropyl ricinoleate IPR by enzymatic Lipozyme 435 means. The percentage of esterification observed was around 91 under the optimized conditions for, temperature, enzyme loading, acid:alcohol ratio and time. 2 Experimental Procedures 2.1 Materials Lipozyme 435 enzyme was obtained as a gift sample from M/s Zytex India Pvt. Ltd., Mumbai, which was produced by fermentation of Candida antarctica and used in immobilized form. Its activity was determined as propyl laurate units/g of immobilized enzyme PLU/g using lauric acid and 1-propanol as substrates according to the manufacturer. Ricinoleic acid was received from M/s Jayant * Correspondence to: Amit P. Pratap, Assistant Professor and Author for Correspondence, Department of Oils, Oleochemicals and Surfactants Technology, Institute of Chemical Technology (University under Section 3 of UGC Act 1956; Formerly UDCT/ UICT), Nathalal Parekh Road, Matunga (East), Mumbai INDIA amitpratap2001@gmail.com, ap.pratap@ictmumbai.edu.in Accepted October 19, 2012 (recieved for review August 12, 2012) Journal of Oleo Science ISSN print / ISSN online

2 N. R. Khan and A. P. Pratap Agro organics Ltd., Mumbai. Isopropyl alcohol was procured from M/s Thomas Baker Pvt. Ltd., Mumbai. Molecular sieves 4 A were procured from M/s Merck Ltd., Mumbai. Ethanol AR grade and petroleum ether AR grade, sodium hydroxide AR grade and potassium hydroxide AR grade were procured from M/s Thomas Baker Pvt. Ltd., Mumbai. Phenolphthalein AR grade was procured from S.D. Fine Chem Ltd., Mumbai. 2.2 Methods Esterification reaction Esterification reactions were carried out in 100 ml stoppered Erlenmeyer flasks using Lipozyme 435 in an orbital shaking incubator at 150 rpm. Molecular sieves 4 A were used for water removal. The esterification reaction was optimized for temperature, enzyme concentration, acid: alcohol ratio, time and enzyme reusability. To elucidate the impact of the temperature on the synthesis of ricinoleic acid ester, the reaction was carried out at 30, 40, 50 and 60. The reaction was carried out at different 1 to 5 enzyme concentrations using different ratios of acid: alcohol i.e. 1:1, 1:2, 1:3 & 1:4. The effect of time on synthesis of Isopropyl ricinoleate was studied for 6 hours. Enzyme being an expensive commodity, its reusability was studied for 5 batches Purification and analysis of sample The samples were taken at regular interval and centrifuged at 6000 rpm for 10 min to recover the immobilized lipase. The IPR was washed free of IPA with warm water and traces of moisture were removed by heating under vacuum. The final product isopropyl ricinoleate was analysed for acid value FT-IR In order to check the esterification process, FT-IR spectrophotometer Perkin Elmer, spectrum 100 was used. IR spectra of the liquid sample was recorded with transmission mode using NaCl plates to show the shift of the carbonyl of carboxylic acid group to the carbonyl of ester group. FTIR spectra were acquired between 4000 cm 1 and 400 cm 1. Fig. 1 Esterification at different temperatures under the following conditions: 3 hrs time interval, 30 g acid g alcohol (as per stoichiometry) & 1% enzyme concentration. 3.1 Effect of temperature The highest conversion 70 was observed at 40 as shown in Fig. 1. Lipase lost its activity above 40 due to thermal deactivation therefore the percentage conversion declined after 40 as shown in Fig. 1. This finding was in line with the result obtained by Mat Radzi, S. et al. who observed that Novozyme 435 Candida antarctica gave maximum conversion at in their research work on high performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica 11. Similar results were also obtained by Syamsul, K. M. W. et al. in their research work on green synthesis of Lauryl Palmitate via lipase-catalyzed reaction Effect of Enzyme loading Reaction was studied at different concentrations from 1 to 5. Conversion increased linearly with enzyme concentration as given in Fig. 2. Maximum conversion was identified at 4 and 5 enzyme concentrations. The difference being not significant, the enzyme loading was optimized at 4 from commercial view point. Wantabe, Y. et al. also observed a plateau formation in the graph beyond 3-4 enzyme Candida antarctica concentration in their work on methyl esterification of waste fatty acids with immobilized Candida antarctica lipase Results and discussions The present research investigation enables to understand the effect of process parameters for synthesis of isopropyl ricinoleate. Fig. 2 % Esterification at different enzyme concentrations under the at 3 hrs time interval, 40 & 30 g acid g alcohol (as per stoichiometry). 154

3 Green Synthesis of Isopropyl Ricinoleate 3.3 Effect of acid: alcohol ratio Reaction was studied at different acid: alcohol ratios 1:1, 1:2, 1:3 & 1:4. It was observed that maximum conversion was seen at 1:1 acid: alcohol ratio Fig. 3 after which there was a decrease in conversion. This may be because the excess Isopropyl alcohol distorts the essential water layer from enzyme. At the same time, the excess Isopropyl alcohol may also hinder the interaction frequency between substrate and lipase 9. Many authors have reported similar behavior that has been attributed to inhibition effects caused by the alcohol on the catalyst. When alcohol is added to the system, due to its high polarity, it undergoes hydrophilic interactions with the aqueous boundary layer on the lipase surface causing modifications of the tertiary structure and therefore enzyme inhibition. Mat Radzi, S. et al. & Syamsul, K. M. W. et al. have observed maximum conversion at 1:2 acid: alcohol ratio in their respective work 9, 13. Fig. 3 % Esterification at different acid: alcohol ratios under the following conditions: 3 hrs time interval, 40 & 4% enzyme concentration. 3.4 Effect of Time A linear increase in the conversion until 4 hours was observed. Plateau formation in the graph indicated there was no significant rise in the conversion after 4 hours Fig. 4. A similar plot of graph which showed a plateau formation after 3-4 hours was obtained by Sekeroglu, G. et al. in their research work on production and characterization of Isopropyl Laurate using Novozyme 435 Candida antarctica Reusability of enzyme Lipozyme 435 enzyme showed over 80 esterification for batches 1-3 as shown in Fig. 5. The percentage conversion turned down to 69 for the next batches. Therefore it was concluded that the Lipozyme 435 can be effectively used for three batches. Similar results were obtained by Sekeroglu, G. et al. in their research work on production and characterization of Isopropyl Laurate using Novozyme 435 Candida antarctica FT-IR Isopropyl ricinoleate was synthesized under the optimized conditions i.e. 40 temperature, 4 enzyme concentration, 1:1 acid: alcohol ratio & 4 hours time interval. The percentage of esterification was around 91. Ricinolic acid has both -OH group and -COOH group in its structure. Candida antarctica which is positionally a non-specific lipase also catalyzed intermolecular esterification of -OH group of ricinoleic acid and its -COOH group resulting in the synthesis of ricinoleic acid estolide along with the product isopropyl ricinoleate 14. Previous studies on synthesis of fatty acid esters using lipases obtained from Candida species gave different yields or percentage of esterification ranging from 90-99, depending on the type of substrate and the process conditions Table 1. The formation of ester was confirmed by FT-IR analysis Fig. 4 % Esterification at different time intervals under the following conditions: 40, 4% enzyme concentration & 1:1 acid: alcohol ratio. Fig. 5 % Esterification at different batches under the following conditions: 40, 4% enzyme concentration, 1:1 acid: alcohol ratio & 4 hours time intervals. 155

4 N. R. Khan and A. P. Pratap Table 1 Percentage of esterification on using immobilised lipase obtained from Candida species in presence of different substrates and reaction conditions. Lipase Acid Alcohol Reaction conditions Candida sp Oleic acid Palmitic acid Palmitic acid Tuna- FFA Palmitic acid Oleyl alcohol Lauryl alcohol 2-ethyl hexanol Methanol 2-ethyl hexanol 40-50, g enzyme concentration, 1:2 acid: alcohol ratio & 5-10 mins time interval 40, 0.4 g enzyme concentration,1:2 acid: alcohol ratio & 10 mins time interval 70, 1.11%(w/w)enzyme concentration, 1:1.25 acid: alcohol ratio & 18 hrs time interval 30, 1%(w/w)enzyme concentration, 2 molar equivalent of methanol & 24 hrs time interval 40, 10%(w/w)enzyme, 1:1 acid: alcohol ratio & 4 hrs time interval % Esterification Reference >90% [11] >90% [9] ~100% [13] >95% [12] >98% [8] Fig. 6 FT-IR spectra of ricinoleic acid. of Ricinoleic acid substrate acid and isopropyl ricinoleate product ester. In the spectra of ricinoleic acid Fig. 6 the O-H of carboxylic group showed absorption band between cm 1 and the O-H group at the 12 th position showed absorption band between cm 1. Taken together, that gave a broad trough in the region between 2500 cm 1 and 3500 cm 1. After esterification there was a marked reduction in the broadness of this trough and a narrow peak was seen which indicated the consumption of O-H of carboxylic acid and indicated only the presence of 12 th position O-H group Fig. 7. In comparison with spectra of the ricinoleic acid Fig. 6, the major change was the presence of a peak at cm 1 corresponding to carbonyl group of the product ester Fig. 7. The intensity of the strong absorption band at cm 1 due to the stretching of the carbonyl of the acid group Fig. 7 was decreased after esterification. The presence of absorption band of small intensity at cm 1 indicated the presence of small amount of unreacted acid. 156

5 Green Synthesis of Isopropyl Ricinoleate Fig. 7 FT-IR spectra of isopropyl ricinoleate. 4 Conclusions A lipase catalyzed process of esterification was developed for the synthesis of novel ester, isopropyl ricinoleate from ricinoleic acid and isopropyl alcohol. The optimized conditions for isopropyl ricinoleate were 40 temperature, 4 enzyme loading, 1:1 acid: alcohol ratio and 4 hours time interval. Under the described conditions, the reusability of lipase was found to be for three cycles and 80 esterification was observed. The percentage of esterification observed was around 91 under the above mentioned optimized conditions. Acknowledgments The authors are thankful to University Grants Commission UGC, Govt. of India, New Delhi and ICT Golden Jubilee Endowment for the financial assistance. References 1 Awang, R., Basri, M. and Sallah, A. B., Enzymatic Esterification of Dihydroxystearic Acid, J. Am. Oil Chem. Soc., 77, , Vulfson, E. N., Enzymatic Synthesis of Food Ingredients in Low Water Media, Trends Food Sci. Tech., 4, , Oliver T., Enzymatic Emollients Sustainability That Gets Under the skin, COSSMA 1-2, 32-33, Aran, H. K., Prasertsan, P. and Sungpud, C., Continuous Production of Fatty Acids from Palm Olein by Immobilized Lipase in a Two-Phase System, J. Am. Oil Chem. Soc., 77, , Unal, M. U., A Study on the Lipase-Catalyzed Esterification in Organic Solvent, Turk J. Agric. Forestry, 22, , Sekeroglu, G., Fadloglu S. and Ibanoglu, E., Production and Characterization of Enzymatically Produced Lauric Acid Esters of Fructose J. Sci. Food Agric., 82, , Garcia T., Sanchez N., Martinez M. and Aracil J., Enzyme Microb. Technol Xiang-ling H., Bi-qiang C., Tian-wei T., Enzymatic synthesis of 2-ethylhexyl esters of fatty acids by immobilized lipase from Candida sp , Journal of Molecular Catalysis B: Enzymatic, 18, , Syamsul K. M. W., Salina M. R., Siti S. O., Hanina M. N., Basyaruddin M. A. R. and Kamaruzaman J., Green Synthesis of Lauryl Palmitate via Lipase-Catalyzed Reaction, World Applied Sciences Journal, 11 4, , Farag H. A., Azza E. M. & Nahla A. T., Optimization of factors affecting esterification of mixed oil with high percentage of free fatty acid, Fuel Processing Tech., 92, , Mat Radzi, S. et al., High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica, Electronic Journal of Biotech., 8 3, , Watanabe, Y., Shimada, Y., Baba, T. et al., Methyl Esterification of Waste Fatty Acids with Immobilized Candida antarctica Lipase, J. Oleo Sci., 51 10, , Brenneis, R. & Baeck, B., Esterification of fatty acids using Candida Antarctica lipase A in water abundant systems, Biotechnology Letters, 34 8, , 157

6 N. R. Khan and A. P. Pratap Horchani, H., Bouaziz1, A., Gargouri,Y. & Sayari, A., Immobilized Staphylococcus xylosus lipase-catalysed synthesis of ricinoleic acid esters, Journal of Molecular Catalysis B: Enzymatic, 75, 35-42,