Applied Catalysis A: General 392 (2011) 136–142 Contents lists available at ScienceDirect Applied Catalysis A: General journal homepage: www.elsevier.com/locate/apcata Catalytic ethanolysis of soybean oil with immobilized lipase from Candida antarctica and 1 H NMR and GC quantification of the ethyl esters (biodiesel) produced Isac G. Rosset, Maria Cecília H. Tavares, Elisabete M. Assaf, André Luiz M. Porto ∗ Institute of Chemistry of São Carlos, University of São Paulo, Av. Trabalhador São-carlense, 400, CEP 13560-970, CP 780, São Carlos, SP, Brazil article info Article history: Received 12 August 2010 Received in revised form 26 October 2010 Accepted 31 October 2010 Available online 5 November 2010 Keywords: Biodiesel Enzymatic ethanolysis Quantitative 1 H NMR analysis Quantitative GC-FID analysis abstract The catalytic ethanolysis of soybean oil with commercial immobilized lipase type B from Candida antarctica to yield ethyl esters (biodiesel) has been investigated. Transesterification was monitored with respect to the following parameters: quantity of biocatalyst, reaction time, amount of water added and turnover of lipase. The highest yields of biodiesel (87% by 1 H NMR; 82.9% by GC) were obtained after a reaction time of 24 h at 32 ◦ C in the presence of lipase equivalent to 5.0% (w/w) of the amount of soybean oil present. The production of ethyl esters by enzymatic ethanolysis was not influenced by the addition of water up to 4.0% (v/v) of the alcohol indicating that it is possible to use hydrated ethanol in the production of biodiesel catalyzed by lipase. The immobilized enzyme showed high stability under moderate reaction conditions and retained its activity after five production cycles. The 1 H NMR methodology elaborated for the quantification of biodiesel in unpurified reaction mixtures showed good correlations between the signal areas of peaks associated with the -methylene groups of the ethyl esters and those of the triacyl- glycerides in residual soybean oil. Monoacylglycerides, diacylglycerides and triglycerides could also be detected and quantified in the crude biodiesel using 1 H NMR spectroscopic and GC-FID chromatographic methods. The biodiesel production by enzymatic catalysis was promising. In this case, was produced a low concentration of glycerol (0.74%) and easily removed by water extraction. © 2010 Elsevier B.V. All rights reserved. 1. Introduction The continued increase in energy consumption, coupled with the imminent exhaustion of fossil fuel reserves in various pro- ducer regions, has promoted worldwide interest in the production of fuels from renewable sources. In identifying alternative energy sources, however, all relevant environmental concerns, includ- ing sustainability, biodegradability and global warming, must be taken into full account [1–3]. Biodiesel, comprising the mono- alkyl esters of fatty acids derived from natural lipids, meets all sustainability requirements and its production is economically viable. Moreover, the use of biodiesel offers a number of environ- mental and social advantages, since it may be produced from a range of renewable sources and the complete process generates employment at all stages [4]. Although current diesel oil-fueled engines can burn pure biodiesel without modification [5], the bio- fuel is often blended with petroleum-derived diesel (PDD). Such fuel mixtures are usually identified by the letter B followed by ∗ Corresponding author. Tel.: +55 16 3373 8103; fax: +55 16 3373 9952. E-mail address: almporto@iqsc.usp.br (A.L.M. Porto). the percentage of biodiesel present; for example, B5 contains 5% biodiesel with 95% PDD, while B100 contains 100% biodiesel [6]. Biodiesel is obtained through the catalytic transesterification of triacylglycerols and alcohols (ethanol or methanol) with glycerol being formed as a byproduct [7]. Efficient methanolysis requires the use of neutral oils with short-chain carboxylic acids as the substrate, a system free of water and typically use of sodium methoxide as the catalyst. Sodium methoxide can be obtained commercially either water-free or in solution at different concen- trations, thus increasing its efficiency and reducing the generation of soaps and the formation of the undesirable emulsions normally associated with the use of in situ preparations employing sodium hydroxide [8,9].Recent studies have focused on the use of enzymes as alternative catalysts in the production of biodiesel since such strategies involve cleaner technology and require a smaller num- ber of operational phases in comparison with chemical catalysis [10]. Immobilized lipases have been widely investigated in the enzymatic-catalyzed ethanolysis and methanolysis of triacylglyc- erols [11–13]. These hydrolytic enzymes primarily promote the fission of the esters bonds of acylglycerol esters to produce car- boxylic acids and glycerol, but they also catalyze esterification, interesterification and transesterification reactions [14–16]. The 0926-860X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apcata.2010.10.035