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Biocatalyzed Acetins Production under Continuous-Flow Conditions: Valorization of Glycerol Derived from Biodiesel Industry Ingrid C. R. Costa1,2, Ivaldo Itabaiana Jr.1,3, Marcella C. Flores3, Ana Clara Lourenço2, Selma G. F. Leite2, Leandro S. de M. e Miranda1, Ivana C. R. Leal2 and Rodrigo O. M. A. de Souza1* 1
Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, CEP22941-909, Brazil 2 Escola de Química, Federal University of Rio de Janeiro, Rio de Janeiro, CEP22941-909, Brazil 3 Faculdade de Farmácia, Federal University of Rio de Janeiro, Rio de Janeiro, CEP22941-909, Brazil
The use of glycerol derived from biodiesel industry is an important development to add value to this actual waste. Several products can be obtained from glycerol, but acetins are very interesting molecules with a wide range of applications in pharmaceutical, cosmetics, food, and fuel industry. Herein we report our results on biocatalyzed batch and continuous-flow process for valorization of glycerol derived from biodiesel industry towards acetin production. Excellent results can be obtained with different selectivities depending on the nature of glycerol used and reaction conditions being able to produce monoacetin, diacetin, or triacetin depending on the reaction condition. Keywords: flow chemistry, lipases, glycerol, packed bed reactor, acetins, triacetin
1. Introduction The race for new bio-based fuels has increased during the recent years, and the research concerning the use of related byproducts such as biomass and glycerol has also been subject of several research papers [1–3]. Among all renewable materials that can be obtained from the production of bio-based fuels, glycerol is the one which attracts more attention, mainly due to the many possibilities that its structure offers and the wide range of applications for derived products. Glycerol can be used as a starting material for the production of 1,2-propanediol [4–7], 1,3-propanediol [8–11], acrolein [12–16], solketal [17–22], hydroxyacetone [23], glyceric acid [24, 25], and glycerol esters [26–28] among others. However, most of the strategies developed still apply commercial glycerol as starting material (Figure 1). Although glycerol is a readily available and cheap raw material easily obtained as a waste from biodiesel industry, working with glycerol obtained directly from the biodiesel hydrolysis production is still a challenge mainly due to several impurities brought from the triacylglycerol hydrolysis, the standard way of producing biodiesel [29, 30]. Among the several products listed before, acetins (mono-, di-, and triacetyl esters of glycerol) are very interesting molecules with a wide range of applications in pharmaceutical, cosmetics, food, and fuel industry [31–33]. Conventionally, acetins are synthesized by mineral or solid acid catalysis under high temperature conditions leading to the desired acetins in moderate to good yields in long reaction times [34–41]. As an alternative, biocatalysis has been also applied to the production of these molecules by lipase-catalyzed hydrolysis and esterification reactions [31–33, 42–44]. The uses of enzymes agree with the green chemistry protocols where benign solvents and reagents are used, affording the desired products under mild reaction conditions with high selectivity and yield. Lipases are special classes of enzyme, which do not need the use of co-factor, have a wide broad application, and are thermo stable and tolerant to organic solvents. Flow reactors are an emerging tool for improving organic chemistry [45–47] and in our continuous work on the development of biocatalytic process under batch and continuous-flow
conditions [48–56]. Herein we report our efforts on the synthesis of acetin mixtures, starting with the use of glycerol obtained from a biodiesel plant without prior purification.
2. Experimental Section 2.1. Chemicals and Materials. All reagents were purchased from Sigma-Aldrich and used without further purification. The immobilized enzyme Novozym® 435 (commercial lipase preparation of Candida antarctica, immobilized acrylic resin) and the free lipase Cal-B were purchased from Novozymes®. The crude glycerol obtained from the biodiesel process was kindly provided by Fertibom® (blond glycerol), obtained by the alkaline hydrolysis of triacylglycerol and named as GlyBio. 2.2. Batch Reactions. In 4-mL vials equipped with a stir bar, 1 mL mixture of glycerol and the acyl donor (1:5) was added followed by the appropriate enzyme (20% w/w). The vials were placed in a silicon carbide plate to have accuracy on the reaction temperature. Conversions in transesterification/esterification reactions were measured by gas chromatography–mass spectrometry (GC–MS) analysis as described below.
Figure 1. Glycerol as starting material for the production of valuable * Author for correspondence:
[email protected]
DOI: 10.1556/JFC-D-13-00001 © 2013 Akadémiai Kiadó
molecules J. Flow Chem. 2013, 3(2), 41–45
Valorization of Glycerol Table 1. Acetins production under batch conditions
Entry
Acyl donora
1 2 3 4 5 6 7 8
Acetic acid Acetic acid Vinyl acetate Vinyl acetate Ethyl acetate Ethyl acetate Acetic anhydride Acetic anhydride
Reaction time (h) 1 24 1 24 1 24 1 24
Conv. (%)a
Selectivity (%)a
(Yield %)
Mono
Di
Tri
6 (2) 56 (50) 100 (92) 100 (92) 44 (38) 67 (60) 67 (58) 100 (94)
6 46 0 0 42 51 50 0
0 10 98 81 2 16 17 54
0 0 2 19 0 0 0 46
a
Reactions were conducted with 1:5 molar ratio between the glycerol and acyl donor at 60 °C with 20% (w/w) of Novozyme. Conversions were measured by GC–MS analysis as detailed in experimental section and yields after product isolation.
2.3. GC–MS Analysis. GC–MS analysis was performed using a modified method from EN 14105. Free glycerol and mono-/diacetins were transformed into more volatile silylated derivatives in the presence of pyridine and N-methyl-N-trimethysilyltrifluoroacetamide (MSTFA). All GC–MS measurements were carried out in duplicate using a DB 5-HT (Agilent, J & W. Scientific®, USA) capillary column (10 m × 0.32 mm × 0.1 μm). The quantifying was done based on calibration curves with internal standards. The GC–MS samples were prepared by dissolving 0.1 g of the final product on 1 mL of n-heptane. One hundred microliters of this solution and pyridine solutions of butanetriol (1 mg/mL) and tricaprine (8 mg/mL), used as internal standards, were added on a flask forward by an addition of 100 μL of MSTFA. After 15 min, these reactants were dissolved on 8 mL of n-heptane. One microliter of this sample was then injected into a Shimadzu CG2010 equipment. 2.4. Continuous-Flow Experiments. A 50-mL tube was equipped with the desired reaction mixture and a stir bar. The starting mixture was vigorously stirred during the entire process. The instrument (Asia Flow Reactor) was equipped with an Omnifit column (2.4 mL, 7 cm length, and 0.34 cm diameter) containing the Novozym® 435 lipase (654 mg). Reaction parameters (60 °C or room temperature; 0.4–3.0 mL/min flow rate) were selected to verify the conversion obtained. Upon processing, conversions for each flow rate were measured the same way as in batch step. The ratios of monoacetin and diacetin isomers were not investigated, but it could be obtained by carbon-13 nuclear magnetic resonance (C13 NMR) experiments [57].
3. Results and Discussion First, we started our studies evaluating the esterification reactions between commercial glycerol and different acyl donors under batch conditions. For this screening, we have selected Novozym® 435 as catalyst at 60 °C, and a proportion of 1:5 between glycerol and acyl donor. Reaction time was 24 h maximum. The results concerning the batch reaction experiments are presented in Table 1 in terms of conversion and selectivity to mono-, di-, and triacetins. As can be seen from the data presented in Table 1, both reaction time and the acyl donor have shown an important influnce in the conversion and product selectivity outcome of the reaction under the conditions studied. Among all acyl donors used, increasing reaction time leads to high conversions. In the case of ethyl acetate, a high selectivity towards the monoacetin product is observed after 1 hour (Entry 5, Table 1), however in moderate conversion. In the case of vinyl acetate, high conversion was achieved with high selectivity between the three possible products, leading to an almost complete conversion towards diacetin after 1 hour of reaction (Entry 3, Table 1). Acetic acid and acetic anhydride lead to a selective reaction towards monoacetin also after 1 hour, however, with moderate conversions (Entries 1, 2 and 9, Table 1). The increase in reaction time for transesterification carried out with acetic anhydride leads to higher conversions and a mixture of di- and triacetins (Entry 10, Table 1). It is important to note that the
Figure 2. Kinetic study on the diacetin production using vinyl acetate as acylating agent 42
I. C. R. Costa et al.
Figure 3. Transesterification reaction using commercial glycerol ((a) reaction between glycerol and ethyl acetate at room temperature; (b) reaction between glycerol and ethyl acetate at 60 °C; (c) reaction between glycerol and vinyl acetate at room temperature; and (d) reaction between glycerol and vinyl acetate at 60 °C)
selectivity obtained for each product is directly related to the reactivity of the acyl donor used, whereas ethyl acetate (less reactive) was more selective to monoacetins, and vinyl acetate/ acetic anhydride was more selective to diacetins/triacetins depending on reaction times. The change in molar ratio of glycerol–acyl donor from 1:5 to 1:12 did not lead to any substantial effect on conversion or selectivity. Other enzymes sources were also tested, but poor results were obtained (from 5 to 12%). In order to explore the excellent selectivity obtained on the diacetin production using vinyl acetate (Table 1), a kinetic study based on the same conditions of the previous reactions was performed. Results are shown in Figure 1.
As a result, high rates of selective diacetin production were achieved where maximum conversion is observed after 10 min. Nevertheless, the increase in reaction time leads to a drop on diacetin content and the concomitant increase on triacetin formation when a 1:5 molar ratio glycerol:acyl donor is used. Longer reaction times (>60 h) can cause a consumption of diacetin to form triacetin as a major product. It is important to analyze that the system is stable (without an increased production of triacetin) under high concentration of diacetin for a periods longer than 6 h. The reasons behind such stability were not evaluated, and we focus our efforts on facing the development of a continuous-flow approach, aiming to improve the productivity towards acetins. Similar reaction conditions obtained in batch were applied to
Figure 4. Transesterification reaction using GlyBio ((a) reaction between glycerol and ethyl acetate at 60 °C; (b) reaction between glycerol and vinyl acetate at 60 °C)
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Valorization of Glycerol
the continuous-flow process; at this point we selected ethyl acetate and vinyl acetate as acyl donors. The results obtained are depicted in Figures 3a–d, and they are related to the use of commercial glycerol as starting material. Figure 1 shows that, under continuous-flow conditions, the selectivity towards the acetins is dependent on the residence time and temperature, for both ethyl acetate and vinyl acetate (Figure 1), leading to a different selectivity when compared to batch conditions. When ethyl acetate was used as acyl donor, the proportions between the products obtained (mono-, di-, triacetin) remained similar in both ambient and 60 °C. It is important to note that under batch conditions, monoacetin is the main product while under continuous-flow conditions diacetin is formed as a major product. As expected, the amount of monoacetin increases with an increase on flow rate, arriving at a maximum of 37% at 3 mL/min (0.8 min residence time). Very low conversions to triacetin were observed independent of the reaction temperature. In the case of vinyl acetate, it is observed that an increase in flow rate leads to an increase in conversion and selectivity toward diacetin for the reaction carried out at 60 °C (Figure 3d). The results show that at 0.4 mL/min and 0.5 mL/min (6 and 4.8 min of residence time, respectively) 50% of triacetin is produced while at 1.5 mL/min only diacetin is observed; these results can be explained as a reduction on the time of contact between diacetin and Novozyme 435 lipase. Interestingly, at ambient temperature (Figure 3c), no triacetin is detected. The continuous-flow process shown above was also applied on the esterification reaction carried out using glycerol obtained as by-product from the biodiesel industry. The use of such glycerol in esterification reactions is not an easy task because of several impurities and the high water content found in this starting material (35% w/w determined by Karl–Fisher analysis). These impurities could drive the reaction equilibrium towards reagents lowering the yield of the intended ester. The conversion and product selectivity on the reactions where the glycerol derived from biodiesel process (GlyBio) was used as starting material, without prior purification, are depicted in Figure 4a and 4b where ethyl acetate and vinyl acetate were used as acylating agents, respectively. Interestingly, a different reaction profile is observed in the function of the acyl donor used. The results presented in Figure 4a show that the use of ethyl acetate as acylating agent on the reaction where GlyBio is the starting material leads to a more selective production of monoacetins with moderated conversions at low flow rates (4.8 min residence time). Increasing the flow rate significantly reduces the conversion towards acetin, with monoacetins still the major product observed (Figure 4). In Figure 4b, it is observed that the vinyl acetate leads the conversion towards triacetin as a major product (84%) at low flow rates (0.5 mL/min–4.8 min of residence time). At higher residence times (14.4 and 28.8 min for 1.5 mL/min and 3.0 mL/min, respectively), the amount of diacetin increases to 70% and 60% (1.5 mL/min and 3.0 mL/min, respectively) on the reaction mixture, becoming the major product. A small amount of monoacetin (8%) can be detected at 3.0 mL/min flow rate. It is also important to note that the packed bed could be recycled 5 times without any loss of activity. 4. Conclusion In conclusion, the biocatalyzed continuous process for acetin production from GlyBio was reported. Using commercial glycerol, such product selectivity depends on the condition in which the reaction is performed. Carrying the reaction under batch conditions, a moderate conversion towards monoacetin with acetic acid or ethyl acetate is observed. Continuous-flow technology led 44
to a high conversion and selectivity towards diacetin, irrespective of the acyl donor employed. The developed methodology is also suitable to valorize the glycerol residue of the biodiesel industry. With such starting material, the proportions between mono-, di-, and triacetin are dependent on the acylating agent used. Less reactive acylating agents such as ethyl acetate can lead to the production of monoacetin-enriched acetin mixtures. When more reactive acylating agents are used, i.e., vinyl acetate, the major product is derived from the residence time used on the continuous-flow reactor where short residence times lead to diacetin-enriched mixtures and long residence times lead to triacetin-enriched mixtures. Acknowledgments. We thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPERJ (Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro), and FINEP (Agência Financiadora de Estudos e Projetos) for financial support.
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