State University of Rio de Janeiro Center for Technology and Science Institute of Chemistry

Sabrina Spagnolo Pedro da Silva

Synthesis of employing lipases dioctyl sebacate

Rio de Janeiro 2012

Sabrina Spagnolo Pedro da Silva

Synthesis of employing lipases dioctyl sebacate

Dissertation submitted to the faculty Graduate Program in Engineering Chemistry Institute of Chemistry, University the State of Rio de Janeiro as a final requirement to obtain the title of Master of Science in Chemical Engineering.

Advisor: Prof.. Dr. Marta Antunes Pereira Langone

Rio de Janeiro 2012

Cataloging AT SOURCE UERJ / NETWORK SIRIUS / NPROTEC

S586

Silva, Sabrina Spagnolo Peter's Synthesis of dioctyl sebacate employing lipase / Sabrina Spagnolo Pedro da Silva. - 2012. 93 f.

Advisor: Marta Antunes Pereira Langone. Thesis (Master) - University of the State of Rio de Janeiro, Institute of Chemistry.

1. Biolubricants - Theses. 2. Esterification (Chemistry) Theses. 3. Lipase - Theses. I. Langone, Marta Antunes Pereira. II. State University of Rio de Janeiro. Institute Chemistry. III. Title.

CDU 665.765.091.3

Authorize only for academic and scientific, the total or partial reproduction this dissertation, provided the source is acknowledged.

Signature

Date

Sabrina Spagnolo Pedro da Silva

Synthesis of employing lipases dioctyl sebacate

Thesis presented as partial requirement for obtain the title of Master, the PostGraduaodeEngenhariaQumica of State University of Rio de Janeiro.

Approved Examining Committee:

____________________________________________________ Marta Antunes Pereira Langone Institute of Chemistry UERJ ____________________________________________________ Aline Machado de Castro Cenpes / Petrobras

____________________________________________________ Antnio Carlos Augusto da Costa Institute of Chemistry UERJ _____________________________________________________ Fatima Maria Zanon Zotin Institute of Chemistry UERJ

Rio de Janeiro 2012

THANKS

For teacher and counselor Martha Langone for their advice, guidance, confidence and patience.

When my manager Wanderley Career, and confidence for the opportunity offered to Throughout the course.

To my colleagues, Fernando de Almeida, Amanda Maciel, Clara Simes, Fbio Ferreira, Renan Nagib, Claudia Sampaio, for friendship and complicity, since without them the completion of the course would be impossible.

By Igor Springs, for their help and collaboration, since without it the path would have been much longer.

Colleagues from the Laboratory of Enzyme Technology, Erika Aguieiras, Natasha, Susana and Marly, demonstrated by fellowship and the good advice.

When Leonardo Lagden, Clelia Jeni, Sergio and Pedro Pereira Eunisia for everything.

To all the other people who somehow were able to collaborate this work.

ABSTRACT

SILVA, Sabrina Spagnolo Peter's. Synthesis of employing dioctyl sebacate lipases. Brazil, 2012. 93f. Dissertation (Master in Chemical Engineering) - Institute of Chemistry, State University of Rio de Janeiro, Rio de Janeiro, 2011.

The growing demand for lubricants derived from renewable sources has stimulated the search for sustainable alternatives. The main objective of this work was to investigate the synthesis of dioctyl sebacate from the reaction Esterification between sebacic acid and 1-octanol and employing biocatalysts Conventional chemical catalyst (sulfuric acid). Some reaction parameters were studied: type of commercial immobilized lipase (Novozym 435, Lipozyme RM IM and Lipozyme TL IM), temperature, molar ratio acid / alcohol concentration lipase, methods of removing water from the reaction medium. The reuse of lipase Novozym 435 was also evaluated. The conversion of the reaction was determined by gas chromatography. The lipase Novozym 435 showed the best

Results: 100% conversion of sebacic acid was employed because when molar ratio acid: alcohol ratio of 1:5 and 5% w / w of lipase after 150 minutes of reaction to 100 C. The use of molecular sieves and vacuum did not increase the conversion of the acid sebacic. The final product was characterized with respect to viscosity index at viscosity, the flash point, the pour point and the neutralization index, and showed similar behavior to a naphthenic oil.

Keywords: biolubricants; esterification, lipase.

ABSTRACT

The growing demand for lubricants derived from renewable sources has Encouraging been the search for sustainable alternatives. The main objective of this study was to Investigate the synthesis of dioctyl sebacate from esterification reaction between sebacic acid and 1-octanol, using conventional chemical and Biocatalysts catalyst (sulfuric acid). Some reactions were Studied parameters: type of commercial immobilized lipase (Novozym 435, Lipozyme RM IM and Lipozyme TL IM), temperature, molar ratio, lipase concentration, and methods of water removal from the reaction medium. The reuse of lipase Novozym 435 was Also Evaluated. The conversion of the reaction was determined by gas chromatography. Lipase Novozym Showed 435 the best results, Achieving 100% conversion of sebacic acid When Employing sebacic acid with 1-octanol molar ratio of 1:5 and 5 wt. lipase after 150% minutes at 100 C. The use of molecular sieve and vacuum did not Enhance the acid sebacic conversion. The end product was Characterized by viscosity, viscosity index, flash point, pour point and neutralization index, and it Showed a pattern similar to the naphthenic oil.

Keywords: Biolubricants; esterification, lipase.

. LIST FIGURES

Figure 1.1 - General formula of esters ........................................... ......................... 21 Figure 1.2 - General scheme of reaction for obtaining esters ........................... 21 Figure 1.3 - Chemical Transformations of castor oil .................................... 23 Figure 1.4 - Scheme of the reactions (A) hydrolysis (B) esterification ................... 25 Figure 1.5 - Reaction for obtaining dioctyl sebacate .................................... 25 Figure 1.6 - Different conformations of the lipase Candida rugosa. (A) Conformation closed. (B) Conformation open ............................................. ................................ 31 Figure 1.7 - Mechanism of interfacial activation of lipases ....................................... 31 Figure 4.1. Effect of the lipase in conversion after 150 sebacic acid minutes of reaction between sebacic acid and 1-octanol (1:5 mole ratio) at 100 C, employing 5% w / w of different commercial lipases immobilized ...................... 53 Figure 4.2. Composition of the reaction medium after 150 minutes in the reaction between the acid 1-octanol and sebacic acid with molar ratio acid / alcohol of 1:5 at 100 C and 5% w / w lipase. (a) Novozym 435. (b) Lipozyme RM IM .................................... ......................... 54 Figure 4.3. Effect of molar ratio on the conversion of acid sebcico/1-octanol acid sebacic after 150 minutes of reaction at 100 C using 5% w / w of lipase commercial immobilized. (A) Lipozyme RM IM. (B) Novozym 435 ............................... 55 Figure 4.4. Effect of the concentration of lipase in the conversion of sebacic acid after 150 minutes of reaction at 100 C sebcico/1-octanol acid molar ratio of 1:5. (A) Lipozyme RM IM (b) Novozym 435 .......................................... ................................. 59 Figure 4.5. Composition of the reaction medium in the reaction between sebacic acid and 1 octanol, employing a molar ratio acid / alcohol of 1:5 at 100 C and lipase Lipozyme RM IM. (A) 3% w / w (B) 5% w / w (C) 7% w / w (D) 9% w / w (E) 11% w / w ................. 61 Figure 4.6. Composition of the reaction medium in the reaction between sebacic acid and 1 octanol, employing a molar ratio acid / alcohol of 1:5 at 100 C and lipase Novozym 435. (A) 3% w / w (B) 5% w / w (C) 7% w / w (D) 9% w / w (E) 11% w / w ....................... 63

Figure 4.7. Effect of temperature on conversion of acid-ic SEBAC after 150 minutes reaction, employing 5% m / m and lipase molar ratio acid sebcico/1-octanol 1:5. (A) Lipozyme RM IM (B) Novozym 435 ................................................ .......... 64 Figure 4.8. Selectivity in dioctyl sebacate obtained after 150 minutes of reaction between sebacic acid and 1-octanol, employing a molar ratio of 1:5 and 5% w / w of the lipase Novozym 435 at different temperatures ........................................... ......... 66 Figure 4.9. Composition of the reaction medium in the synthesis reaction of sebacate dioctyl to 100 C, with sebcico/1-octanol acid molar ratio of 1:5, with 5% m / m Novozym 435. (A) free evaporation. (B) Reactor Closed ........................................... 68 Figure 4.10. Composition of the reaction medium in the synthesis reaction of sebacate dioctyl to 100 C, with sebcico/1-octanol acid molar ratio of 1:5, with 5% m / m Novozym 435. (A) Void (b) Reactor Closed ......................................... ................... 70 Figure 4.11. Composition of the reaction medium in the synthesis reaction of sebacate dioctyl to 100 C, with sebcico/1-octanol acid molar ratio of 1:5, with 5% m / m Novozym 435. (A) 50 mg of molecular sieve (b) Reactor Closed ......................... 72 Figure 4.12. Mole fraction of dioctyl sebacate obtained in reactions between acid sebacic acid and 1-octanol at 100 C, using a molar ratio of acid sebcico/1-octanol 1:5 and 5% w / w Novozym 435. In closed reactor () in open reactor (free evaporation) (), employing vacuum () and adding 50 mg of molecular sieve 73 Figure 4.13. Conversion of sebacic acid in the synthesis reactions sebacate dioctyl being conducted at 100 C with molar ratio of acid sebcico/1-octanol 1:5, 7% w / w Novozym 435 lipase in various uses .............. 74 Figure 4.14. Composition of the reaction medium of the synthesis reaction of sebacate dioctyl employing a molar ratio of 1:5 sebcico/1-octanol acid and 7% w / w lipase Novozym 435 to 100 C (A) Initial reaction (B) First reuse (C) Second 76 Figure 4.15. Composition of the reaction medium of the synthesis reaction of sebacate dioctyl to 100 C, employing a molar ratio of 1:5 sebcico/1-octanol acid and 7% m / m of catalyst. (A) Novozym 435 (B) Sulfuric acid ......................................... 77

LIST OF TABLES

Table 3.1 - Solubility of sebacic acid in 2-octanol, ethanol, and the mixture octanol / ethanol at different molar ratios at temperatures of 75 C, 80 C, 85 C, 90 C and 100 C, in the proportions used ......................................... 45 .......... Table 4.1 - Solubility of 0.1 g of sebacic acid in different solvents (3ml) and 51 Table 4.2. Solubility of 1 mole of sebacic acid in different solvents and temperatures 52 Table 4.3. Results of the characterization of the product of reaction between sebacic and 1-octanol (94.2% of sebacate and dioctyl sebacate 5.8% of monooctila) compared to a paraffinic mineral base oil and an oil base mineral 79 Table 4.4. Comparative results of characterization tests of basic mineral paraffinic and naphthenic mineral basic ............................................ ..... 81

LIST OF ABBREVIATIONS AND ACRONYMS

ASTM CONAMA CEPED Uba EMBRAPA CCS

American Society for Testing and Materials National Council for the Environment Center for Research and Development State University of Bahia Empresa Brasileira de Pesquisa Agropecuaria Cold Cranking Simulator

SUMMARY INTRODUCTION Literature Review ................................................ .............................. 16 ............ 1. LUBRICANTS .................................................. ............................................ 16

1.1. Biolubricants .................................................. ........................................ 18 ..... 1.2. Esters 20 ........ 1.2.1. Dioctyl sebacate ............................................... ........................................ 21 1.3. Reactions for obtaining sebacate diocila ........................................... 24 1.4. Catalysts used in esterification reactions ......................... 26 .. 1.4.1. Chemical catalysts ................................................ ................... ............... 26 1.4.2. Biocatalysts ................................................. ............................................ 27 1.4.2.1. Lipases and their mechanism of action ............................................ ........... 29 1.4.3. Enzymes in non-aqueous media ............................................. ......................... 32 1.5. Influence of some variables in esterification reactions .................. .36 2. OBJECTIVES 3. EXPERIMENTAL .................................................. ................................................ 42 3.1. Materials .................................................. ................................................. 42 ........ 3.2. Equipment .................................................. ......................................... 42 ....... 3.3. Reactor 42 ........ 3.4. Determination of the enzymatic activity of commercial lipases ................. 43 3.5 The solubility tests .................................................. ............................. 44 ...... 3.6. Esterification reaction between sebacic acid and alcohol employing lipases 45 ....... 3.6.1. Effect of the lipase ............................................. ...................................... 46 3.6.2. Effect of molar ratio of reactants ............................................ .................. 46 3.6.3. Effect of enzyme concentration .............................................. .... ............... 46 3.6.4. Effect of temperature ............................................... ........................................ 46 3.6.5. Methods for removing water ............................................. ........................... .47 3.6.6. Reuse of lipase ............................................... ..................................... .47

3.6.7. Effect of chemical catalyst .............................................. ......................... 48 .. 3.7. Chromatographic analysis .................................................. ............................... 48 .. 3.8. Standard curves .................................................. .............................................. .49 4. RESULTS AND DISCUSSION .................................................. ........................ .50 4.1. Preliminary results .................................................. ...................... 50 ......... 4.1.1. Solubility tests ............................................... ..................................... 50 4.2. Influence of some reaction parameters on the conversion of the reaction esterification of sebacic acid with 1-octanol employing lipase ................ 52 4.2.1. Influence of lipase .................................................. ....................... 52 4.2.2. Influence of molar ratio of sebacic acid / alcohol reaction medium ............... 56 4.2.3. Influence of lipase concentration in the reaction medium ................. ............... 58 4.2.4. Influence of temperature ............................................... ................. ............... 64 4.2.5. Influence of water removal from the reaction medium ......................................... 66 4.2.6. Reuse of lipase ............................................... ..................................... .74 4.2.7. Chemical catalysis ................................................ ............................................. 77 4.2.8. Characterization of biolubrificante ............................................... ..... ............... 78 5. CONCLUSIONS AND SUGGESTIONS .................................................. 83 ........................ 5.1. Conclusions 83 5.2. Suggestions for future work .............................................. ............... 84 ........ REFERENCES 86 ANNEX I. Chromatographic analysis .................................................. ....................... 90

13

INTRODUCTION

The increasing fleet of automobiles around the world makes the demand in production of lubricants also come on increasing

considerably in recent years. According to report in The Globe February 1, 2011, it is estimated that only in Brazil, the automobile fleet has increased 114% in the last ten years. Much of lubricants currently used to meet this demand Vehicle used as a source of raw mineral base oil, from processes of refining oil. It is estimated that only 10% of lubricants produced worldwide are entirely synthetic (GRYGLEWICZ et. al. 2005). However, products arising from the oil, as well as concepts widespread, have been replaced with sustainable alternatives. Mineral-based lubricants, which are currently used in large scale, they have some undesirable features, such as, for example, are derived from nonrenewable sources after their use are considered toxic and dangerous, and its recycling the only correct destination for this, besides not being biodegradable. There are also studies that suggest a major shortage of oil in the near future, which would lead to a consequent increase in the price this raw material, which can be considered a source of the incentive search for sustainable alternatives (SALIH et. al. 2011). The bio-lubricants are characterized by being biodegradable lubricants and non-toxic to humans and the environment. These lubricants can be obtained from renewable sources such as vegetable oils (such as oil castor bean) or from synthetic ester oils made from renewable modified 2. The biolubricants show up as an alternative to lubricants mineral-based due to a number of factors: they are biodegradable, have low or no toxicity, have a higher lubricity and are derived from sources
1

Site http://www.globo.com. Accessed on 02/02/2011 at 20:00.

14

renewable. Also, allow for a greater range of trade, less downtime for engine maintenance, and have better physical and chemical properties, as better pour point, improved viscosity index, low volatility and so on. Among the highlights are the biolubricants synthetic esters, which are considered promising alternatives for the replacement of lubricant mineral origin, or oil arising because of the characteristics esters synthetic meet the challenges posed by the modern machinery and engines, both the technical issue as the environmental (GRYGLEWICZ et al. 2005). An ester may be used as synthetic lubricant is sebacate dioctyl. The dioctyl sebacate is a colorless, low viscosity emollient with good penetration power. It is also film forming properties 3 plasticizers. According to the company Dhaymers, supplier of synthetic lubricant dioctyl sebacate, it is produced on an industrial scale from direct esterification between the alcohol and sebacic acid (which may be n-octanol or 2 ethyl hexanol). It is usually produced in large industrial reactors, with capacity of 15 to 50 tons batch using acid catalyst and vacuum for removal of reaction water. The yield is normally between 90 and 98% of stoichiometric. An alternative route for obtaining the dioctyl sebacate is from the esterification reaction between the acid and the alcohol employing sebacic acid as Commercial immobilized lipase catalyst. Lipases are enzymes classified as hydrolases (triacylglycerol ester hydrolases EC. 3.1.1.3.) And have several advantages over catalysts conventional chemical, such as sulfuric acid, for example, which is usually used in esterification reactions. Examples of these advantages: increase reaction rate, higher specificity, acting on smaller tracks

Site: http://www.biolubrificantes.com. Accessed on 18/12/2011 at 16:00.

Site http://www.dhaymers.com.br/Defaultb.asp?area=11&produto=125. Accessed 17/10/2011 19:00.

15

temperature and pressure, as well as performance in a wide pH range (DALLA VECCHIA et al. 2004). In this case, it may also be noted that the immobilized lipases are heterogeneous catalysts. Thus, the separation of this biocatalyst reaction products becomes easier and it is not necessary to use unit operations complex at the end of the process. In addition, the lipase does not causes corrosion in reactors, as may occur when using the Conventional chemical catalysts, such as sulfuric acid. The above factors have increased the number of research about the use of lipases as catalysts for the production of those substances, for example, work Akerman et al (2011), Torres et al (2000); Gryglewicz (2003), Drmo et al (2004). Based on the above, this dissertation aims to obtaining dioctyl sebacate by enzyme catalysis. Thus, the present work will be presented as follows: the first part will be presented Chapter I in the literature review, which aims to provide theoretical as well to enable the analysis of the results obtained in the literature on relevant points the issue at hand. In Chapter II are explicit objectives of this thesis. On Chapter III will be described in the experimental part, which allows clear methods and materials used in the research in question, in Chapter IV will describe the Results found for the systems studied, and finally be presented in Chapter V the conclusions and suggestions for future studies.

16

LITERATURE REVIEW

1. LUBRICANTS

A lubricant acts to reduce accelerated wear of the parts a motor having the ability to form a protective film which surrounds these parts, keeping them separated (American Petroleum Institute, 1996). To meet the these requirements, some properties are required in the final product. Some these properties are listed below.

Pour point - is the lowest temperature at which the lubricant can flow freely, indicating the ability to work low temperatures (American Society for Testing and Materials ASTM D 97). However, it is observed that some oils are still able to flow even at temperatures below the pour point.

"In the basic paraffinic formation occurs a structure of wax crystals which prevents the free flow of the oil [...]. If this structure is altered or broken, the oil as a whole flows again. "(Lubricants Industry. RUNGE, 1989, p.22).

Viscosity - It is a measure of resistance to fluid flow. The measurement method most used is the kinematic viscosity. In this method, the control is done by the time in which a fluid flows on a given area under the gravitational action. It is a measure important as through it is possible to estimate conditions storage, handling, besides the operating condition, since the correct engine operation depends on the viscosity of the fluid employed (ASTM D 445). The viscosities more usually adopted to lubricants are measured at 100 C and 40 C.

Viscosity Index - is an empirical number that relates the change in viscosity with temperature variation, within a range of

17

40 C to 100 C in lubricating oils (ASTM D 2270). Higher the viscosity index, the less the change in viscosity with increasing temperature. Cold Cranking Simulator (CCS) - Measurement of apparent viscosity at low temperatures (between -5 C and -35 C), simulating a cold start of a vehicle (ASTM D 5293). Flash Point - It is the lowest temperature at which, with the passage of a flame test (spark), the vapors of the fluid under test become is flammable. This analysis is an important parameter for security since the lower the flash point of a product, more dangerous it becomes for handling and storage (ASTM D 93).

"The distinction between flammable and combustible products, for insurance purposes, transport security etc.., based on the flash point. A product submit a flash point below 65.6 C, by the ASTM D 93, considered flammable [...], above this value is considered combustible. "(RUNGE, 1989, p.21).

Other properties such as color, aniline point, tendency to foam and alkaline reserve, are important in characterizing a lubricant. The base oil Pure generally does not meet all of these requirements are then required to Addition of additives to achieve the final lubricant specifications. The big challenge for researchers in the field of lubrication is to balance the use of non-biodegradable products in the midst of so many programs to improve environment, where all efforts together to achieve reduction targets Products pollutant emissions of greenhouse gases and many other actions. The lubricating oil from fossil material after use and / or contaminated is considered a hazardous waste and dangerous for the population and for the environment. An attempt to manage this waste is to send it for re-refining, this being the only correct destination, according to CONAMA resolution (CONAMA 362, 2005). However, it is a costly process, since it is a complex industrial process and even this process generates byproducts. Other big problem besides the cost is to have control of all the used lubricating oil that

18

is dropped, since the actual population even disposes of the waste oil in soils and waters. The idea of using a biodegradable product will be essential for coming years, since the term biodegradable refers to products that do not harm the environment because they can be used as a substrate by microorganisms, that employ these materials to perform their cellular metabolism 4 (Total lubricants). In this context fall into the biolubricants. The biolubricants

represent the possibility of manipulating low or no lubricant toxicity, and the end of its life cycle, can be easily discarded because it is a biodegradable material. Moreover, due to the fact that biolubricants present good, or even better results for the properties listed above, such as flash point, pour point, among other, more research is being conducted to develop new technologies for obtaining the same.

1.1.

Biolubricants

The

biolubricants

are

defined

second

the business

Portuguese

Biolubricants - Total 5 on their homepage, a reference in the production of thereof, such as:
"The term biolubrificante applies to all the lubricants that are rapidly Biodegradable and non-toxic to humans and the environment. [...] Can be based on vegetable oils or synthetic ester oils made from renewable modified [...]. "

Currently, the constant demand for renewable energy sources has become increasingly frequent surveys about biolubricants. The biolubricants are considered a good alternative to traditional lubricants,

Site: http://www.total.pt. Accessed on 23/01/2010 at 19:30. Site: www.total.pt Accessed on 23/01/2010 at 21:00.

19

arising from fossil materials, which present good physicochemical properties, as low volatility, have excellent lubricity, are biodegradable materials and not toxic can be considered environmentally friendly products

(Quinchia et al. 2009). Furthermore, biolubricants are obtained from renewable sources, which causes decrease dependence of oil reserves, more scarce in the world - one of its great advantages over those derived from oil. Because they are synthetic lubricants, the biolubricants present several Advantages arising in relation to the lubricating oil. As a result of good physicochemical properties shown above lubricants synthetics have some benefits, such as:

- Easy starting the machine; - Smaller and smaller shear viscosity loss; - Less formation of gums and deposits; - Lower risk of health and safety; - Lower risk of fire; - Longer service life of the lubricant.

From the point of view of cost-effectiveness, also can highlight some advantages:

- Fewer maintenance shutdowns; - Lower consumption; - Greater efficiency.

Some esters are widely used as raw materials for obtaining biolubricants. This is due to its excellent properties physicochemical, and high flash point, good thermal stability and low

20

volatility (GRYGLEWICZ, 2000).

"Specific properties of esters enable them to meet the challenges of lubrication imposed by modern machines, both technically and in respect to environmental protection. Compared to oils derived from hydrocarbons, esters exhibit a strong dipole moment. This improves their lubricicidade, since they adhere strongly to the metal surface at the point of friction. " (GRYGLEWICZ et al. 2005, p.560, our translation).
6

1.2.

Esters

The esters are substances found in nature and may be found in liquid or solid depending on the number of atoms carbon and condition of the environment. Have the same boiling points of aldehydes and ketones relative molecular mass similar. Due to the presence of the C = O group, the esters have a polar character. However, as the do not possess hydrogen bonds, usually poorly soluble in water and soluble in alcohol. For esters, the solubility in water covers only Compounds of three to five carbon atoms (Morrison, 1983). These products are commonly used as flavoring, by having characteristic smell of certain fruits and flowers. Esters are being also widely used as lubricants. Figure 1.1 shows the formula Overall esters. The esters are obtained by the direct reaction of an alcohol (or even a phenol) and an organic acid (or acid derivative) in the presence of a catalyst. This reaction results in the replacement of an-OH group by an acid, the radical-OR ', generating the ester and water, the latter resulting Hydrogen alcohol and hydroxy acid. Figure 1.2 represents this reaction. It is noteworthy that the reactivity of the alcohol used meets the following order: Primary> Secondary> Tertiary (Morrison, 1983).

The text is in a foreign language: "Specific properties of esters enable Them to meet the vagaries of lubrication challenges posed by modern machines Both technically and with respect to environmental protection. In comparison to hydrocarbon oils, esters exhibit strong dipole moments. This Improves Their lubricity by adhering strongly to the metal surface at the friction point. "

21

Figure 1.1. Esters of the formula

Figure 1.2. General reaction scheme for obtaining esters

Among the esters that can act as bio-lubricants, highlights the dioctyl sebacate, sebacic acid, from which in turn is coming from the castor oil. The sebacates are products of great interest as an alternative to lubricating oils, because they have excellent characteristics for this purpose; exhibit good results for the tests already cited: pour point, high level viscosity and stability at high temperatures (Center for Research and Development of the State University of Bahia - Ceped / Uba) 7. Previous works, such as Gryglewicz (2005), show some esters obtained from dicarboxylic acids which can be used for this purpose, for example, esters derived from sebacic acid and adipic acid.

1.2.1. Dioctyl sebacate

The dioctyl sebacate (C26H50O4), the product of interest to the present work, which is also known as Decanodiico bis (2-etillhexil) ester can be obtained from the transesterification reaction of sebacic acid, this latest from the alkaline fusion of castor oil (Empresa Brasileira de

Available at: http://www.biodiesel.gov.br/docs/ofuturodaindustria% 20 -% 20Biodiesel.pdf.

Accessed on 28/02/2010.

22

Agricultural Research Corporation - EMBRAPA, 2010). Specific reactions to sebacate obtaining this will be discussed later. Castor oil, which is extracted from the seeds of the plant Ricinus communis has in its composition 90% triglyceride. Of these triglycerides, the is the main ricinolena that after suffering a reaction with methanol in alkaline (NaOH) at 45 C gives rise to methyl ricinoleate. From a reaction conducted at a temperature range of 250-275 C using 2 moles of NaOH per 1 mol of the methyl ricinoleate, one obtains the product alcohol caprylic and sebacic acid. Figure 1.4 illustrates this achievement, as well as all other chemical transformations of castor oil. The dioctyl sebacate is derived from sebacic acid diester, which is a dicarboxylic acid. It is in the form of a viscous, colorless or slightly yellowish, with a flash point of around 210 C (Merck Index, 1976). As any ester derived from dicarboxylic acids, sebacate dioctyl characterized by being thermally stable, have excellent lubricicidade and be biodegradable (GRYGLEWICZ, 2005). The dioctyl sebacate already been pointed out by other studies as a good alternative products of mineral origin. At work "Lipase catalyzed synthesis of sebacic and phthalic esters "Gryglewicz (2003) studied the synthesis of this, as well as the synthesis of other esters (di-2-ethylhexyl phthalate and di-3 ,5,5-trimetilhexil phthalate). The Results were evaluated by taking into consideration the type of lipase used as catalyst. Among the enzymes studied, Novozym 435, Lipozyme Im and PPL the first two, which are immobilized, were the most efficient in the synthesis of esters when compared with the use of the latter enzyme, PPL, lying in free form. Gryglewicz and coworkers (2005) compared esters derived from with sebacic acid esters obtained from adipic acid, always in the same reaction conditions. It was observed that the former had better physicochemical properties than the other, supporting the proposal

23

this work.

Figure 1.3. Chemical transformations of castor oil Source: EMBRAPA, 2010. 8

The sebacate didecila presented a viscosity index of 167, while didecyl adipate ester obtained a viscosity index of 117. To evaporation loss test, although both esters have shown
8

Available in:

http://www.sistemasdeproducao.cnptia.embrapa.br/FontesHTML/Mamona/SistemaProducaoMamona/i ndex.htm. Accessed on 23/01/2010.

24

good results, it can be seen that after a temperature of 350 C as didecyl sebacate showed a mass loss less than didecyl adipate. It is noteworthy that the evaporative loss test (Noack - ASTM D 5800) is an important parameter for lubricating oils requiring work higher temperatures. Once the lubricant has a large mass loss when subjected to high temperatures, no significant loss of fluid in use, beyond the issue of toxic substances to the environment. Also tested in this study was the addition of these esters in lubricating oils minerals in a proportion of 10% m / m. This addition led to improved physicochemical conditions of pure mineral oil. The pour point of the mineral oil was reduced by 5 C, the CCS (cold cranking Simulator - Simulator cold start) was reduced from 8900 cP to 7800 cP, the viscosity index increased from 176 to 184. In addition, the flash point had no significant reduction.

1.3.

Reactions for obtaining dioctyl sebacate Emil Fischer was the chemist who discovered in 1895 that the

Esters are formed by heating a carboxylic acid in a solution alcohol with a small amount of a strong acid as catalyst. In tribute to him, this reaction is known as Fischer esterification (Morrison, 1983). A Fischer esterification reaction is a nucleophilic substitution of the acyl grouping usually catalyzed by an acid. As the carboxylic acids are not sufficiently reactive to undergo attack, uses a strong acid (Usually HCl or H2SO4). The esterification reaction is considered to be the inverse of hydrolysis, where the water and ester react to form an alcohol and an organic acid (Reactions presented in Figure 1.5). All reaction steps are reversible, Then, it becomes possible to shift the direction of reaction. To encourage the formation of ester can work with excess alcohol, to promote the formation of acid carboxylic acid, works with large excess of water in the middle (McMurry, 2005).

25

The esterification reactions are extremely important in the chemical industry, since, as previously mentioned, the esters are used in various applications, such as in the production of flavors and essential oils, solvents, plasticizers for polymers, pesticides, herbicides, and the actual use as lubricants. Estimated that due to the extensive use of esters in these industries, the number of ester commercial exceed 500 (ALI, et al. 2007). The ester of interest, dioctyl sebacate was synthesized in this work the reaction of esterification of sebacic acid with octanol. The scheme reaction is shown in Figure 1.6. This reaction is also a reaction gradual polymerization, since the raw material is a dicarboxylic acid.

The II R-C-O-R '+ H2O

The II R-C-OH + HO-R '

(A) Hydrolysis

The II R-C-OH + HO-R '

The II R-C-O-R '+

(B) Esterification

H2O

Figure 1.4. Scheme of the reactions (A) hydrolysis (B) esterification. Source: Oak et al. 2003

HO2 C-(CH2) 8-CO 2 H + 2 CH 3 OH (CH2) 6CH3

Sebacic acid + 2 Octanol

C26H50O4
Dioctyl sebacate

2 H2O

2 Water +

Figure 1.5. Reaction for obtaining dioctyl sebacate.

Griglewicz et al. (2005) suggested another way to obtain esters dicarboxylic acids (such as sebacic acid and adipic acid). This could occur through transesterification from methyl esters with alcohols appropriate in the presence of basic catalysts. In this case, were studied and comparing the physicochemical properties of the two esters, such as the use of these lubricants.

26

1.4.

Catalysts used in esterification reactions

Catalysts are substances that accelerate the rate of reaction. This acceleration is due to the decrease in activation energy of the reaction, promoted by this catalyst. It is noteworthy that a catalyst only speeds a reaction thermodynamically possible by reducing its activation energy, But it does not allow a reaction impossible to happen (and STUMPF CONN, 1980). The catalysts generally are divided into homogeneous catalysts and heterogeneous catalysts. What distinguishes these two types of catalysts is the fact that the homogeneous catalysts to form a single phase mixture reaction, while still forming heterogeneous catalysts another phase during the reaction process. This feature allows us to affirm that the heterogeneous catalysts can be more easily separated at the end of the reaction, and can be reused. In the esterification reactions may be used acidic catalysts or enzyme. The acid catalysts have several drawbacks for use. Of According to Ali et al (2006), the recovery of these catalysts is not profitable, plus the fact that high concentrations of acid increase the rate of system corrosion, resulting in an increase in operating costs. Furthermore, as acid catalysts are homogeneous catalysts, there is still the difficulty Treatment of these wastes which must be neutralized for separating the product.

1.4.1. Chemical catalysts The catalysts used in esterification reactions may be acidic minerals. As carboxylic acids are reactive enough not to suffer an attack of an alcohol, there is a need of adding a strong acid (for example, HCl or H2SO4) to increase the reactivity of these carboxylic acids. These acids accelerate the esterification process so as hydrolysis by

27

protonarem fact that the carbonyl oxygen and thus become carbon carbonyl more susceptible to nucleophilic attack (McMurry, 2005). Although the yield of reactions using this type of catalyst is relatively high, the use of acid catalysts in industrial scale becomes limited because large amounts of these catalysts lead to processes corrosion in the system by requiring appropriate materials to withstand this corrosion, which increases production costs (ALI SAMI et al. 2007) Also according to Ali et al. (2007), other problems associated with the use of acid catalysts can be listed as follows:

- Products with a lower degree of purity; - The formation of byproducts is more pronounced; - There is a need for more complex operations for the separation of catalyst from the final product.

1.4.2. Biocatalysts

The use of biocatalysts is the subject of ongoing research due to its high catalytic activity, compared with the conventional chemical catalysts. Moreover, the catalyzed reactions occur in biocatalysts milder conditions, without losing its efficiency (DALLA-VECCHIA et al. 2004; LINKO et al. 1998). In this context is enzymatic catalysis. Some of the advantages of enzyme catalysis against conventional chemical catalysts are (BON, FERRARA and CROW, 2008):

- Can increase the speed of the reactions of 106 to 1012 times; - Its specificity is much higher; - Have high enantioselectivity; - They act in reaction temperatures lower than 100 C; - The reactions are conducted at atmospheric pressures;

28

- They are more environmentally acceptable.

According to Stumpf and Conn (1980) Enzymes are proteins which catalyze or accelerate reaction thermodynamically possible. The function of a catalyst enzyme has a high specificity due to its protein nature. Structure highly complex protein enzyme provides both the environment for particular mechanism of reaction as the ability to recognize a group limited substrates (E CONN STUMPF, 1980). The conversion of substrate to product occurs at a region of the protein which participates directly in this conversion. This region is known as the active site (Conn and Stumpf 1980). Among the enzymes, hydrolases are the most used industrially. Its use is due to the fact that the hydrolases have a higher specificity of substrates, wide availability, low cost, among others (DALLA-VECCHIA et al. 2004). Among the hydrolases, lipases are a large group of interest and come the subject of research for use as catalysts in several areas. In addition to the use these in detergent industries, other industries are gaining prominence as pharmaceutical, cosmetics, fine chemicals, biofuels, and Branch interest to this work, which is to obtain biolubricants. (CASTRO et al. 2003; CRESPO et al. 2004; GRYGLEWICZ, 2000; REETZ et al. 1998; REETZ, 2002).
"In general, lipases are capable of catalyzing esterification reactions and tioesterificao, and the hydrolysis of triglycerides. Features like catalytic versatility, commercial availability, low cost, stereoselectivity, and the possibility to use lipases in wide pH ranges and temperatures between 20 and 70 C, increased their applications. " our translation).
9

(CRESPO et al. 2004, p.401,

Duan and colleagues (2010) used the lipase Novozym 435 (Novozymes) to catalyze the reaction of synthesis of 1,3-diacylglycerol in the esterification reaction
9

The text is in a foreign language: "In general, lipases are suitable to catalyze esterification and thioesterification reactions and hydrolysis of triglycerides. Characteristics such as catalytic versatility, commercial availability, low cost, stereoselectivity and the Possibility of using Them Within the large

29

between oleic acid and glycerol in medium containing t-butanol. The authors observed that the enzyme showed high activity and stability for a long period. At the Best test conditions, it was possible to obtain 40% of the product interest. Gomes et al (2004) studied the synthesis of butyl butyrate, ester important for the food industry. The tests were made from esterification reactions between acid and butyric acid n-butanol, using lipase commercial immobilized Candida rugosa (Candida rugosa lipase type VII). It was found that even with low concentrations of lipase obtained an acid conversion almost 56%. Already Torres and Otero (2000) evaluated the performance of the esterification reaction lactic acid using lipase Novozym 435 (Novozymes). The yield obtained was 35%, employing the best test conditions. This in turn yields around the maximum efficiency that can be obtained by using lactic acid commercial.

1.4.2.1. Lipases and their mechanism of action

Lipases classified as hydrolases (triacylglycerol ester hydrolases, EC. 3.1.1.3) are enzymes that catalyze the breaking of the bonds ester different compounds, releasing fatty acids and glycerol (CASTRO et al. 2003; DALLA-VECCHIA et al. 2004). Can be found in nature, being obtained from renewable plant, animal or microbial (CASTRO et al. 2003; Rodrigues, 2009) and the microbial the most commonly used, since the point of economically and industry are more viable (DALLA-VECCHIA et al. 2004). Beyond of microbial lipases present isolation procedure simpler from the fermentation broth are also generally more stable than lipase from other sources (plants and animals) (OAK et al. 2003). In lipases, their active sites are characterized by a catalytic triad
range of pH and at temperatures between 20 and 70 C Increases Their application. "

30

comprises amino acids serine, histidine and aspartic or glutamic acid (Ser-HisAsp). These amino acids are essential for the reaction, since it records a loss of catalytic action by modifying these (VILLENEUVE et al. 2000). This triad is repeated throughout the entire structure of lipases. Lipases have their active sites protected by a lid peptide form of -helix in aqueous medium remains closed (closed conformation), causing the site to become totally isolated from the reaction medium. The active site of lipase is constantly protected by this cover hydrophobic described above, called "lid". When in contact with the hydrophobic interface or organic solvent, this cover is opened due to a conformational change experienced by the structure, thereby exposing the active site (Figure 1.7 and Figure 1.8) (VILLENEUVE et al. 1999). Lipases have been classified into two groups according to their specificity with respect to the substrate: 1) 1,3 specific lipases - catalyze the release of fatty acids specific primers, namely at position 1 or 3 of the glycerides. 2) non-specific lipases - not demonstrate with any specificity the nature of the acyl group or the position in which it is esterified in glycerol. Releases fatty acids in position 1 (3) 2 or Acylglycerol.

31

Figure 1.6. Different conformations of lipase Candida rugosa. (A) closed conformation (b) Open conformation.

Figure 1.7. Mechanism of interfacial activation of lipases.

May be used as biocatalysts immobilized lipases or even its free form. The advantage of using the lipase is in immobilized form the fact that when immobilized support provides greater stability and life of the enzyme. In addition, the carrier may provide better dispersion of the enzyme in reaction medium (GOMES et. al. 2006). Gomes et. al. (2006) studied the following types of situation: reactions Esterification between different alcohols and acids by lipase-catalyzed Candida rugosa in immobilized form and the same reaction using the aforementioned lipase, but in its free form. The reactions were studied in organic media and aqueous medium. The results obtained from the experiments show that the

32

enzyme in immobilized form starred in temperatures and pH values more extreme the enzyme in the free form distorted when applied to the same values pH and temperature (45 C and 8.0, respectively).

1.4.3. Enzymes in a nonaqueous medium

For many years it was believed that the use of enzymes was only possible in aqueous medium. The use of enzymes in aqueous media has been extensively used in catalytic processes, but their use has been limited, since many substrates are little or not soluble in aqueous media, necessitating large volumes reactive and complex separation procedures (GOMES et al. 2004). Despite reports that some enzymes exhibit catalytic activities in organic media datem the beginning of the century, from the 1980s that began to use the enzymes in catalytic processes in nonaqueous media (LIMA and Angnes, 1999). Water plays an important role in the properties of the enzymes, and like other proteins, in the absence of water, the enzymes have their conformation changed, causing the same to lose their activity, stability, and selectivity, causing the enzymes become inactive (ZACKS and Klibanov, 1985). The water acts as a kind of lubricant molecular without which enzymes become very rigid. With the hydration of enzymes, the active center is an increase in mobility due to an increase in polarity and flexibility (Klibanov, 2001). To maintain the active enzyme, it should provide water in the reaction. So the question is not whether the media should contain water, but the as Water the medium should contain. And furthermore, what the availability of water for enzyme (LIMA and Angnes, 1998). Some of the advantages of using enzymes in biocatalysis with middle nonaqueous are listed below (Garcia, 2005; Lee and Dordick, 2002; SERDAKOWSKI and Dordick, 2007; Zaks and Klibanov, 1985; Zaks and Klibanov,

33

1987): - Increased solubility of some substrates and / or products, allowing some reactions are possible to happen; - Increased thermal stability of enzymes; - Possibility of some reactions occur, since in these aqueous reactions are restrictions order kinetics and / or thermodynamics; - Because enzymes are insoluble in organic media, the removal of these reaction medium and subsequent reuse of the same becomes easier. - Displacement of the equilibrium reaction towards synthesis, since there is a reduced amount of water.

Currently conventionally be considered as the non-aqueous media organic solvents, supercritical fluids, gas phase or solid phase, in addition to ionic liquids, the latter being used for this purpose only more recently (AIRES-Barros, 2002; GARCIA, 2005). With these advantages the use of enzymes in non-aqueous media, it is becoming increasing number of studies and researches presented in this context. After pioneering work by Zaks and Klibanov 1984 (Garcia, 2005), more Articles are published in this area (GOMES et al. 2004; Halling et al. 1998; LEE and Dordick, 2002; Angnes and LIMA, 1998; YANG et al. 2004). Examples of reactions obtained by enzyme catalysis in an organic medium some organic syntheses (separation of racemic alcohols or acids, synthesis peptides), industries (synthesis of aspartame, regioselective interesterification of oils and fats), beyond the analytical (and Angnes LIMA, 1998). There are also some disadvantages to the use of organic solvents as a medium reaction, for example, mass transfer limitations (especially catalysts immobilized) and toxicity of solvents. However, the great problem of using enzymes in organic solvents and the yield of reaction. It is known that the yields of reactions in aqueous solution are higher than in organic media - about four or five orders of

34

magnitude higher (SERDAKOWSKI and Dordick, 2007). According Klibanov (1997), this lower yield in organic solvents can be explained by the fact that in aqueous medium, the enzyme forms a solution (since that it is soluble in water). When the enzyme is added in solvents Organic form a suspension. Therefore, it is quite plausible that catalytic activity is lower in organic solvents because of the limitations in diffusional substrates. Also according Klibanov (1997), another possible cause for this performance lower is the conformational change of the enzyme. As you know, when proteins are in contact with mixtures of water and organic solvent takes place denaturation thereof. When in contact with pure organic solvents, it is expect an even worse situation. But actually, it is not properly contact with an organic solvent which causes this enzyme denaturation but its dehydration which alters the structure of enzymes and consequently reduces their catalytic activity. With the need to use organic solvents as reaction medium, for reasons mentioned earlier, new research nowadays are about how to improve the yield of these reactions. Thus, emerge increasingly efficient alternatives to improve the performance of reactions in media organic (Halling et al.; 1998; LEE et al. 2002; Klibanov, 1997; Klibanov, 2001; SERDAKOWSKI et al. 2007; YANG et al. 2004). As some alternative can quote (Klibanov, 2001;

SERDAKOWSKI et al. 2007):

- Use of hydrophobic solvents preferentially to hydrophilic. Solvents Hydrophilic can remove the minimum amount of water from the surface of the enzyme, this water is essential for the activation of the same; - Use of more vigorous homogenization of the middle, so as to eliminate possible problems of mass transfer. - Lyophilization of the enzyme. This is a dehydration process in which

35

Enzymes are frozen and subsequently placed in a vacuum. Molecules sublimating water present in the enzyme without the melting of the same. For this, The temperature of the enzyme is cooled during the freezing step. In step Next, the enzymes are subjected to a vacuum and higher temperatures. Soon then the temperature is further raised to break any interaction that may have occurred between the water molecules and the frozen material. One of the ways to protect the enzyme interaction with the organic solvent the reaction medium is to immobilize the enzyme, that efficiency is maintained catalysis, in addition to providing long-term use of this biocatalyst. The immobilized enzymes are stable under conditions adverse pH and temperature, and facilitate its recovery from the reaction medium and reuse. Moreover, the immobilized enzymes can be used in conducting an ongoing process (OAK et al. 2006).

The main interest in immobilizing an enzyme is to obtain a biocatalyst with activity and stability that are not affected during the process, compared to its free form. Ideally, the immobilized enzyme should display a higher catalytic activity (DALLA-VECCHIA, et al. 2004).

According Aires-Barros (2002), reactions in organic media directly depend on the correct choice of solvent for each reaction. This choice should take into account the physical-chemical (solubilizing capacity Substrate and product density, viscosity, etc.), biological (aggressive for biocatalyst), safety (toxicity, flammability), logistics (easy acquisition, disposal), and economic (cost feasible). Also according to studies by Aires-Barros (2002) as main Disadvantages related to the use of organic solvents as a means of bioconversion can be mentioned: increase in transfer limitation mass products and / or reactants is introduced as a further step (Organic), and the aqueous phase and the solid phase itself (if the enzyme is immobilized), using the solvent can mean an adverse environment for the

36

biocatalyst, may also occur the formation of stable emulsions generally due to interfacial effects, should also consider the additional costs for process safety, considering the wastewater treatment process, including the actual reuse of the organic solvent.

1.5.

Influence of some variables in esterification reactions

Some variables directly interfere in obtaining the product in case reactions employing lipases as biocatalyst. Can highlight:

Effect of lipase concentration in the reaction medium: As with any catalyst, increasing the concentration of

enzyme increases the rate of reaction. With the low initial concentration of substrate, the active sites of the enzyme are not blocked. As increases the concentration of substrate, the active sites becomes saturated, blocking these sites, causing the speed to be independent of substrate concentration (conn and Stumpf 1980). According to Crespo and collaborators (2004), increasing the reaction yield with increasing lipase concentrations can be related to the fact that there is greater amount of available biocatalyst to form the complex "enzyme-substrate". However, some articles mentioned that in so far as increasing the amount of enzyme in the reaction medium, one can reach a point where the reaction yield decreases. Studies such as Duan et al (2010) to esterification to obtain 1,3-diacylglycerol show that behavior. With 0.50 g of enzyme in the reaction medium, the concentration of 1,3 diacylglycerol is around 25% after 12 hours of reaction, to increase the amount to 1g of enzyme in 50 mL observed in a maximum concentration of 1,3 diacylglycerol (35% after the same 12 hour reaction), to further enhance the enzyme concentration, this time to 1.25 g in 50 ml, there is a decrease the concentration of the product of interest (32.8% after 12 hours of reaction).

37

Article Torres and Otero (2000) also demonstrates behavior similar reaction in the enzymatic esterification of lactic acid with fatty acids (Such as caprylic acid) using Lipase Novozym 435. The higher yield response was obtained when the amount used of 100 mg of Novozym 435 (were tested the following amounts of lipase in the reaction medium: 25, 50, 100, 200 and 300 mg). By using greater or lesser amounts of lipase in the reaction medium, the reaction yield decreased. The authors attributed these results to the fact that with large amounts of biocatalyst in the reaction medium increases the fraction of Lactic acid molecules that form acyl-enzyme complexes. This formation of complex prevents further reactions involving caprylic acid, and This condition favors the formation of lactic acid oligomers.

Effect of temperature The reaction when catalyzed causes the value of the activation energy (EA)

reagent molecule is reduced before undergoing chemical transformation, thereby increasing reaction rate. Thus, the reactions catalyzed occur more quickly than non-catalyzed. Besides using catalysts to accelerate a chemical reaction, can also be used to increase the reaction temperature. With an increase in temperature of the chemical reaction, provides a greater kinetic energy of the molecules of the reagents, causing a greater quantity of production shocks between these molecules per time unit (Gomes et al. , 2006), so that there is an acceleration in reaction speed. Thus, increasing temperature increases the speed catalyzed reactions or even non-catalyzed reactions. The reactions catalyzed by enzymes present similar behavior to chemically catalyzed reactions, with regard to increasing temperature. However, the reactions biocatalisadas, there is a point temperature known as "Optimum" reaction, as it increases the temperature of the reaction medium, there was a marked increase in speed reaction. However, due to the nature of the enzyme protein, there may be

38

thermal denaturation at elevated temperatures, causing decrease the concentration effective enzyme, with consequent reduction of the reaction rate (and CONN STUMPF, 1980). Several studies in the literature demonstrate widespread this behavior, being the optimum always found justified by the denaturation of the enzyme in certain temperature. There may be mentioned the study by Wu and colleagues (2001) for esterification catalyzed by lipase from Candida rugosa in organic solvents. This article investigated among other factors, the influence of temperature. The best yield in the reaction was 30 C, which was considered the optimum temperature for the reaction. From there, small increases in temperatures have caused rapid denaturation of lipase. Already Gomes et al (2006) investigated the dependence of ezimtica temperature in activity, compared to the lipase of Candida rugosa in their free and immobilized. It was observed that for the free form of lipase activity maximum occurred at 37 C, whereas the lipase in the immobilized form had activity maximum 40 C. From these temperatures, observed a sharp drop in Relative activity (%) of lipases. The authors explained this fact by the denaturation enzyme, and, when it undergoes a process of immobilization can increase the optimum temperature of the enzyme, allowing jobs wider ranges of temperature. Gomes and colleagues (2006) justified the denaturation of the enzyme by the fact that the enzyme, by having a protein nature, requires its tertiary structure is maintained, primarily by large numbers of non-covalent bonds (eg hydrogen bonds, bonds disulfide) to ensure its catalytic activity. If the molecule absorbs excess energy, the tertiary structure is disrupted, denaturing the enzyme and thereby causing it to lose its catalytic activity.

Presence of Water The presence of water in the environment is a minimum amount needed to

ensure solvation of the enzyme or the substrates and products. This quantity

39

Minimum retained water also allows the enzymes retain their conformation dimensional active (DALLA-VECCHIA et al. 2004; Lortie, 1997). However, when excessive, the ester reacts with water produced, resulting in the displacement reaction in the opposite direction to the esterification reaction hydrolysis. Drm et al (2004) in studies on obtaining biolubrificante from esterification catalysis, enzymatic pathway, suggest the use of a pervaporation system to remove the water formed during the reaction between oleic acid and isoamyl alcohol (1:2). In this work, the output of the reactor was connected the pervaporation unit which used hydrophilic membrane to remove continuous water. The yield of the ester without the use of pervaporation system was 92%. When using the pervaporation unit, the yield of interest ester increased to 99.8% Linko et al. (1995) suggested the use of vacuum to remove water reaction system in the reaction of synthesis of poly (1,4-butyl sebacate), formed from polyesterification reaction between sebacic acid and 1,4-butanediol. The result the use of vacuum when the reaction is conducted in the presence of a solvent organic (diphenyl ether) was visible improvement in obtaining the product of interest, When compared with reaction elapsed without the aid of vacuum. When elapsed 160 minutes of reaction, the experiment that used vacuum obtained a average molecular weight of poly (1,4-butyl sebacate) of about 40,000 gmol-1. But the experiment did not use vacuum obtained an average molecular mass of product of interest of 9000 gmol-1.

40

2. OBJECTIVES

The aim of this study is to investigate the synthesis of dioctyl sebacate using commercial immobilized lipase. To this end, we determined the following specific objectives, in order to evaluate the best synthesis conditions between reactants sebacic acid and 1-octanol, evaluating the influence of various parameters: - Test the solubility of sebacic acid to identify what would be the best solvent used to analyze the reactions of synthesis gas chromatography, and to verify the composition of the reaction medium for the reaction between the acid sebacic acid and octanol. To this end, the following solvents were used in proportions of 0.1 g of sebacic acid in 3 ml of solvent: water, n-hexane, ethyl acetate, acetone, ethanol, methanol, ethyl ether, petroleum ether, diisopropyl ethyl methyl ketone, diisobutilcetona, 1-hexanol, diphenyl ether and 2-octanol. - To evaluate the solubility of sebacic acid in the solvents mentioned above in different temperatures (25 C, 40 C, 50 C and 60 C). - Check the influence of commercial immobilized lipase (Novozym 435, Lipozyme RM IM and Lipozyme TL IM) conversion of the esterification reaction between sebacic acid and 1-octanol. - To investigate the effect of temperature (90 C, 100 C and 110 C) the conversion of esterification reaction between sebacic acid and 1-octanol. - To study the influence of the molar ratio between the acid sebcico/1-octanol (1:4, 1:5, 1:6 and 1:7) in the conversion reaction. - Evaluate the effects of the concentration of biocatalyst (Novozym 435 and Lipozyme RM IM), 3, 5, 7, 9 and 11% w / w in the synthesis of dioctyl sebacate. - Investigate methods of removing the water formed in the reaction of esterification: using open reactor (free evaporation), adding sieve Molecular reaction system and the use of a vacuum. - Check the feasibility of reusing the biocatalyst in the synthesis of dioctyl sebacate.

41

- Compare the enzymatic pathway with chemical route, performing the reaction Esterification between sebacic acid and 1-octanol in the presence of 7% w / w acid sulfuric acid as catalyst. - Characterize the reaction product obtained by the following tests: viscosity of 100 C and 40 C, viscosity, pour point, flash point and acid number.

42

3. EXPERIMENTAL

3.1. Materials

Sebacic acid 99% 99% 1-octanol, 2-octanol 97% bis (2-ethylhexyl) sebacate and 97% molecular sieve 3 were supplied by Si gma-Aldrich. The Commercial lipase Novozym 435 (lipase specific 1.3 Pseudozyma antarctica, also known as Candida antarctica, immobilized on acrylic resin macroporous), Lipozyme RM-IM (1,3 specific lipase Mucor miehei, immobilized on ion exchange resin) and Lipozyme TL-IM (1,3 specific lipase Termomyces lanuginosus) were all kindly donated by Novozymes Latin America Ltda (Araucaria, Brazil). PA 99.9% absolute ethanol was supplied by Merck.

3.2. Equipment

The equipment used in this work were:

- Digital Analytical Balance METTLER TOLEDO; - Thermostatic Bath HAAKE DC 10; - Vacuum Pump FISATON model 825; - Gas Chromatograph Varian model CP 3380; - Board of agitation and heating IKA C-MAG HS 4.

3.3. Reactor

The esterification reactions were carried out in a batch reactor closed 15 ml capacity, fitted with condenser and magnetic stirrer. The temperature of the reaction medium was kept constant by circulating ethylene glycol from the water bath (HAAKE DC10) by the shirt reactor.

43

Monitoring the performance of esterification was conducted by gas chromatography. 20 l aliquots of the medium were removed reaction, after stopping the stirring, when then occurred to decanting the lipase reaction medium at different reaction times.

3.4. Determination of the enzymatic activity of commercial lipases

The activity of commercial immobilized lipases Novozym 435, Lipozyme RM IM and Lipozyme TL IM in esterification reactions was determined by initial reaction rate of esterification between oleic acid and butanol, with stoichiometric ratio of the reagents (0.03 mmol oleic acid and 0.03 mmol of butanol), using 3% w / w immobilized lipase at a temperature of 45 C. This esterification reaction was conducted in open reactor with capacity 20 mL, equipped with magnetic stirring and connected to a thermostatic bath (HAAKE D10). Was accompanied by the progress of the reaction by withdrawing aliquots from reactional medium (100 mL in duplicate) at the following times: 0, 5, 10, 15, 20, 30, 40, 50 and 60 minutes of reaction. These aliquots were analyzed by titration of neutralization. Samples were dissolved in 30 mL of acetone / ethanol with molar ratio of 1:2 and titrated against NaOH solution, 0.02 mol / l, using Mettler DL25 automatic titrator model. One unit of activity esterification was defined as the amount of enzyme which consumes 1 mol of oleic acid per minute under the experimental conditions described above. Below the equation that describes the calculation used to determine the activity Enzymatic esterification.

Where: A = activity of esterification (mols / min.g);

44

V1 = volume of NaOH consumed in titration of the sample taken at time zero reaction (mL); V2 = volume of NaOH consumed in titration of the sample taken at time t minutes of reaction (mL); C = concentration of the NaOH solution (mol / L); t = reaction time (min); m = mass of the enzyme preparation used in the reaction (g); Vm = volume of the reaction medium (mL); Va = volume of sample (ml).

3.5. The solubility tests

The solubility of sebacic acid was tested using 0.1 g of sebacic acid in 3 ml of the solvents listed below:

- Water - N-Hexane - Ethyl acetate - Acetone - Ethanol - Methanol - Ethyl ether

- Petroleum ether - Diisopropyl ether - Ethylmethylketone - Diisobutilcetona - 1-hexanol - 2-octanol - Diphenyl ether

The solubility was observed at different temperatures as follows: room temperature (approximately 25 C), 40 C, 50 C and 60 C. The mixture was maintained in an open reactor under constant stirring and the temperature of the medium reaction was kept constant by circulating ethylene glycol from thermostatic bath (HAAKE D10), the jacket of the reactor. Additionally, we tested the solubility of 0.02 mole of sebacic acid (Molecular weight = 202.25 g / mol) in octanol, ethanol and ethanol / n-octanol at various proportions and temperatures, as described in Table 3.1.

45

Table 3.1. Sebacic acid solubility in 1-octanol, ethanol, and the mixture octanol / ethanol in different molar ratios at temperatures of 75 C, 80 C, 85 C, 90 C and 100 C, in proportions used.

Solvent 2-octanol 2-octanol 2-octanol 2-octanol 2-octanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol and 2-octanol (1:2) Ethanol and 2-octanol (2:2) Ethanol and 2-octanol (3:2) Ethanol and 2-octanol (4:2) Ethanol + 2-octanol (5:2)

Molar ratio of reactants (Sebacic acid: solvent) 1:1 1:2 1:3 1:4 1:5 1:1 1:2 1:3 1:4 1:5 1:6 1:7 1:1:2 1:2:2 1:3:2 1:4:2 1:5:2

The solubility in ethanol was only tested at 75 C, considering the point This boiling alcohol (78.5 C). For octanol were evaluated temperatures 75 C, 80 C, 85 C, 90 C and 100 C, since its boiling point is 195 C.

3.6. Esterification reaction between sebacic acid and alcohol employing lipases

The effects of reaction temperature, concentration of enzyme, the ratio molar ratio of reactants, the type of enzyme, removing water from the reaction medium of reuse of lipase, plus the use of a chemical catalyst were investigated the esterification of sebacic acid.

46

3.6.1. Effect of type lipase

The effect of the type of lipase in esterification reactions between 0.02 mol sebacic acid and 0.1 mol of 1-octanol, using 5% w / w of lipase in 100 C. Lipases were studied: Novozym 435, Lipozyme RM IM and Lipozyme TL IM.

3.6.2. Effect of molar ratio of the reactants

The effect of molar ratio of reactants was studied in reactions between acid sebacic acid and 1-octanol, using 5% w / w of lipase (Novozym 435 and Lipozyme RM IM) at 100 C. The molar ratios of sebacic :1-octanol were investigated: 1:4 1:5, 1:6 and 1:7 for lipase Lipozyme RM IM and Novozym 435. The lower limit of molar ratio of 1:4 was used, considering the results of tests solubility.

3.6.3. Effect of enzyme concentration

The effect of enzyme concentration was studied in the reaction between 0.02 mol sebacic acid and 0.1 mol of 1-octanol, which corresponds to a molar acid: alcohol ratio of 1:5, employing lipase (Novozym 435 and Lipozyme RM IM) at 100 C. In these assays the enzyme concentration is expressed in% w / w in relation to mass sebacic acid. We studied the following concentrations Enzyme: 3, 5, 7, 9 and 11% w / w of Lipozyme RM IM and Novozym 435.

47

3.6.4. Effect of temperature

To evaluate the effect of temperature on esterification of sebacic acid, the reactions were conducted using 0.02 mole of sebacic acid (4.045 g) and 0.1 mol of ethanol (16.06 mL 1-octanol/2-octanol), the equivalent molar ratio acid / ethanol 1:5, and 5% w / w of lipase (Novozym 435 and Lipozyme RM IM) the mass of sebacic acid, corresponding to a mass of 0.2832 g. We studied the following reaction temperatures: 90 C, 100 C and 110 C.

3.6.5. Methods for removing water

The effect of the removal of the water formed in the esterification reaction between sebacic acid and 1-octanol was studied in experiments conducted with 0.02 mol sebacic acid and 0.1 mol of 1-octanol, using 5% w / w of lipase Novozym 435-100 C for 150 minutes of reaction. The forms of the water removal reaction medium used in this work were free evaporation, addition of molecular sieves in the reaction medium and vacuum. The molecular sieve used was previously heat treated in an oven at 200 C for 24 h. After elimination of adsorbed water to the molecular sieve (About 20% w / w of water), this was immediately added to the reaction medium, at the time the reaction started. The following amounts of sieve 3 molecular Sigma were added to the reaction medium: 50, 100, 250 and 500 mg. The reactions conducted under vacuum were carried out with the aid of a Fisaton vacuum pump model 825, creating constant vacuum during 150 minutes of reaction. The reactions that used a system of free evaporation were conducted in open batch reactor (capacity 15 mL), equipped with agitation magnetic. Was made weighing the reaction system at time zero (immediately after the addition of lipase) and the end time of the reaction (after 150 minutes), so monitoring the mass loss after evaporating the reaction mixture.

48

3.6.6. Reuse of lipase

The effect of reuse of lipase Novozym 435 in the reactions of 0.02 mol esterification of sebacic acid and 0.01 mol of 1-octanol, molar ratio acid / alcohol of 1:5, using 7% w / w of lipase studied to 100 C. Was this concentration of lipase chosen to facilitate the handling of lipase, since concentration of 5% w / w would be a very small amount, hindering manipulation. Recovery of the lipase to the end of the reaction, the enzyme was separated from the culture broth by filtration using filter paper and vacuum assistance. After this step, the lipase was washed with 50 ml of 1-octanol, filtered and dried in vacuum in a desiccator for 24 hours before each reuse.

3.6.7. Effect of chemical catalyst

We investigated the use of a chemical catalyst (sulfuric acid) in esterification reactions between 0.02 mole of sebacic acid and 0.01 mol of 1-octanol molar ratio acid / alcohol of 1:5, using 7% w / w sulfuric acid relative the mass of sebacic acid, 100 C.

3.7. Chromatographic Analysis

20 l aliquots of the reaction medium were taken at times 0, 10, 20, 30, 60, 90, 120 and 150 minutes and then diluted with ethanol (1:25) and injected in the Varian gas chromatograph, model CP 3380, equipped with ionization detector flame (FID) and a capillary column CP WAX 52 CB 30m x 0.25 mm x 0.25 microns in injection system with flow split 1:20. The temperatures of the injector and detector were maintained at 250 C and 280 C. The oven temperature was initially 80 C for 30 seconds, and then increased at a rate of 20 C / min final temperature of 150 C. Subsequently, the furnace temperature reached

49

300 C at a rate of 20 C / min. Hydrogen was the carrier gas used in the flow 2 mL / min and the column pressure was kept constant at 20 psi. A computer equipped with the Star Workstation 6.2 software was connected to the chromatograph through the Star interface module 800 for automatic integration of peaks obtained. An example of chromatogram for the analysis of the culture broth by mentioned method is shown in Appendix I. Note that all analyzes chromatographic analyzes were performed in duplicate.

3.8. Standard curves

We performed standard curves for the chromatography method analysis of the gaseous sebacic acid and bis (2-ethylhexyl) sebacate, a product of interest. The concentrations used for both products were: 0.007, 0.016; 0.033, 0.066, 0.099, 0.132 and 0.165 mol / L. The results of these analyzes are presented in Appendix I. Aliquots of 2 L of the solutions were prepared injected into the gas chromatograph according to the method previously described (Item 3.6).

50

4. RESULTS AND DISCUSSION

This chapter will show the results obtained for the synthesis of the ester dioctyl sebacate and the effect of certain variables on the conversion of the reaction. Will also show preliminary results of solubility reagent sebacic acid in different solvents, which allowed the choice of best solvent for the assay of chromatographic analysis, in addition to checking viability of the reaction proposed in this paper. The variables studied in this work were: type of lipase, temperature, concentration of lipase in the reaction medium, the molar ratio of acid sebacic acid: alcohol, a method of removing water in the reaction medium, re-use of lipase, in addition to the comparison of the results obtained employing reaction biocatalyst with that using chemical catalyst (sulfuric acid). Note that along the observations of the results obtained, treat as reaction products, not only the diester dioctyl sebacate, but also the product mono-octyl sebacate. The latter is believed to be there is formed before replacing the second hydroxyl sebacic acid. A Since there was a standard for this monoester, it was compared in Chromatograms using the same response factor obtained with standard dioctyl sebacate.

4.1. Preliminary results

4.1.1. The solubility tests Initially, we tested the solubility of the reagent in various sebacic acid solvents (as proposed in the literature), whose results are shown in Table 4.1. It was found that the reagent is soluble in ethanol at temperatures tested,

51

which enabled it to be used as a solvent for dilution analysis chromatography. As the objective of this work was the product of the esterification reaction between sebacic acid and octanol was tested solubility of 0.02 mole of sebacic acid with 1-octanol at higher temperatures as shown in Table 4.2. In addition to the 1-octanol was verified sebacic acid solubility in ethanol and mixture of ethanol and 1-octanol. The results are presented in Table 4.2.

Table 4.1. Solubility of 0.1 g of sebacic acid in different solvents (3 mL) and temperatures

Solvent water n-hexane ethyl acetate acetone ethanol methanol Ethyl ether Petroleum ether Diisopropyl ether Ethylmethylketone diisobutilcetona 1-hexanol 1-octanol Diphenyl ether

T environment insoluble insoluble insoluble insoluble Soluble Soluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble

40 C insoluble insoluble insoluble insoluble soluble soluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble

50 C insoluble insoluble insoluble soluble soluble soluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble

60 C insoluble insoluble insoluble soluble soluble soluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble

From the solubility tests it was concluded that the esterification reaction between sebacic acid and octanol may be conducted employing reasons molar acid: alcohol ratio of 1:4 and above temperatures above 100 C or ratios molar acid: alcohol ratio of 1:5 above, when employed temperatures above 90 C. The reaction between sebacic acid and octanol could also be conducted at lower temperatures (75 C) was also added since the ethanol in the sebcico/etanol/1-octanol acid molar ratio equal to 1:5:2. Considering the possibility of reaction between sebacic acid and ethanol also be catalyzed by lipase, observed by chromatographic analysis (results not shown), we chose

52

by studying the reaction of synthesis of dioctyl sebacate only in the reaction medium containing sebacic acid and octanol.
Table 4.2. Solubility of 1 mole of sebacic acid in different solvents and temperatures

Solvent
1-octanol 1-octanol 1-octanol 1-octanol 1-octanol ethanol ethanol ethanol ethanol ethanol ethanol ethanol ethanol and 1-octanol ethanol and 1-octanol ethanol and 1-octanol ethanol and 1-octanol ethanol and 1-octanol

Quanti -Ity (Moles)

1 2 3 4 5 1 2 3 4 5 6 7 1:2 2:2 3:2 4:2 5:2

75 C
Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble Soluble Insoluble Insoluble Insoluble Insoluble Soluble

80 C
Insoluble Insoluble Insoluble Insoluble Insoluble -

85 C
Insoluble Insoluble Insoluble Insoluble Insoluble -

90 C
Insoluble Insoluble Insoluble Insoluble Soluble -

100 C
Insoluble Insoluble Insoluble Soluble Soluble -

4.2. Influence of some reaction parameters on the conversion of the reaction esterification of sebacic acid with 1-octanol employing lipase

4.2.1. Influence of lipase

Initially, the esterification reactions between sebacic acid and 1 Octanol was tested three commercial immobilized lipase (Novozym 435, Lipozyme RM IM and Lipozyme TL IM) in the following test conditions: molar ratio acid :1-octanol sebacic acid of 1:5 at 100 C using 5% w / w (relative to the mass of sebacic acid) lipase. The results are shown in Figure 4.1.

53

Figure 4.1. Effect of the lipase in the conversion of sebacic acid after 150 minutes of reaction between sebacic acid and 1-octanol (1:5 mole ratio) at 100 C using 5% w / w Different commercial lipases immobilized.

It is noted from Figure 4.1, the highest conversion was obtained by using lipase Novozym 435 (100%) followed by lipase Lipozyme RM IM (68.6%) and lipase Lipozyme TL IM (7.81%). Due to the low conversion value obtained in the reaction employing lipase Lipozyme TL IM, this was not assessed in most other tests investigation of esterification reactions for the synthesis of dioctyl sebacate. These results are consistent with the activity values determined for the three commercial lipases (item 3.4). According to the determination of esterification activity performed, Novozym 435 showed the highest (3824 mols acid / min.g), followed by Lipozyme RM IM (1909 mols acid / min.g) and lower potential was actually observed for Lipozyme TL IM (699 mols of acid / min.g). When analyzing the specificity of lipases, one can conclude that the Novozym 435 is also the one with the best result, as shown in Figure 4.2.

54

The acid co sebci

100

Mono-sebacate octi l di octyl sebacate l

80
Molar fraction (%)

60

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165 180 195

Reaction time (minutes)

(A) Novozym 435

100
Sebacic acid

80 Molar Fraction (%) 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165 180 195

Mono-sebacate octyl Dioctyl sebacate

Reaction time (minutes)

(B) Lipozyme RM IM
Figure 4.2. Composition of the reaction medium after 150 minutes in the reaction between sebacic acid and 1 octanol, with a molar ratio acid / alcohol of 1:5 at 100 C and 5% w / w lipase. (A) Novozym 435. (B) Lipozyme RM IM.

Figure 4.2 (a) for the reaction with lipase Novozym 435, shows that the mole fraction of the product of interest (dioctyl sebacate) was 94.2% after 150 minutes of reaction, being, after 15 minutes reaction, as sebacic acid had been consumed nearly all. These results show the efficiency of lipase for the synthesis reaction of dioctyl sebacate. Already in Figure 4.2 (b), for the reaction conducted with lipase Lipozyme RM IM,

55

it can be seen that although the conversion was shown to have around 60%, the mole fraction of dioctyl sebacate indicates a low yield for the same (5.8 %), Still can be concluded that apart from low selectivity for the product of interest, conversion of sebacic acid also occurs more slowly. According to Figure 4.2, we note the occurrence of another product which initially has its appearance so increasingly being consumed along the reaction. It is believed to be the mono-ester substituted; since sebacic acid is a dicarboxylic acid first to occur of a carbonyl substitution for then be completely converted to the diester of interest. The lipase Lipozyme RM IM showed a lower conversion compared to Novozym 435. Being a 1,3-specific lipase as the dicarboxylic acid and used in this study presents the carbonyl at position 1, was expected to best performance of Lipozyme RM IM, even considering the results of Esterification activity, since this enzyme is widely used in reactions of esterification (and RODRIGUES FERNANDES-LAFUENTE, 2010). Studies using lipases suggest that the amount of carbon sum reagents may exert an influence on the activity of the enzyme. The size of the carbon chain may restrict the access of reactants to the active site of the enzyme. This explanation is reported in the work of Gomes et al (2006), on the determination of catalytic properties in organic and aqueous lipase Candida rugosa immobilized in cellulignin chemically modified by

carbonyldiimidazole. The authors concluded that for the sum of reactants carbons is equal to or greater than 14, there may be a gradual reduction in converting the corresponding ester. Already Castro et al (2000), the study on the influence coefficient Partition the substrate in the performance of lipase in the synthesis of ethyl citronella by reactions alclise, found the same influence on the lipase Mucor miehei. The conclusion is that for esterification reactions using octanol, the Ideally the acid has a carbon number equal to seven. Thus, the sum ideal for the number of carbons of the substrates would be equal to 15, for the reactions

56

employing Lipozyme RM IM. Bruno et al (2004), the study of ester synthesis catalyzed by lipase from Mucor miehei immobilized on magnetic particles alcohol polysiloxane-polyvinyl also found that the lipase becomes more active esterification reactions such as alcohol reagent having octanol, when acid used has a number of carbon atoms equal to seven. Thus, the sum of the number of carbons ideal reagents for optimum activity enzyme would be 15. These studies may explain the behavior of lipase Lipozyme RM IM the reactions of synthesis of dioctyl sebacate, since reagents This work possess a sum of the number of carbon atoms equal to 18, sebacic acid having 10 carbons and octanol 8 carbons. Thus, the synthesis of dioctyl sebacate exceeds 14 carbon atoms and even number 15 optimal, as reported in previous work. Thus, only the output of mono-octyl sebacate whose carbon number is equal to 16, is favored when employed lipase Lipozyme RM IM.

4.2.2. Influence of molar ratio of sebacic acid / alcohol reaction medium

To verify the effect of the molar ratio of the reactants in the synthesis sebacate dioctyl was tested in the following molar ratios of acid sebcico/1-octanol 1:4, 1:5, 1:6, 1:7 for reactions using Lipozyme RM IM and Novozym 435. The Results can be viewed in Figure 4.3. All reactions were conducted at 100 C using 5% w / w lipase.

57

(A) Lipozyme RM IM

(B) Novozym 435

Figure 4.3. Effect of molar ratio on the conversion of sebcico/1-octanol acid, sebacic acid after 150 minutes of reaction at 100 C using 5% w / w commercial immobilized lipase. (A) Lipozyme RM IM (b) Novozym 435.

Note that for both lipases, the molar ratio does not strongly influence Conversion of the reaction. According to Figure 4.3 (a), which illustrates the effect of molar ratio of the reactants with the use of lipase Lipozyme RM IM, it was observed that the 1:5 molar ratio offered conversion sebacic acid (68.6%) very similar the conversions obtained with other molar ratios: acid molar ratio to the alcohol / 1:6, gave 67.3% conversion and the molar ratio acid / alcohol of 1:7, the

58

Conversion was 59.8%. The same occurs for the least amount of 1-octanol: with the mole ratio acid / alcohol of 1:4, the conversion of sebacic acid was 62.3%. The increase of the reagents worked on the principle of Le Chatelier: the greater the quantity of a reagent, the equilibrium will shifted in the forward direction of the reaction. Therefore, to increase the concentration of alcohol in the reaction medium, the higher must be the conversion of sebacic acid to product. However, the excess alcohol was probably inhibition of the enzyme as reported in the literature (Kraai et. al. 2008; DRM et. al. 2004; GHAMGUI et. al. 2004). According to Trubiano et al (2011) when Alcohol is added to the system due to their polarity, are created interactions with the hydrophilic layer of water essential for lipase causing changes the structure of the same protein, resulting in the inhibition of this enzyme. For Novozym 435 lipase, as described in Figure 4.3 (b), the lower quantity of 1-octanol (molar ratio acid / ethanol 1:4) provided a conversion of 78.5%, this being the lowest conversion found. As there was an increase the amount of alcohol, was also increased conversion, reaching a up to 100% conversion in molar ratios 1:5, 1:6 and 1:7, with no observed its denaturation by excessive alcohol. According to these results, it was adopted as the molar ratio of acid sebcico/1-octanol ideal of 1:5 for both lipases, considering the highest sebacic acid conversion and the best cost-benefit condition.

4.2.3. Influence of lipase concentration in the reaction medium

The effect of the amount of lipase in the reaction medium was evaluated in the Experiments conducted at 100 C with molar ratio sebacic acid / alcohol 1:5, with the following amounts of lipase 3, 5, 7, 9 and 11% w / w with respect to acid sebacic for both Lipozyme RM IM and for Novozym 435. Results are shown in Figure 4.4.

59

(A) Lipozyme RM IM

(B) Novozym 435

Figure 4.4. Effect of the concentration of lipase in the conversion of sebacic acid after 150 minutes reaction at 100 C sebcico/1-octanol acid molar ratio of 1:5. (A) Lipozyme RM IM (b) Novozym 435.

In reactions using lipase Lipozyme RM IM, conversions acid sebacic acid, in concentrations of 3%, 5%, 7%, 9% and 11% w / w of lipase were, respectively, 0%, 63.6%, 68.6%, 40.5% and 30.8% after 150 minutes of reaction.

60

These values characterizing a peak of conversion when used if 7% w / w of lipase Lipozyme RM IM. Lower concentrations of lipase resulted in lower conversions, as well as concentrations above 7% w / w. As one increases the amount of lipase in the reaction medium, the is also a tendency to increase the conversion of the acid (limiting reactant) a Since there will be more available to form the catalyst complex enzyme substrate. However, as enzyme concentration was increased by large amounts to the reaction medium, there was a decrease in the conversion reagent. This decrease in the conversion of the reactant can be explained by the fact that formed a cluster of biocatalyst that can lead to problems diffusional. The catalyst that is within these "lumps" turns out not participate in the reaction, thus decreasing their catalytic ability. There was then a decrease in efficiency per unit weight of the catalyst. Similar behaviors related to enzyme concentration were reported in previous studies, such as Duan et al (2010), where studied the esterification reaction to obtain 1,3-diacylglycerol using Lipase Novozym 435. With the increase in the concentration of lipase in the reaction medium, also observed a decrease in conversion to product. However, according to Figure 4.4 b, in reactions using the lipase Novozym 435, sebacic acid conversions to concentrations of 3%, 5%, 7%, 9 % And 11% w / w of lipase were, respectively, 39.6%, 100%, 100%, 99.9% and 100%. For this lipase is observed that the maximum conversion was obtained from the using 5% w / w lipase. Because of the cost-benefit ratio, it was considered best reaction conditions that would, therefore, using 5% w / w Novozym 435. From Figure 4.5 one can see the mole fractions of sebacic acid, sebacic acid monoester and diester, sebacic acid during the reaction of dioctyl sebacate synthesis employing the Lipozyme RM IM.

61

100 80 F m the la (% ra 60 r) 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Sebacic acid Sebacate mono-octyl Sebacate dioctyl

Reaction time (minutes)

(A) 3% w / w
Sebacic acid

100 80 F O M la (% ra r) 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Mono-sebacate octyl Dioctyl sebacate

Reaction time (minutes)

(B) 5% w / w
100
Acid sebico Mono-sebacate octyl Dioctyl sebacate

Molar fraction (%)

60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

80

Reaction time (minutes)

(C) 7% w / w
100 80 Molar fraction (%) 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165 Sebacic acid Mono-sebacate octyl Dioctyl sebacate

Reaction time (minutes)

(D) 9% w / w
100
sebacic acid

80 F the la m () r CO r% 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Sebacate mono-octyl Sebacate dioctyl

Te mpo rea tion (minutes)

(E) 11% w / w Figure 4.5. Composition of the reaction medium in the reaction between sebacic acid and 1-octanol, employing a molar ratio acid / alcohol of 1:5 at 100 C and lipase Lipozyme RM IM. (A) 3% w / w (B) 5% w / w (C) 7% w / w (D) 9% w / w (E) 11% m / m.

62

In Figure 4.5, it is observed that the reaction conducted with lipase Lipozyme RM IM did not show good selectivity for the ester of interest, even in better molar ratio of reactants and concentration of lipase in reaction medium (7% w / w). The mole fraction of dioctyl sebacate (diester) at the end of reaction, ie after 150 minutes was 7.0%. The income sebacate ester mono-octyl was approximately 62%. Moreover, acid is consumed slowly until the first hour of reaction, the conversion was only 35%. The Initial reaction rates, as expected, increased with increasing lipase concentrations in the reaction medium. However, this also was not observed until concentration of 7% w / w of lipase Lipozyme RM IM in the reaction medium; above this concentration, the initial conversion rate also decreased. For the reaction conducted with lipase Novozym 435 (Figure 4.6), and the excellent conversion (100%), there is a great selectivity for sebacate dioctyl (94%). At the end of the reaction there are only 5.8% of mole fraction of monoester. The higher the amount of enzyme added to the reaction medium, higher the conversion rate of sebacic acid. 5% w / w Novozym 435, the Conversion after 15 minutes of reaction corresponded to 89%. Already with the addition of 7% w / w 9% m / m 11% w / w Novozym 435, sebacic acid conversion after 15 minutes the reaction was already 100%.

63

100
Sebacic acid

80 Molar fraction (%) 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Mono-sebacate octyl Sebacate dioctyl

Reaction time (min)

(A) 3% w / w
100 F the la 80 m () r CO r% 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165
Sebacic acid Mono-sebacate octyl dioctyl sebacate

Reaction time (minutes)

(B) 5% w / w
Sebacic acid Mono-sebacate octyl Dioctyl sebacate

100 F m the80 la (% ra r) 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (minutes)

(C) 7% w / w
100 80
Sebacic acid Mono-sebacate octyl Dioctyl sebacate

F m the la (% ra r) 60
40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (minutes)

(D) 9% w / w
Sebacic acid

100 Molar 80 fraction (%) 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Mono-sebacate octyl Dioctyl sebacate

Reaction time (minutes)

(E) 11% w / w Figure 4.6. Composition of the reaction medium in the reaction between sebacic acid and 1-octanol, employing a molar ratio acid / alcohol of 1:5 at 100 C and lipase Novozym 435. (A) 3% w / w (B) 5% w / w (C) 7% w / w (D) 9% w / w (E) 11% m / m.

64

4.2.4. Effect of temperature

The effect of temperature on the conversion of the reaction between sebacic acid and 1 octanol was investigated in reactions employing molar ratio acid: alcohol 1:5, 5% w / w commercial immobilized lipase at temperatures of 90 C, 100 C and 110 C. The results can be seen in Figure 4.7.

(A) Lipozyme RM IM

(B) Novozym 435 Figure 4.7. Effect of temperature on conversion of sebacic acid after 150 minutes of reaction, employing 5% m / m and lipase sebcico/1-octanol acid molar ratio of 1:5. (A) Lipozyme RM IM (B) Novozym 435.

65

It was observed that the highest conversion for both sebacic acid lipase occurred at 100 C. Note also that for the lipase Novozym 435, the results were very similar at all three temperatures tested. According to Figure 4.7a, which shows the conversion reaction using lipase Lipozyme RM IM, it is observed that the conversion of sebacic acid at 90 C was 54.3%, while the conversion obtained at 100 C was 62.2% and 110 C was only 22.8%. The selectivity of this lipase for dioctyl sebacate, was around 10% for the temperatures tested. A reaction, even when not catalyzed, can increase its speed due to an increase in the temperature of the reaction system, until a providing high temperature conversion of reactants. Increasing temperature provides a greater kinetic energy of the molecules of the reagents, causing a greater number of collisions between these molecules. According with Ananthanarayan and Iyer (2008), an increase in reaction temperature provides some advantages, such as: decrease the viscosity of the reaction medium and diffusional limitations, thus providing higher rates of reaction. However, when a reaction is catalyzed by enzymes, due to their proteinaceous nature, they may denature at temperatures very high. Behaviors similar were identified in reactions of

esterification, for example, in the work-Jin Wu et al Chuam (2001), who studied esterification catalyzed by lipase Candida rugosa in organic solvents. The reaction between lauric acid and lauryl alcohol in presence of iso-octane showed higher enzyme activity in the temperature 30 C (about 300 mmol / ming), whereas at 20 C the enzyme activity was 200 mmol / ming and at 40 C was 240 mmol / ming. For reactions conducted with the lipase Novozym 435, as b shown in Figure 4.7 it can be seen that the conversion rates are similar. The conversion of sebacic acid obtained was 97%, 100% and 99% for temperature 90 C, 100 C and 110 C, respectively.

66

Selectivity in dioctyl sebacate obtained in reactions employing Novozym 435 is illustrated in Figure 4.8. It is observed that the highest value was obtained at 100 C: 94%. At temperatures of 90 C and 110 C were observed the same values of selectivity for the diester formed: 70%.

Figure 4.8. Selectivity in dioctyl sebacate obtained after 150 minutes of reaction between acid sebacic acid and 1-octanol, employing a molar ratio of 1:5 and 5% w / w Novozym 435 lipase in different temperatures.

4.2.5. Influence of water removal from the reaction medium

It is known that the amount of water in the reaction medium exerts great influence in esterification reactions. As stated previously, the enzyme needs a minimum amount of water to become active, and becomes inactive in media completely anhydrous. However, the increase in the amount of water present in the medium reaction in esterification reactions can cause the reverse reaction - hydrolysis the ester formed. With this, it becomes important to control the amount of water in the reaction proposed in this work. To evaluate the influence of the amount of water formed during the reaction

67

esterifying the conversion of sebacic acid, we tested the removal of this three methodologies: free evaporation (using open reactor), and the addition of a vacuum molecular sieves to the reaction medium. Whereas the highest values of conversion were obtained with Novozym 435, the results of the experiments will be described below, were performed only with this enzyme. The reactions were conducted at 100 C, using 5% w / w Novozym 435, and sebacic acid molar ratio of 1:5 :1-octanol.

a) free evaporation The reaction conducted with free water evaporation, by employing a batch reactor open allowed to obtain 99.8% conversion of the acid sebacic, after 150 minutes of reaction. The progress of the reaction, as well as reaction in closed reactor, can be observed in Figure 4.9. When compared to the conversion obtained in the reaction with the reactor closed in which yielded 100% conversion, note a negligible reduction. To verify the loss of weight occurred in the reaction system experiment in open reactor, the system was weighed at time zero (immediately After addition of enzyme) and after 150 minutes of reaction. The expected was that the evaporated mass was only corresponding to the mass of water formed during the esterification reaction. But when weighing the system before and after the reaction, mass lost by evaporation did not match the stoichiometry of the reaction. To each mole of sebacic acid generated is converted to two moles of water. Thus, the it is expected that by using 0.02 mol of sebacic acid, 0,04 mol was generated water, which corresponded to 0.72 g. But the mass of the reaction system of the reaction conducted with the open reactor decreased from approximately 3.5 g. That is, only about 20% of the mass evaporated water corresponded evaporated. The remainder believed to be corresponding to 1-octanol also must have evaporated from the reaction medium. It is noteworthy that the boiling point of 1 Octanol is 195 C, while the boiling point of the dioctyl sebacate is 248 C versus 294 C which is the boiling point of sebacic acid. These data

68

lead to the conclusion that a substance possibly evaporated system actually was 1-octanol. It is noticeable, the occurrence of two phenomena occurring simultaneously: removing water from the reaction medium and the decrease of the molar ratio as alcohol also evaporates from the system, the latter being responsible for the possible product formation of interest more slowly.
100
Sebacic acid Mono-sebacate octyl Sebcato of dioctyl

80

Molar fraction (%)

60

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (min)

(A) free evaporation

100
Sebacic acid Mono-sebacate octyl dioctyl sebacate

Molar fraction (%)

60

80

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (min)

(B) Reactor Closed Figure 4.9. Composition of the reaction medium in the synthesis reaction of dioctyl sebacate at 100 C, sebcico/1-octanol acid with molar ratio of 1:5 with a 5% w / w Novozym 435. (A) Free Evaporation (b) Reactor closed.

With respect to the synthesis of dioctyl sebacate, when compared to the progress of the reaction with time, it can be noted that in Figure 4.9 with the reactor

69

closed as there was a large formation of dioctyl sebacate in the first few 15 minutes reaction, in contrast to the reaction with the reactor open, only the 60 minutes of reaction showed considerable training of the diester. The mole fraction of diester of interest after 15 minutes the reaction with open reactor was 9%, while at the same time the reaction closed reactor, the mole fraction of dioctyl sebacate was 70%. This longer time to form the product of interest with the reactor open can be explained by evaporation of the reagents, which justifies say that the best condition for obtaining the ester interest between the two compared conditions, the reactor is closed.

b) Vacuum In the reaction performed under vacuum, the conversion of sebacic acid obtained after 150 minutes of reaction was 99.7%. Comparing this result with that obtained by the conventional method (reactor closed), it is noted that the conversion was only slightly lower. The same occurred when compared to the conversions in the first 15 minutes of reaction. Obtained 88% conversion in 15 minutes the reaction with vacuum and 89% conversion in 15 minutes in the reaction with the reactor shut down. However, when comparing the fractions molars dioctyl sebacate 15 minutes of both reactions, it is noted by Figure 4.10 that for the reaction with the closed reactor, the selectivity for the ester interest is greater, as its mole fraction is 70%, compared to only 46% of mole fraction in the reaction under vacuum. The selectivity of the lipase for sebacate dioctyl after 15 minutes of reaction is 52% in the reaction under vacuum to 78% when using the closed reactor.

70

Sebacic acid

100

Mono-sebacate octyl Dioctyl sebacate

80

Molar fraction (%)

60

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (min)

(A) Vacuum
100

Sebacic acid Mono-sebacate octyl dioctyl sebacate

Molar fraction (%)

60

80

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (min)

(B) Reactor Closed Figure 4.10. Composition of the reaction medium in the synthesis reaction of dioctyl sebacate at 100 C, sebcico/1-octanol acid with molar ratio of 1:5 with a 5% w / w Novozym 435. (A) Vacuum (b) Closed reactor.

c) Molecular sieve Was also tested with addition of molecular sieves to the reaction medium for Removal of the water produced in the reaction. The molecular sieve used was a sieve of 3, supplied by Sigma, and was previously dried in an oven at 200 C for 24 h before being added to the reaction. Weighing the sieve before and after the period of drying, it was found that it contained about 20% w / w water.

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Tests using molecular sieve were conducted at 100 C, with sebacic acid molar ratio of 1:5 :1-octanol and 5% w / w of lipase Novozym 435, with the reactor shut down. The following amounts of molecular sieve were tested: 50, 100, 250 and 500 mg. Conversions of sebacic acid were obtained as follows: 99.7% with 50 mg sieve, 100%, 100 mg sieve, 99.8%, 250 mg screen; 99.7% with 500 mg sieve. Thus, the results obtained were similar for all conditions tested. However, when observed at molar fractions reactions along with several additions sieve, note that the reaction there was the addition of 50 mg of molecular sieve was the reaction that converted the acid in less time. After 15 minutes of reaction the conversion of sebacic acid was 81% and selectivity of the diester 83%, for the addition of 100 mg sieve values of conversion and selectivity sebacic acid diester to the 15 minutes of reaction were 72% and 42% respectively, for the addition of 250 mg values were 94% conversion but the selectivity for sebacate dioctyl was 43% and for the addition of 500 mg conversion values acid sebacic acid and selectivity to the product of interest were 98% and 0.42%; respectively. That probably happened because of that, add larger quantities of sieve layer minimal essential for water enzyme activity was also removed (Klibanov, 1997). As the reaction was the addition of 50 mg of molecular sieve was that showed a better selectivity for lipase and consequently the increased yield of product of interest at the end of the reaction (after 150 minutes) this was chosen as the best reaction conditions used when the sieve molecular. With respect to the molar fraction dioctyl sebacate, it has the value of 93% with the addition of 50 mg sieve while in the closed reactor obtained 94%. The molar fractions of dioctyl sebacate along the reaction using 50 mg of molecular sieve and reaction with closed reactor are presented Figure 4.11.

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100
Sebacic acid

80 Molar fraction (%) 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Sebacate mono-octyl Sebacate dioctyl

Reaction time (min)

(A) 50 mg of molecular sieve

100

Sebacic acid Mono-sebacate octyl dioctyl sebacate

Molar fraction (%)

60

80

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (min)

(B) Reactor Closed Figure 4.11. Composition of the reaction medium in the synthesis reaction of dioctyl sebacate at 100 C, sebcico/1-octanol acid with molar ratio of 1:5 with a 5% w / w Novozym 435. (A) 50 mg molecular sieve (b) Reactor closed.

The Figure 4.12 summarizes the conditions tested and shows that there there was an increase in the conversion of sebacic acid or an increase in rate of formation of the ester of interest from the use of open reactor, vacuum or addition of molecular sieve. On the contrary, according to Figure 4.12, it can be seen that in closed reactor obtained with a higher mole fraction of acid

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sebacic 15 minutes of reaction. Thus, according to Zaks et al (1985), the methodologies used to remove water from the reaction medium may also be contributing for the removal of the water layer essential for the activation of the enzyme. This so, it becomes less active during the reaction by retarding the conversion the reactant into a product. The study by Carlos Torres and Cristina Otero (2001) showed behavior when similar amounts of added molecular sieves in the reaction of Enzymatic esterification of lactic acid. In this study, the larger the amount Sieve added (over 200 mg), the lower the yield of ester. The fact was attributed to the affinity of the molecular sieve for adsorption of polar molecules. Adsorption with this substrate, there are fewer molecules of lactic acid in the free reaction mixture. How sebacic acid has polar character, there may also be an impact the interaction of the acid with a sieve.

100 Closed reactor 80 Free evaporation Vacuum Molecular sieve (50mg)

Molar fraction (%)

60

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (min)

Figure 4.12. Mole fraction of dioctyl sebacate obtained in reactions between sebacic acid and 1 octanol at 100 C, using a molar ratio of 1:5 sebcico/1-octanol acid and 5% w / w Novozym 435. In closed reactor () in open reactor (free evaporation) (), employing vacuum () and adding 50 mg of molecular sieve (x).

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4.2.6. Reuse of lipase

In the study of esterification to obtain sebacate dioctyl also tested reuse of lipase Novozym 435, since this lipase provided the best results of conversion and selectivity for the product of interest. Proceeded with reuse of lipase after it has been removed the first reaction, filtered and washed with 50 ml of 1-octanol with the aid of a vacuum pump. After this procedure, the biocatalyst was kept desiccator during 24 h. Although the results with a 5% w / w of lipase have been shown more Advantageous, the amount of enzyme in 5% w / w is very small making it difficult to Manipulation of the lipase. Thus, we chose to use the concentration of 7% m / m lipase in the reaction medium. We tested two reuses this enzyme, when carrying out washing and drying the same as explained above. Results can be seen in Figure 4.13.

100

80

Conversion (%) 60

40

20

0 1 2 3

Use of lipase

Figure 4.13. Conversion of sebacic acid in the reactions of synthesis of dioctyl sebacate, being conducted at 100 C with molar ratio of 1:5 acid sebcico/1-octanol, with 7% w / w Novozym 435 lipase in various uses.

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In figure 4.13 we can see that conversion values were obtained sebacic acid similar for all three reactions. In the first reaction, sebacic acid conversion achieved was 100%, while after the first reuse, the conversion was 98.4% and in the second reuse, the conversion was 97.8%. However, in the first reuse, the mole fraction of dioctyl sebacate declined significantly relative to the molar fraction obtained in the initial system, which was obtained value of 63.4%. In the second reuse, the yield was even lower, 34.4% (Figure 4.14.) It is noted large decline in mole fraction of dioctyl sebacate over the first and second reuse. As increased numbers of Reuse of lipase, increased selectivity for monooctila sebacate, decreasing its selectivity to the product of interest. The result can be explained by an accumulation of products on sites active enzyme, altering its selectivity along Reuse by enzyme denaturation caused by elevated temperature, by washing with octanol, loss of minimum water required to maintain native conformation by changing the structural conformation of the enzyme caused by interactions with the substrates and products of the reaction medium or even by desorption of the supported lipase. Ghamgui et al (2004) tested the reuse of lipase Rhizopus oryzae the esterification reaction between oleic acid and butanol for obtaining 1-butyl oleate. From the sixth reuse, the conversion of the reaction fell from 73.8% to 60% and the enzyme activity decreased. The reduced activity enzyme was attributed to desorption of the lipase support.

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Sebacic acid

100 80 60 40 20

Sebacate mono-octyl Sebacate dioctyl

Molar fraction (%)

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (minutes)

(A) Initial reaction

Sebacic acid 100 Mono-sebacate octyl Sebacate dioctyl

80

Molar fraction (%)

60

40

20

0 0 15 30 45 60 75 90 105 120 135 150 165

Reaction time (min)

(B) first reuse

Sebacic acid 100 Sebacate mono-octyl Sebacate dioctyl

80

Molar fraction (%)

60

40

20

15

30

45

60

75

90

105

120

135

150

165

Reaction time (min)

(B) Second reuse Figure 4.14. Composition of the reaction medium of the synthesis reaction of dioctyl sebacate employing a molar ratio of 1:5 sebcico/1-octanol acid and 7% w / w of lipase Novozym 435 to 100 C (A) Initial reaction (B) First reuse (C) Second reuse.

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4.2.7. Chemical catalysis

The synthesis reactions dioctyl sebacate were also performed using as catalyst sulfuric acid. The reactions were conducted under the same conditions employed towards Enzyme DE100 temperature C, with sebcico/1-octanol acid molar ratio of 1:5, using 7% w / w of catalyst. The results obtained for these two catalysts are shown in Figure 4.15.

Sebacic acid Sebacate mono-octyl Sebacate dioctyl

100 80 60 40 20 0 0 15 30 45 60 75 90 105 120 135 150 165

Molar fraction (%)

Reaction time (minutes)

(A) Novozym 435

100

Sebacic acid Mono-sebacate octyl Dioctyl sebacate

Molar fraction (%)

60

80

40

20

15

30

45

60

75

90

105 120 135 150 165

Reaction time (min)

(C) Sulphuric acid Figure 4.15. Composition of the reaction medium of the synthesis reaction of dioctyl sebacate, 100 C, employing a molar ratio of 1:5 sebcico/1-octanol acid and 7% m / m of catalyst. (A) Novozym 435 (B) Sulfuric acid.

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Both reactions had a conversion of 100% of sebacic acid. Comparing the selectivity of the catalyst for the diester of interest can be complete according to Figure 4.15, the reaction catalyzed with the catalyst Chemical achieved a value of 90.7% dioctyl sebacate end of the reaction, while the reaction catalyzed with the biocatalyst obtained a value of 94.1%. However, the reaction with sulfuric acid is faster and the balance seems to have was reached 15 minutes after initial reaction, while the Novozym 435 biocatalyst, equilibrium was reached only after 60 minutes reaction (Figure 4.15a). It is worth noting the advantages of enzyme catalysis front of catalysts Chemicals: Since lipases are immobilized heterogeneous catalysts, the they can be easily removed from the reaction medium and reuse. The Use of biocatalysts, instead of using conventional chemical catalysts (Sulfuric acid, hydrochloric acid, etc.). Also prevents the occurrence of corrosion reaction system (ALI et. al. 2006). In addition, several other advantages can be associated with the use of enzyme catalysts, as can be discarded without pre-treatment (unit operations).

4.2.8. Characterization of biolubrificante

The characterization of biolubrificante produced from the reaction between sebacic acid and 1-octanol, employing a molar ratio of 1:5 at 100 C using a 5 % W / w of the biocatalyst Novozym 435 was made by the following methods:

Kinematic viscosity at 40 C - ASTM D 445 Kinematic viscosity at 100 C - ASTM D 445 Viscosity Index - ASTM D 2270 Pour point - ASTM D 97 Flash Point - ASTM D 92

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Neutralization - ASTM D 974

The results were compared with a paraffinic base oil mineral and a naphthenic base oil, also mineral origin (both oil coming through their fractionation). The results are presented in Table 4.3.

Table 4.3. Results of characterization of the reaction product between sebacic acid and 1 octanol (94.2% of sebacate and dioctyl sebacate 5.8% of monooctila), compared to a paraffinic mineral base oil and an oil naphthenic base mineral.

Product reaction Test Methodologically the (Sebacate and dioctyl sebacate mono-octyl)
Viscosity to 100C Viscosity to 40C Point fluidity

Oil mineral paraffinic (Spindle 09)

Oil mineral naphthenic (NH10)

ASTM D 445 ASTM D 445 ASTM D 97

2,000 cSt 7,370 cSt - 63 C 128 C 8.64 mg KOH / g

2,741 cSt 10.68 cSt -9 C 184 C 0.01 mg KOH / g

2,365 cSt 9,805 cSt -51 C 150 C 0.01 mg KOH / g

Point blaze Index acidity Index viscosity and

ASTM D 92 ASTM D 974

ASTM D 2270

39

95

30

As noted in Table 4.3, the product obtained has the pour point smaller than the paraffinic mineral base, and is lower than the base oil naphthenic mineral too. This is an advantage of biolubrificante generated because of According to the definition pour point is described in Chapter I of this work, the lower the pour point, the better are the characteristics of the lubricant

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negative temperatures. This means that, even at temperatures up to -63 C, the lubricant seep into the lubrication system, and may exit the sump and reach the system cylinders without major problems. The result for the flash point is below the results for the two basic minerals. This is not considered a problem since, according to Norm NR 20 10, a product is only considered flammable in its flash point is below 70 C. That is, the product containing dioctyl sebacate can be handled and stored without special care. With respect to the results obtained for the acid, it can be noted acid present in a large dioctyl sebacate produced, which is not present in the basic minerals. This acidity is a drawback, because if used Thus, the lubricant may cause corrosion process in the parts of motor. Therefore, it is suggested that the biolubrificante pass through at least one of the three possibilities below:

a) The biolubrificante can be treated with anti-additives corrosive to inhibit their tendency to cause corrosion on systems. Note that there will be an extra cost with the addition of additives; b) The biolubrificante can undergo a neutralization process, which also means that there will be additional costs involved. A process usually employed in industries re-refining lubricants is a washing product in a bed containing oxide calcium; c) biolubrificante may be used together with a lubricant mineral base, thus losing its potential corrosive, free of charge
10

Site http://www.mte.gov.br. Accessed on 26/12/2011, 21:30.

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Additional. Already viscosity index shows a value close to that found in the oil naphthenic, being well below the result obtained in paraffinic base. This property is also slightly lower when compared to the biolubrificante paraffinic mineral base oil. However, caters to applications where It is typically employs a naphthenic mineral oil. For a better understanding of the difference between naphthenic oils and paraffinic can be seen in Table 4.4, where there are arranged the results of some comparative tests.

Table 4.4. Comparative results of characterization tests of basic minerals paraffinic and naphthenic mineral basic. leoleo EnsaioMetodologia parafniconaftnico
Density Index viscosity Aniline point ASTM D 4052 ASTM D 2270 ASTM D 611 0.8517 95 90 0.8914 30 60.3

The Naphthenic mineral base oils are generally chosen for the Soluble lubricants manufacture of products, which have additives such as the emulsifying agents, which have in their structure a polar and other nonpolar, causing the formation of an emulsion is possible to add Oil to water. These soluble lubricants are usually employed in industries machining. From Table 4.4 it is understood why this choice. As the density of the naphthenic base mineral is higher than that of paraffinic mineral base, the difference in density between the naphthenic oil and water is lower. Thus, the stability of More emulsion is hardly affected when subjected to shear forces, guaranteeing that there is no separation of the emulsion, leaving the cut pieces unprotected. The index of the lowest viscosity naphthenic oils means that they suffer a sudden drop in viscosity over when there is an increase

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temperature, as explained in Chapter I. This makes these oils are most interesting for machining applications, because they are most the effective heat transfer. With respect to the aniline point, the lower this value, the greater the aromaticity (and polarity) of oil. How naphthenic mineral oil has the point aniline about 30 C lower than the paraffinic mineral oil, it can be said the naphthenic has greater polar character, being more suitable for use in emulsions.

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5. CONCLUSIONS AND SUGGESTIONS

5.1. Conclusions

In the present work the synthesis of dioctyl sebacate from Esterification reactions using immobilized lipases commercial as well as Effect of various process variables. This work allowed us to assess the influence of lipase, the molar ratio acid: alcohol, the concentration of lipase in the reaction medium, temperature, type catalyst (chemical and enzymatic), in the manner of removal of water from the reaction, the reusability of the enzyme, and characterizing the product of interest can be concluded that:

a) The use of the lipase Novozym 435 gave the best results, both in As regards the conversion of the reactant, the yield in dioctyl sebacate, when compared to other lipases tested (Lipozyme RM IM and Lipozyme TL IM); b) the best condition for obtaining a product of interest when used lipase Novozym 435 was: molar ratio acid: alcohol 1:5 with 5% m / m of lipase, 100 C; c) The reaction employing Novozym 435 conversion values obtained sebacic acid similar to those obtained using sulfuric acid and greater selectivity dioctyl sebacate, than that observed for the catalyzed chemical reaction conducted under the same conditions experimental; d) Although the conversion values have been shown to be equal when biocatalyst used and acid catalyst using a chemical catalyst formation provides a faster product of interest; e) The methodologies used to remove water from the reaction medium were unsuccessful in forming the product of interest,

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delaying the appearance thereof; f) The reuse of lipase Novozym 435 was not effective, whereas even although still presenting converes satisfactory selectivity for the product of interest has decreased considerably during reuses; g) the lipase Novozym 435 can be used for the reaction for obtaining dioctyl sebacate to replace conventional chemical catalyst sulfuric acid. h) testing for characterization of the product obtained show that the Lubricant can be used as pure (with the addition of additives), or even added to other base oils of mineral origin.

5.2. Suggestions for future work

Based on the results obtained in this study, new goals can be outlined:

a) To study the reuse of the lipase, after checking some properties their use as morphological structure, amount of water, amount of protein in the reaction (to check if desorption of lipase support), etc..; b) Evaluation of stepwise addition of alcohol, once the literature cites conversion values varied depending on the way in which the alcohol is added to the reaction medium; c) Improvement of the physico-chemical sebacate dioctyl in order to improve its performance when used as a Single oil. d) Characterization of a lubricating semisynthetic using sebacate of dioctyl fractions mixed with a mineral oil base. e) Removal of acid present in the product. f) Attempted conversion of mono-ester di-ester to slow the rate

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acid reaction product. g) Use of continuous system in the synthesis of dioctyl sebacate. h) use of a lipase laboratory.

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REFERENCES

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AKERMAN, Cecilia Orellana et. al. Biolubricants synthesis using immobilized lipase: Process optimization of trimethylolpropane oleate production. Sweden. Process Biochemistry V.46, p.2225-2231, 2011. ALI, Sami H. et al. Synthesis of esters: Development of the expression rate for the Dowex 50 W x 8-400 of propionic acid catalyzed esterification with 1-propanol. Kuwait. 2007. Chemical engineeing v science. 62, p.3197-3217, 2007. AYRES-Barros, Maria R. Biocatalysis in organic solvents. B.Biotecnol. Lisbon n.72, p. 2-13, 2002. CARVALHO, Patricia O. et al. Application of microbial lipases in obtaining concentrates of polyunsaturated fatty acids. Bragana Paulista. 2002. Chemistry New, vol. 26, n.1, p.75-80, 2003. CARVALHO, Walter et al. Use of immobilized biocatalysts: an alternative for driving bioprocesses. 2006. Analytica Magazine, n. 23, p.60-70, 2006. CRESPO, Janaina et al. The use of lipases immobilized on poly (ethylene oxide) is the preparation of alkyl esters. Santa Catarina. 2004. Process Biochemistry v.40 p. 401-409, 2005. DALLA-VECCHIA, Roberto et al. Synthetic applications of immobilized lipases polymers. Itaja. 2004. New Chemistry, vol. 27, n. 4, p.623-63, 2004. DRM, N. et al. Manufacture of an environmental-safe biolubricant from fusel oil by enzymatic esterification in solvent-free system. Oberhausen. Germany. 2004.

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Biochemical Engineering Journal. v. 21, p. 229-334, 2004. DUAN Zhang-Qun et al. Novozym 435 1,3-diacylglicerol preparation via esterification in t-butanol system. Beijing. China. 2009. Process Biochemistry. (2010) 10.1016/j.procbio.2010.03.007. GHAMGUI, Hanen et. al. 1-Butyl synthesis by immobilized lipase from Rhizopus oryzae: a comparative study between n-hexane and solvent-free system. Tunisia. Enzyme and Microbial Technology v.35, p.355-363, 2004. GOMES, Fabricio M. et al. Evaluation of reaction conditions for the synthesis Enzymatic butyl butyrate employing lipase Candida rugosa. Lorena. 2004. Rev. Bras. Cienc. Farm. v. 40, n.2. , 2004. GOMES, Fabricio M. et al. Determination of the catalytic properties among Aqueous and organic Candida rugosa lipase immobilized on cellulignin chemically modified with carbonyldiimidazole. So Paulo. New Chemistry, v. 29, n. 4, p. 710-718, 2006. GRYGLEWICZ, Stanislaw. Enzyme catalysed synthesis of some adipic esters. Wroclaw. Journal of Molecular Catalysis B: Enzymatic v.15, p.9-13, 2001. GRYGLEWICZ, Stanislaw. Lipase catalysed synthesis of sebacic acid and phthalic esters. Wroclaw. Enzyme and microbial technology v. 33, p. 952-957, 2001. GRYGLEWICZ, Stanislaw. et al. Esters of the acids dicarboxilic additives for lubricating oils. Wroclaw. Tribology International v.39, p. 560-564, 2006. Halling, Peter J. et al. Inactivation of enzymes at the aqueous-organic interface. Glasgow. Stability and stabilization of Biocatalysts. p. 365-372, 2006. IYER, V. Padma; Ananthanarayan, Laxmi. Enzyme stability and stabilization Aqueous and non-aqueous environment. Mumbai. Process Biochemistry. 43, (2008) From 1019 to 1032. Klibanov, Alexander M. Improving enzymes by using Them in organic solvents. Massachusetts. Nature, Vol 409 (2001) 241-246. Klibanov, Alexander M. Why are enzymes less active in organic solvents than in water? Massachusetts. Tibtech, Vol 15 (1997) 97-101].

Kraai, G. N. et. al. Kinetic studies on the Rhizomucor miehei lipase catalyzed reatem esterification of oleic acid with 1-butanol in a biphasic system. Netherlands. Biochemical Engineering Journal 41 (2008) 87-94.

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LEE, Moo-Yeal; Dordick, Jonathan. Enzyme activation for nonaqueous media. New York. Current Opinion in Biotechnology 13 (2002) 376-384. LIMA, Anthony W.; Angnes, Lucio. Biocatalysis in aqueous media constrained: fundamentals and applications in analytical chemistry. So Paulo. New Chemistry, Vol 22. 2 (1999). LINKO, Yu-Yen et al. Biodegradable products by lipase biocatalysis. Raisio. Journal of Biotechnology 66 (1998) 41-50. LINKO, Yu-Yen et al. Lipase-catalyzed synthesis of its derivates with 1,4-butanediol. Espoo. Finland. Journal of Biotechnology. 40 (1995) 133-138. Lortie, Robert. Enzyme Advances. 15 (1997) 1-15. catalyzed esterification. Montreal. Biotechnology

OLIVEIRA, Peter C. et al. Synthesis of n-butyl butyrate, employing lipase Microbial immobilized on styrene-divinylbenzene copolymer. Lorena. Chemistry New, Vol 23, No 5, 2000, 632-636. Quinchia, L. A. et al. Viscosity modification of high-oleic sunflower oil with polymeric additives for the design of new formulations biolubricant. Huelva. Environ. Sci Technol. 43 (2009) 2060-2065. REETZ, Manfred T. Lipases the practical Biocatalysts. Kaiser. Current opinion in Chemical Biology, 6 (2002) 145-150. REETZ, Manfred T.; JAEGER, Karl-Erich. Overexpression, immobilization and biotechnological application of Pseudomonas lipases. Germany. Chemistry and Physics of Lipids 93 (1998) 3-14. RODRIGUES, Rafael C. Synthesis of biodiesel through the transesterification Enzymatic Enzymatic vegetable oils by lipase-catalyzed bond multipoint covalent [dissertation]. Porto Alegre. Federal University of Rio Grande do Sul Chemical Engineering. Department of Engineering Chemistry, 2009. SALIH, Nadia; Salimon, Jumat; YOUSIF, Emad. The physicochemical and tribological properties of oleic acid based biolubricants trimester. Baghdad. Industrial Crops and Products 34 (2011) 1089-1096. SERDAKOWSKI, Anne L.; Dordick, Jonathan S. Enzyme activation for organic solvents made easy. New York. 2007.

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TORRES, Carlos; OTERO, Cristina. Part III. Direct enzymatic esterification of lactic acid with fatty acids. Madrid. Enzyme and Microbial Technology 29 (2001) 3-12. TRUBIANO, G.; BORIO, D.; Errazu, A. Influence of the operating conditions and the external mass transfer limitations on the synthesis of fatty acid esters using the Candida Antarctica lipase. Baha Blanca. Enzyme and microbial technology 40 (2007) 716-722.

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ANNEX I - CHROMATOGRAPHIC ANALYSIS

This appendix will present the chromatograms obtained as methodology described in item 3.7. The chromatograms obtained for the reactions of sebacic acid with 1-octanol Reaction (using 0.02 mol of acid, 0.120 mol of 1-octanol and 5% w / w of lipase Novozym 435 at 100 C) are shown below, points 0 (Figure A) and 150 minutes (Figure B). According to the following chromatograms of pure products: acid sebacic (Figure C), bis (2-ethylhexyl) sebacate (Figure D), and comparing their times retention with retention times of peaks obtained in the chromatograms of esterification reaction, the spots were identified as follows:

___________________________________________________________________________
Figure A. Chromatogram of esterification of sebacic acid with 1-octanol in time 0 min, 100 C, which is used 5% w / w Novozym 435, molar ratio acid / alcohol 1:5.

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Figure B. Chromatogram of esterification of sebacic acid with 1-octanol in time 150 min, 100 C, which is used 5% w / w Novozym 435, molar ratio acid / alcohol 1:5.

Figure C. Standard chromatogram to sebacic acid.

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Figure D. Standard chromatogram for the bis (2-ethylhexyl) sebacate