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 Post- GraduaçãodeEngenhariaQuímica of State University of Rio de Janeiro.

Approved

Examining Committee:

____________________________________________________

Marta Antunes Pereira Langone Institute of Chemistry UERJ

____________________________________________________

Aline Machado de Castro Cenpes / Petrobras

____________________________________________________

Antônio 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

Simões, Fábio 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 º 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 sebácico/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 sebácico/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 sebácico/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 º C, with sebácico/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 º C, with sebácico/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 º C, with sebácico/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 sebácico/1-octanol 1:5 and 5% w / w Novozym 435. In closed reactor (◊) in open reactor (free evaporation) (□), employing vacuum (Δ) 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 sebácico/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 sebácico/1-octanol acid and 7% w / w lipase Novozym 435 to 100 º 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 sebácico/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 º C, 80 º C, 85 º C, 90 º C and 100 º 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

Uêba

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

2

3 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), Dörmo 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 º C and 40 º C.

• Viscosity Index - is an empirical number that relates the

change in viscosity with temperature variation, within a range of

17

40 º C to 100 º 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 º C and -35 º 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,

4

5

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. "

6

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 / Uêba) 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 Decanodióico 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

7 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 ricinoleína 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 tioesterificação, 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

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

9

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-His-

Asp). 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 α-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

ezimática 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.

Dörmö 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 º C, 40 º C, 50 º C and 60 º 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 sebácico/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 º C and 40 º 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 Ǻ 3 were supplied by Sigma-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 (μmols / 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 º C, 80 º C, 85 º C, 90 º C and 100 º 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 º C, 80 º C, 85 º C, 90 º C and 100 º C, since its boiling point is 195 º 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 º 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 º C, 100 º C and 110 º 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 º 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 Ǻ 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 ìL 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 º C insoluble insoluble insoluble soluble soluble soluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble

60 º 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 º 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

sebácico/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

μmols acid / min.g), followed by Lipozyme RM IM (1909 μmols acid / min.g) and

lower potential was actually observed for Lipozyme TL IM (699 μmols 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 sebáci

100

80 Molar fraction (%)

Mono-sebacate octi l di octyl sebacate l

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

Molar Fraction (%)

80

60

40

20

0

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

Reaction time (minutes)

Mono-sebacate octyl Dioctyl sebacate

(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 alcólise, 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 sebácico/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 sebácico/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; DÖRMÖ 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

sebácico/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 sebácico/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

Sebacic acid Sebacate mono-octyl Sebacate dioctyl

F ç m the la (% ã ra r) 60

40 20

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

Reaction time (minutes)

(A) 3% w / w Sebacic acid

100 F O M la (% ã ra r)

80 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 sebáico

Mono-sebacate octyl Dioctyl sebacate Molar fraction (%) 80

60 40 20

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

Reaction time (minutes)

(C) 7% w / w 100

Sebacic acid Mono-sebacate octyl Dioctyl sebacate

Molar fraction (%) 80 60

40 20

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

Reaction time (minutes)

(D) 9% w / w 100

Á sebacic acid F the ã la m () r CO r%

80 60 40 20

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

Te mpo rea tion (minutes)

Sebacate mono-octyl Sebacate dioctyl

(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 Molar fraction (%) 80

60 40 20

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

Reaction time (min)

Sebacic acid Mono-sebacate octyl Sebacate dioctyl

(A) 3% w / w

100 F the ã la m () r CO r% 80

60 40 20

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

Reaction time (minutes)

Sebacic acid Mono-sebacate octyl dioctyl sebacate

(B) 5% w / w

100 Sebacic acid Mono-sebacate octyl Dioctyl sebacate F ç m the la (%

ã ra r)

80 60 40 20

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

Reaction time (minutes) (C) 7% w / w

100 Sebacic acid Mono-sebacate octyl Dioctyl sebacate F ç m the la (%

ã ra r)

80

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 fraction (%) 80 60

40 20

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

Reaction time (minutes)

Mono-sebacate octyl Dioctyl sebacate

(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 sebácico/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

80

60

40

20

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

Sebacic acid

Mono-sebacate octyl Sebcato of dioctyl Molar fraction

(%)

Reaction time (min)

(A) free evaporation

100

80

60

40

20

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

Sebacic acid

Mono-sebacate octyl dioctyl sebacate Molar

fraction (%)

Reaction time (min)

(B) Reactor Closed

Figure 4.9. Composition of the reaction medium in the synthesis reaction of dioctyl sebacate at 100 º C, sebácico/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

80

Mono-sebacate octyl Dioctyl sebacate

Molar fraction (%)

60

40

20

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

Reaction time (min)

(A) Vacuum

100

80

60

40

20

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

Sebacic acid

Mono-sebacate octyl dioctyl sebacate Molar

fraction (%)

Reaction time (min)

(B) Reactor Closed

Figure 4.10. Composition of the reaction medium in the synthesis reaction of dioctyl sebacate at 100 º C, sebácico/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 3Ǻ, supplied by Sigma, and was previously dried in an oven at 200 º 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.

71

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.

72

100 Sebacic acid

Molar fraction (%)

80

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

80

60

40

20

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

Sebacic acid

Mono-sebacate octyl dioctyl sebacate Molar fraction

(%)

Reaction time (min)

(B) Reactor Closed

Figure 4.11. Composition of the reaction medium in the synthesis reaction of dioctyl sebacate at 100 º C, sebácico/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

73

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 60

Molecular sieve (50mg)

Molar fraction (%)

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 sebácico/1-octanol acid and 5% w / w Novozym 435. In closed reactor (◊) in open reactor (free evaporation) (□), employing vacuum (Δ) and adding 50 mg of molecular sieve (x).

74

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 sebácico/1-octanol, with 7% w / w Novozym 435 lipase in various uses.

75

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.

76

Sebacic acid

100

Molar fraction (%)

80

60

40

20

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

Sebacate mono-octyl Sebacate dioctyl

Reaction time (minutes)

(A) Initial reaction Sebacic acid

100

80

60

40

20

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

Molar fraction (%)

Mono-sebacate octyl Sebacate dioctyl

Reaction time (min)

(B) first reuse

Sebacic acid 100

Sebacate mono-octyl Sebacate dioctyl

Molar fraction (%)

80

60

40

20

0 0 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 sebácico/1-octanol acid and 7% w / w of lipase Novozym 435 to 100 º C (A) Initial reaction (B) First reuse (C) Second reuse.

77

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 º C, with sebácico/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

100

Molar fraction (%)

80

60

40

20

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

Sebacate mono-octyl Sebacate dioctyl

Reaction time (minutes)

(A) Novozym 435

Sebacic acid Mono-sebacate octyl Dioctyl sebacate

100

80

60

40

20

0

Molar fraction (%)

0 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 º C, employing a molar ratio of 1:5 sebácico/1-octanol acid and 7% m / m of catalyst. (A) Novozym 435 (B) Sulfuric acid.

78

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

79

• 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 100ºC Viscosity to 40ºC Point fluidity

Point blaze

Index acidity

Index viscosity and

ASTM D 445

ASTM D 445

ASTM D 97

ASTM D 92

ASTM D 974

ASTM D 2270

2,000 cSt

7,370 cSt

- 63 ºC

128 ºC

8.64 mg KOH / g

39

Oil

mineral

paraffinic

(Spindle

09)

Oil

mineral

naphthenic

(NH10)

2,741 cSt

10.68 cSt

-9 ºC

184 ºC

0.01 mg KOH / g

95

2,365 cSt

9,805 cSt

-51 ºC

150 ºC

0.01 mg KOH / g

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

80

negative temperatures.

This means that, even at temperatures up to -63 º 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 º 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.

81

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. ÓleoÓleo EnsaioMetodologia parafíniconaftênico

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

82

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.

83

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,

84

delaying the appearance thereof;

f) The reuse of lipase Novozym 435 was not effective, whereas even

although still presenting converões 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

85

acid reaction product.

g) Use of continuous system in the synthesis of dioctyl sebacate.

h) use of a lipase laboratory.

86

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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