Rapid Epoxidation Of Palm Acid Oil With Lipase Biology Essay

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In view of growing environmental concerns and tightened regulations over contaminants and pollution in the environment in recent years, calls for biodegradable and nontoxic vegetable oil-based lubricants are abound. They have very low volatility due to the higher molecular weight of the triacylglycerol molecule and a narrow range of viscosity changes with temperature. Polar ester groups in the molecule are able to adhere to metal surfaces, and therefore, possess good boundary lubrication properties. In addition, vegetable oils have high solubilising power for polar contaminants and additive molecules. However, vegetable oils show poor oxidative and thermal stability primarily due to the presence of unsaturation. The presence of ester functionality also renders these oils susceptible to hydrolytic breakdown. The proposed modification of the vegetable oils is an important manner to obtain potentially useful products using a renewable feedstock. In designing a 'green' process to effectively carry out the epoxidation reaction, we report herein, an inexpensive, practical, safe and environmentally friendly method to epoxidize palm acid oil under extremely mild conditions. This work highlights the increased reaction rate of the epoxidation process when microwave irradiation is introduced. The starting material used is Palm Acid Oil, a by-product of the alkali refining process of palm oil. Acid oil can serve as an inexpensive raw materials and are very good substitute for neat vegetable oil such as palm oil for the production of biolubricant. It is high in Free Fatty Acids (FFA) and is the ideal material for the epoxidation process due to the importance of FFAs in producing peroxy-acids as an oxygen carrier. The double bonds the triglycerides are reacted with a peracid, generated for safety reasons in situ using hydrogen peroxide. Novozym 435 acts as the catalyst in the process and with its good selectivity, the occurrence of by-products is controlled. The method and materials used are rapid, non-toxic and easily available while the incorporation of low powered microwave irradiation during the reaction process greatly reduces the time, from a typical 16 hour to 1 hour. The microwave irradiation increases the reaction rate and decreases inactivation of the enzyme during the reaction.


1.1 Using vegetable oil

In recent years, there has been a surge in interest in bio-based oleo-chemicals, seen in the switching of mineral to vegetable based epoxidised oils, as the latter is very much environmentally friendly and rapidly biodegradable. It is rightly so, as vegetable based oils often exhibit superior attributes and as in the case of lubricants, vegetable oil based products have better anti-wear properties, higher lubricity, higher viscosity index, non-toxic and ultimately, causes no pollution. Epoxidized oils can be the starting material to many of the common oleochemical products. They are used as plasticizers, crosslinking agents, stabilizers, pre-polymers and are intermediates for polyol production, providing more areas of modification in terms of lubricant properties by addition of functional groups onto the epoxide ring. However, they also have drawbacks that must be overcome, including poor low-temperature properties-such as opacity, precipitation, poor flowability and/or solidification at relatively moderate temperature, their susceptibility to oxidative degradation and their tendency to undergo hydrolysis in acid media. [Hwang and Erhan, 2001; Hwang et al., 2003] As such, chemical modifications to the vegetable oil must be done to improve usability.

1.2 Current Industrial Practices

Epoxidized oils are currently produced on an industrial scale by the Prileshajev epoxidation reaction in which a peracid is used for oxygen transfer to the double bonds.

Fig. Epoxidation reaction of vegetable oils

A short-chain peroxy acid, usually peracetic acid is prepared from hydrogen peroxide and acetic acid either in a separate step or in situ. Large scale epoxidation usually avoid the handling of peroxy acids by using the in situ method but this requires strong mineral acid to act as a catalyst in the forming of the peroxy acid. The presence of strong mineral acid causes side reactions and thus the selectivity rarely exceeds 80%.

1.3 Innovations

1.3.1 Chemo-enzymatic Epoxidation

Enzymes are capable of lowering activation energy by temporarily combining with the chemicals involved in the reaction, acting as a reaction site. Klaas et. al [] had showed that Novozym 435 is capable of catalyzing the conversion of fatty acids with hydrogen peroxide to peroxy fatty acids. In this method of 'self-epoxidation' of unsaturated fatty acids, up to 91% yield and more than 98% selectivity has been reported.

Fig. Chemo-enzymatic 'self'-epoxidationof unsaturated fatty acids; conversion of oleic acid to epoxystearic acid via peroxy oleic acid, as reported by Klaas et. al[]

Only a minimum amount of enzyme was necessary to show the catalytic effect. Up to six rounds of usage can be achieved with reported optimum enzyme activity. [Emanuel Ankudey, Horacio F. Olivo and Tonya L. Peeples, Lipase-mediated epoxidation utilizing urea-hydrogen peroxide in ethyl acetate, 2006 Green Chem.8, 1-5 |The Royal Society of Chemistry (DOI: 10.1039/b604984b)]

1.3.2 Non-thermal microwave effects

With microwave irradiation, it was found that higher yields, milder reaction conditions and shorter reaction times can be used for organic chemical reactions and many of these processes can be improved. Even reactions that do not occur by conventional heating can be performed using microwaves. There exists a so-called 'non-thermal microwave effect' apart from simple thermal heating. Hence, acceleration or changes in reactivity and selectivity could be explained by a specific radiation effect and not merely by a thermal effect [Selectivity under microwave irradiation. Benzylation of 2-pyridone: an experimental and theoretical study, Antonio de la Hoza, María Pilar Prieto, Michel Rajzmann, Abel de Cózara, Angel Díaz-Ortiza, Andrés Morenoa and Fernando P. Cossío; doi:10.1016/j.tet.2008.06.052 ]

1.3.3 Availability of Palm Acid Oil

Palm Acid Oil is a by-product of the refining process of palm oil. It is used for making laundry soaps and for producing calcium soaps for animal feed formulations. The main components of palm acid oil (PAO) are free fatty acids, neutral oil and moisture. Palm Acid Oil proves to be a suitable candidate for the base material for lubricant as it has very high Free Fatty Acid contents, is not edible and is cost-beneficial as the current uses are few.

Fatty Acid has been reported to increase lubricity of a material. The film-forming properties of triacylglycerol molecules are believed to inhibit metal-to-metal contact and progression of pits and asperities on the metal surface. Strength of the protective fluid film and extent of adsorption on the metal surface dictate the efficiency of a lubricant's performance. In chemical terms, native and synthetic esters exhibit the same structure. Lubricity is greatly increased because the fatty acids tend to migrate from the oil to the surface of the part where vacant sites are available. They adsorb and assemble a protective film till they are removed by subsequent contact with counter-surfaces. The assembly and desorption or wear and reassembly is a dynamic process. Furthermore, some other works report a reduction in friction for the fatty acids with humidity. However, if a group of fatty acids in the adsorbed state is removed by the sliding of the surfaces and the adsorption kinetics does not allow the reassembly to occur before the next contact occurs, the film is not protective and the friction tends to be that corresponding to bare metallic contact [Rashmi R. Sahoo and S.K. Biswas, Frictional response of fatty acids on steel, Journal of Colloid and Interface Science doi:10.1016/j.jcis.(2009).01.046].

1.3.4 Food vs Fuel Debate

Food prices between 2005 to 2008 had rose dramatically, at the same time that biofuel production increased. In major crop producing countries like Brazil, (although food production stayed the same) corn has tripled in price since biofuel has become a common energy source [Beatty, B. and Aubele , M. 2008]. Like biofuels, biobased lubricants are derived from vegetable oils, thus there always exists an ethical debate on the idea of using edible oils for non-edible products. Likewise, Palm Oil is widely used for cooking in Southeast Asia and the tropical belt of Africa. However, as opposed to using Palm Oil, the use of Palm Acid Oil, which is a by-product, to produce biolubricant is adding value by diversifying its uses.


Mean value of 27 samples

Standard Deviation


Moisture Content (%)




Free Fatty Acid (%)




Peroxide Value(meq/kg)




Iodine Value




Saponification Value




Unsaponifiable matter




Fig. 1 Properties of Palm Acid Oil

According to above properties of Palm Acid Oil [Characterization of Palm Acid Oil, Ainie Kuntom, Wai-Lin Slew and Yew-Ai Tan, 1994, JAOCS, Vol. 71, no. 5 ], it is then calculated that



% Neutral Oil (after removal of moisture)


Unsaturation (C=C)

0.187 mol-1

Molecular Weight of PAO

468.93 g/mol

Fig. 2 Ppercent Unsaturation, Molecular Weight, Free Fatty Acid content of Palm Acid Oil


2.1. Materials

Palm Acid Oil (kindly supplied by UNItata Refinery) was used as the starting material for chemical modification. Formic acid (99.81%), Ethyl Acetate, Novozym 435, and Hydrogen Peroxide (30%) were ACS grade and purchased from Sigma-Aldrich and J.T. Baker. Water was doubly distilled (DD) and deionized.

2.2. Preparation of reactants before incorporating Microwave irradiation

Palm Acid Oil (40g) was first heated to remove moisture and left to cool until 40-C. Next, 80ml of Ethyl Acetate and 1.47 g of Formic Acid were added with 50 mg Novozym 435, under moderate stirring. The mixture was so kept for 15 minutes, after which Hydrogen Peroxide (total 10.85 ml) was added dropwise for 15 minutes (2.7 ml). Hydrogen Peroxide was then added periodically every 15 minutes (2.7 ml) as the concentration of the peroxy fatty acid ester formed is detrimental to enzyme activity. [Stability of immobilized Candida antarctica lipase B during chemo-enzymatic epoxidation of fatty acids, Ulrika T¨ornvall, Cecilia Orellana-Coca, Rajni Hatti-Kaul, Dietlind Adlercreutz, Enzyme and Microbial Technology 40 (2007) 447-451]-doi:10.1016/j.enzmictec.2006.07.019

2.3 Microwave Irradiation

**The reaction vessel was placed into a microwave oven (2.45 GHz) and heated for 3 minute cycles at 150 W setting. However the reaction should not exceed 60-C as the enzyme activity will greatly decrease upon exposure to higher temperatures. Hence, the reaction vessel was removed from the oven intermittently and placed in a water bath to reduce its temperature to 20-C. The cycle is repeated for 20 times with hydrogen peroxide added every 15 minutes.

2.4 Oxirane Oxygen Titration

Epoxy content is the most important property of epoxy materials. Dry epoxidized samples were analyzed for their percents (by weight) of epoxy functional groups by an official method AOCS Cd 9-57 (Oxirane Oxygen in Epoxidized Materials).

According to percentages of unsaturated fatty acids listed in Fig.**, full epoxidation should yield 0.433% [Using Klaas' equation for oxirane percent] of epoxy content for Epoxidized Palm Acid Oil. Moreover, the following equation was used to calculate the epoxy functionality of Epoxidized Palm Acid Oil where the molecular weights of EPAO are approximately []. *HOW TO CALCULATE mw of EPAO?

Results and Discussions

Fig. 1 Oxirane oxygen content vs. time

Fig. 2 Reaction Rate

Determination of Ea and other Thermodynamics of Epoxidation