nowadays , people are concerned about green technology that is more environmental friendly and can save our environment .
green technology concept is good for industry which can be realized with sustainable and renewable resources .
epoxidized oils are currently produced by epoxidation of unsaturated plant oils , soybean , linseed oil , and jatropha curcas seed oil .
although there are several methods available to epoxidize the double bonds of unsaturated fatty acids , the only method applied on industrial scale is the prileshajev epoxidation reaction . in this reaction ,
a peracid from a short - chain fatty acid and hydrogen peroxide under strong acidic conditions is used as the oxidizing agent .
the presence of the strong acid in the reaction mixture , however , causes the formation of side products such as vicinal diols , estolides , and other dimmers .
although a carful choice of the peracid and the reaction conditions can help to minimize the epoxides loss , the selectivity of industrial epoxidation of plant oils rarely exceeds 80% .
monoepoxidized oil has gained widely application including functional fluids , fuel additives , polyol replacements , pharmaceutical molecules , reactive diluents in cationic and uv cure applications , surfactants , adhesives , sealants , coatings , and biolubricants .  
. this study will be one of the solutions to obtain cheaper biolubricant to replace the existing epoxy biolubricant .
a milder and more selective epoxidation process has been suggested , wherein a lipase is used to catalyze the peracid formation . in the lipase - mediated epoxidation of unsaturation fatty acid , the acid itself reacts with hydrogen peroxide to form the peracid , which then epoxidized the double bond .
the reaction is therefore often referred to as  self - epoxidation reaction ,  in spite of the fact that the second step proceeds predominantly via an intermolecular process . among the various lipases studied so far , novozym 435 ,
a commercial preparation of candida antarctica lipase b , has been shown to be the most effective .
recently , lipase - mediated epoxidation in a solvent - free medium has also been reported .
this study focuses on the physicochemical characteristics such as pour point ( pp ) , flash point ( fp ) , viscosity index ( vi ) , and oxidative stability ( ot ) of the monoepoxidation process , with the aim to determine optimal reaction conditions with regard to reaction efficiency and enzyme stability using d - optimal design . in this paper , producing of meoa over diepoxide as biolubricants has been studied for improvement of the physicochemical characteristics ( i.e. , pp , fp , and vi ) .
linoleic acid ( la ; 18  :  2 ) was used as the model substrate .  
monoepoxidation of la results in the mixture of two monoepoxides ( cis-9 , 10-epoxy 12  c- 18  :  1 and cis-12 , 13 epoxy 9  c- 18  :  1 ) .
the enzymatic monoepoxidation was carried out using novozym 435 , a commercial catalyst made up of lipase , from candida antartica , immobilized on a polyacrylate resin . 3 factors ( variables ) such as hydrogen peroxide ( l , x1 ) ,
enzyme ( mg , x2 ) , and ration time ( h , x3 ) were obtained according to .  
table 1 shows different ratios of hydrogen peroxide , enzyme , and reaction time using d - optimal design . in a typical chemoenzymatic monoepoxidation of la 9(12)-10(13)-monoepoxy 12(9)-octadecanoic acid ( meoa ) , the la ( 1.4  g )
was dissolved in 10  ml of toluene , and the lipase was added . after stirring for 15  min
, 30% h2o2 were added , and every 15  min the addition was repeated .
afterwards , the lipase was removed by filtration , the mixture was washed with water to remove excess h2o2 , and the organic phase was dried over anhydrous sodium sulfate , and solvent was evaporated in a vacuum rotary evaporator .  
after the laboratory reaction was completed , the actual yield to the theoretical yield was compared to determine the percent yield 
 ( 1)actual  yield  ( in  grams)theoretical  yield  ( in  grams)100%=percent  yield .
iodine value was determined according to . to explore the effect of the operation variables on the response in the region of investigation ,
hydrogen peroxide ( l , x1 ) , amount of enzyme ( mg , x2 ) , and reaction time ( h , x3 ) were selected as independent variables .
the range of values and coded levels of the variables are given in table 1 .
a polynomial equation was used to predict the response as a function of independent variables and their interactions . in this work ,
the number of independent variables was three , and therefore the response for the quadratic polynomials becomes 
 ( 2)y=0+ixi+iixi2+ijxixj , 
 where 0 ; i ; ii ; ij are constant , linear , square , and interaction regression coefficient terms , respectively , and xi and xj are independent variables .
usa ) was used for multiple regression analysis , analysis of variance ( anova ) , and analysis of ridge maximum of data in the response surface regression ( rsreg ) procedure .
the goodness of fit of the model was evaluated by the coefficient of determination r  and its statistical significance that was checked by the f - test .
ftir of the products was recorded by using perkin elmer spectrum gx spectrophotometer in the range of 4004000  cm a very thin film of products was covered on nacl cells ( 4  mm thickness ) and was used for analysis .
nuclear magnetic resonance spectroscopy for proton h and c nmr has been carried out according to .
c and h nmr spectra were recorded using a 400  mhz bruker 300  nmr spectrometer . for these analysis ,
20  mg of samples were dissolved in 1  ml of cdcl3 solvent and introduced into the nmr tube .
the pour point ( pp ) is defined as the lowest temperature at which a liquid remains pourable ( meaning it still behaves as a fluid ) .
this method is routinely used to determine the low - temperature flow properties of fluids .
pp values were measured according to   using a phase technology analyzer , model psa-70s ( hammersmith gate , richmond , bc , canada ) .
each sample was run in triplicate , and average values rounded to the nearest whole degree are reported . for a greater degree of accuracy ,
pp measurements were done with a resolution of 1c instead of the specified 3c increment .
generally , materials with lower pp exhibit improved fluidity at low temperatures than those with higher pp .
the flash point of a volatile liquid is the lowest temperature at which it can vaporize to form an ignitable mixture in air .
flash point determination was run according to the american national standard method using a tag closed tester .
each sample was run in triplicate , and the average values rounded to the nearest whole degree are reported .
viscosity index ( vi ) is an arbitrary measure for the change of kinematic viscosity with temperature .
automated multirange viscometer tubes hv m472 obtained from walter herzog ( germany ) were used to measure viscosity .
measurements were run in a temp - trol ( precision scientific , chicago , ill , usa ) viscometer bath set at 40.0 and 100.0c .
pressurized dsc ( pdsc ) experiments were accomplished using a dsc 2910 thermal analyzer from ta instruments ( newcastle , del , usa ) .
typically , a 2  l sample , resulting in a film thickness of < 1  mm , was placed in an aluminum pan hermetically sealed with a pinhole lid and oxidized in the presence of dry air ( gateway airgas , st .
louis , mo , usa ) , which was pressurized in the module at a constant pressure of 1378.95  kpa ( 200  psi ) .
the oxidation onset ( ot , c ) was calculated from a plot of heat flow ( w / g ) versus temperature for each experiment .
the sample was run in triplicate , and average values rounded to the nearest whole degree are reported .
in this study , following the reaction experiments , the response surface is approximated by d - optimal design .
hydrogen peroxide is an important reactant for the formation of peracids from fatty acids ; hence , the influence of its amount on the monoepoxidation reaction was studied .
the addition of h2o2 solution to the reaction medium ( toluene with la ) leads to the formation of two distinct phases : an organic phase and an aqueous phase .
the designs and the responses yield% of meoa ( y1 ) , ooc% ( y2 ) , and iv  mg / g ( y3 ) are given in table 2 .
the novozym 435 , being adsorbed on a hydrophobic carried , is mainly present in the organic phase , while h2o2 will be partitioned in both the aqueous and the organic phases , with the concentration being higher in the aqueous phase ( figure 2 ) .
this was determined performing varying amounts of h2o2 ( 15 , 17.5 , and 20  l ) which has been added every 15  min , and the addition was repeated 24 times , novozym 435 ( 80 , 100 , and 120  mg ) , and different times ( 6 , 7 , and 8  h ) . a stoichiometric excess of the required amount of the peroxide was used to compensate for its possible decomposition by light and temperature .  

table 2 showed that the yield percentage of meoa , y1 , has increased to 82.14% , while ooc% , y2 , 4.91 and iodine value , y3 , 66.65 which are considerably compared to the theoretical ( ooct ) 9.02% and initial iodine value ( iv ) 157.35 
subsequent experiments were performed using different amounts of h2o2 15 , 17.5 , and 20  l for every 1.4  gm of la in one single step . as seen in table 2 , there was a clear increase in the reaction rate ( ooc% ) and a decrease ( iv  mg / g ) with increasing h2o2 amount . with 15  l , monoepoxidation was achieved at 7  h using 120  mg novozym 435 , while total epoxidation was observed within 10  h using 30  l .
increasing the peroxide amount used for the reaction results in increasing peracid formation . in the state of partial epoxidation ,
the amount of peracids accumulated is not significant ( less than 2% of the total ) , because the chemical reaction in which they are consumed is very fast .
but once all the double bonds are epoxidized , the remaining peracid is not consumed .   the quadratic regression coefficient obtained by employing a least squares method technique to predict quadratic polynomial models for the yield% of meoa ( y1 ) , ooc% ( y2 ) , and iv  mg / g ( y3 ) is given in tables 3 , 4 , and 5 . for the yield% of meoa ( y1 ) ,
the linear term of novozym 435 catalyst amount ( x2 ) , quadratic terms of h2o2 ( x11 ) and novozym 435 catalyst amount ( x22 ) were significant ( p < 0.05 ) .
the interaction between h2o2 ( x11 ) and novozym 435 catalyst amount ( x12 ) and the interaction between h2o2 ( x11 ) , and reaction time ( x13 ) were significant ( p < 0.05 ) , while its quadratic term of reaction time ( x33 ) was highly significant ( p < 0.01 ) .   highly significant ( p < 0.01 ) terms of ooc% ( y2 ) and iv  mg / g ( y3 ) for the h2o2 ( x1 ) were linear , while linear term of iv  mg / g for the reaction time ( x3 ) was significant ( p < 0.05 ) .
the coefficients of independent variables ( h2o2 ; x1 , catalyst novozym 435 ; x2 , and reaction time ; x3 ) were determined for the quadratic polynomial models tables 3 , 4 and 5 .
the lack of fit f - value for all the responses showed that the lack of fit is not significant ( p > 0.05 ) relatively to the pure error .
regression models for data on responses y1 , y2 , and y3 were highly significant ( p < 0.01 ) with satisfactory r. however , r for y2 ( 0.69 ) was lower although the model was significant .  
table 6 summarizes the analysis of variance ( anova ) of all the responses of this study .  
these results suggest that linear effect of hydrogen peroxide is the primary determining factors for meoa .
reference   also concluded that this variable had a very large effect on the results of their monoepoxidation study .
final equations in terms of actual factors are :  
 ( 3)y1=+87.532.82x14.50x20.14x39.06x12  5.43x229.81x329.74x1x27.14x1x3  7.80x2x3 , 
 ( 4)y2=+5.59 + 1.00x1 + 0.58x2 + 0.51x30.37x12  0.099x22 + 0.31x320.22x1x20.55x1x3  0.27x2x3 , 
 ( 5)y3=+56.2418.82x18.91x210.57x3 + 8.06x12  + 4.15x224.41x32 + 11.59x1x2 + 10.81x1x3  + 5.42x2x3 . 
 
rsm is one of the best ways of evaluating the relationships between responses , variables , and interactions that exist .
significant interaction variables in the fitted models ( tables 3to 5 ) were chosen as the axes ( amount of h2o2 ; x1 , catalyst novozym 435 ; x2 and reaction time ; x3 ) for the response surface plots . the relationships between independent and dependent variables are shown in the three - dimensional representation as response surfaces . in a contour plot ,
curves of equal response values are drawn on a plane whose coordinates represent the levels of the independent factors .
each contour represents a specific value for the height of the surface above the plane defined for a combination of the levels of the factors .
therefore , different surface height values enable one to focus attention on the levels of the factors at which changes in the surface height occur .   figures 3to 5 are the design - expert plots for all the responses . in the meoa , performing the technique using low amount of h2o2 would give the desired ooc% of meoa as shown in figure 4 , while iv ( figure 5 ) was higher at this condition .
as shown in figures 4 and 5 , the increasing amount of h2o2 led to an increase in the ooc% and reduction of iv  mg / g .
high ooc% could be obtained by using high amount of h2o2 at high reaction time .
experimental variables should be carefully controlled in order to recover a medium percentage of meoa of interest with reasonable yield .
optimum conditions of the experiment to obtain higher yield% of meoa and medium ooc% were predicted at amount of h2o2  l of 15 , catalyst novozym 435 of 120  mg , and 7  h of reaction time . at this condition ,
mg / g of iv . in order to prove the present oxirane ring of meoa
the comparison between la ( a ) and meoa ( b ) , ftir spectra is shown in figure 6 .
for the carboxylic acid carbonyl functional groups ( c = o ) , ftir spectrum showed absorption bands of la and meoa at 1719 and 1711  cm , respectively , while stretching vibration peak of c = c can be detected at wavenumber 3009  cm , and peaks at 29272856  cm indicated the ch2 and ch3 scissoring of la and meoa based on figures 6(a ) and 6(b ) .
ftir spectrum also showed absorption bands at 722 , 723 for ( c - h ) group .  

the c spectroscopy shows the main signals assignment of the la as shown in table 8(a ) .
the signals at 180.49  ppm refer to the carbon atom of the carbonyl group ( carboxylic acid ) .
the signals at 128.27130.38  ppm refer to the unsaturated carbon atoms ( olefinic carbons ) ; 24.8629.79  ppm due to methylene carbon atoms in fatty acid moieties of la .   figures 7(b ) and 7(c ) can be confirmed by the oxirane ring of meoa 54.5957.29  ppm and diepoxide la at about 54.6157.32  ppm .
indeed , it appeared that the signals were present in the meoa , as four peaks of roughly equal intensity ( 132.89 , 132.72 , 130.15 , and 124.02  ppm ) were observed in the alkenic carbon region in the c nmr spectrum figure 7(b ) , while they disappeared in the diepoxide la ( figure 7(c ) ) .
the c - nmr spectra indicate the existence of carbonyl group ( carboxylic acid ) in their structure meoa 179.32  ppm and diepoxide la at about 178.79  ppm .
the other distinctive signals were aliphatic carbons meoa at about 22.6929.38 ppm and di - epoxide la at about 22.7729.44  ppm , which are common for these types of compounds .  

h nmr spectroscopy shows the main signals assignments in la , meoa and di - epoxide la as shown in table 9 .
the h nmr spectra for the products show some of the key features for a typical ( -ch - o - ch- ) at about 2.923.12  ppm of meoa , and about 2.993.13  ppm of di - epoxide la ( figure 8(b ) ) .
the distinguishable groups are the protons of the terminal methyl of the fatty acid chain .
the signals at 0.880.86  ppm referred to the methylene group ( -ch3 ) of la figure 8(a ) which also appear in meoa 0.860.88  ppm and di - epoxide la 0.880.92  ppm ( figures 8(b ) and 8(c ) ) next to the terminal methyl ( -ch2 ) at 1.302.77  ppm of la , 1.292.33 of meoa , and 1.342.36 ppm of di - epoxide la . however , the methane proton signals ( -ch = ch- ) were shifted upfield at about 5.355.36  ppm of la and 5.385.49  ppm of meoa , while they disappeared in di - epoxide la .
another distinctive feature is the hydroxyl proton ( -cooh ) of the carboxylic acid at about 7.27  ppm .
this approach is used here to improve the low - temperature flow behavior of fatty acids by monoepoxide ring .
monoepoxidation to produce meoa was the most effective decreasing the pp to 41c ( table 10 ) .
it can be assumed that the presence of oxirane ring on the fatty acid creates a steric barrier around the individual molecules and inhibits crystallization , resulting in lower pp .  
flash point is another important factor in determining how well oil will behave as a potential biolubricant .
the flash point is often used as a descriptive characteristic of oil fuel , and it is also used to describe oils that are not normally used as fuels .
there are various international standards for defining each , but most agree that oils with a flash point less than 43c are flammable , while those having a flash point above this temperature are combustible .
table 10 has shown the improvement in flash point of meoa which increased to 128c comparing with 115c of la that means the result agrees with the various international standards .   the efficiency of the biolubricant in reducing friction and wear is greatly influenced by its viscosity .
generally , the least viscous biolubricant which still forces the two moving surfaces apart is desired .
if the biolubricant is too viscous , it will require a large amount of energy to move ; if it is too thin , the surfaces will rub and friction will increase . the viscosity index highlights how a biolubricants viscosity changes with variations in temperature .
many biolubricant applications require performing across a wide range of conditions , for example , in an engine .
automotive biolubricants must reduce friction between engine components when it is started from cold ( relative to engine operating temperatures ) as well as when it is running ( up to 200c ) .
the best oils ( with the highest vi ) will not vary much in viscosity over such a temperature range and therefore will perform well throughout . in meoa , decreased viscosity index ( vi ) to 130.8 of meoa is the result of less double bond ( table 10 ) .  
the ability of a substance to resist oxidative degradation is another important characteristic of biolubricants .
therefore , meoa was screened to measure their oxidation stability using pdsc through determination of ot .
pdsc is an effective method for measuring oxidation stability of oleochemicals in an accelerated mode .
the ot is the temperature at which a rapid increase in the rate of oxidation is observed at a constant , high pressure ( 200  psi ) .
ot was calculated from a plot of heat flow ( w / g ) versus temperature that was generated by the sample upon degradation and by definition . in this chapter , oxirane ring of meoa
did not show improvement in the oxidation stability of ot for meoa at 168c comparing with la at 189c ( table 10 ) .
rsm and 3-factor d - optimal design were employed for optimization of meoa . from the present study ,
it is evident that hydrogen peroxide is the most critical parameter influencing the chemoenzymatic monoepoxidation reaction .
increase in the hydrogen peroxide amount has a strong effect on the reaction kinetics ; however , a large excess of hydrogen peroxide results in accumulation of peracid in the final product . based on the results obtained ,
 linoleic acid ( la ) is converted to per - carboxylic acid catalyzed by an immobilized lipase from candida antarctica ( novozym 435 ) . 
 this per - carboxylic acid is only intermediate and epoxidized itself in good yields and almost without consecutive reactions . 
 monoepoxide linoleic acid 9(12)-10(13)-monoepoxy 12(9)-octadecanoic acid ( meoa ) was optimized using d - optimal design . at optimum conditions , 
 higher yield% ( 82.14 ) and medium oxirane oxygen content ( ooc ) ( 4.91% ) of meoa were predicted at 15  l of h2o2 , 120  mg of novozym 435 , and 7  h of reaction time . in order to develop better - quality biolubricants , 
 pour point ( pp ) , flash point ( fp ) , viscosity index ( vi ) , and oxidative stability ( ot ) were determined for la and meoa . 
 the results showed that meoa exhibited good low - temperature behavior with pp of 41c . 
 fp of meoa increased to 128c comparing with 115c of la . in a similar fashion , vi for la was 224 generally several hundred centistokes ( cst ) more viscous than meoa 130.8 . 
 the ability of a substance to resist oxidative degradation is another important property for biolubricants . 
 therefore , la and meoa were screened to measure their ot which was observed at 189 and 168c , respectively . 
1. Introduction
2. Methodology
3. Results and Discussion
4. Conclusion