OXIDATIVE AND STORAGE STABILITY STUDIES OF BIODIESEL USING COMMERCIALLY AVAILABLE ANTIOXIDANTS

Transcription

1 CHAPTER IV OXIDATIVE AND STORAGE STABILITY STUDIES OF BIODIESEL USING COMMERCIALLY AVAILABLE ANTIOXIDANTS 1. Introduction The word antioxidant has become increasingly popular in modern society as it gains publicity through mass media coverage of its health benefits. The dictionary definition of antioxidant is rather straightforward but a traditional annotation would define antioxidant as a substance that opposes oxidation or inhibits reactions promoted by oxygen or peroxides, many of these substances (as the tocopherols) being used as preservatives in various products (as in fats, oils, food products, and soaps for retarding the development of rancidity, in gasoline and other petroleum products for retarding gum formation and other undesirable changes, and in rubber for retarding aging). A more biologically relevant definition of antioxidants is synthetic or natural substances added to products to prevent or delay their deterioration by action of oxygen in air [1]. Biodiesel, defined as fatty acid mono-alkyl esters made from vegetable oil or animal fat, is an alternative fuel for combustion in compression ignition (diesel) engines. Several recent reviews have reported on the technical characteristics of biodiesel. In short, biodiesel is made from domestically renewable feedstock, is environmentally innocuous, is relatively safe to handle (high flash points), and has an energy content, specific gravity, kinematic viscosity (KV), and cetane number (CN) comparable to petroleum middle distillate fuels (petro diesel) [2, 3]. With a production of almost one million tons in Europe, fatty acid methyl ester (FAME) more generally called biodiesel, has become a fast growing renewable liquid biofuel within the European Community. Just like vegetable oils or fats, fatty acid methyl esters undergo degradation over time, mainly influenced by temperature and oxygen. Degradation products of biodiesel, such as insoluble gums and sediments, or the formation of organic acids and aldehyde may cause engine and injection problems [3-5]. 140

2 The bis-allylic configurations, where the central methylene group is activated by the two double bonds (i.e., -CH=CH-CH 2 -CH=CH-), react with oxygen via the autoxidation mechanism, with the radical chain reaction steps of initiation, propagation, chain branching, and termination. During these reaction steps, several products can be formed, such as peroxides and hydro peroxides, low molecular weight organic acids, aldehydes and keto compounds, alcohols, as well as high molecular weight species (dimers, trimers, and cyclic acids) via polymerization mechanisms. The use of antioxidant additives can help slow the degradation process and improve fuel stability up to a point [6-9]. Fuel properties degrade during long-term storage as follows: (i) oxidation or autoxidation from contact with ambient air; (ii) thermal or thermal-oxidative decomposition from excess heat; (iii) hydrolysis from contact with water or moisture in tanks and fuel lines; or (iv) microbial contamination from migration of dust particles or water droplets containing bacteria or fungi into the fuel [10]. Monitoring the effects of autoxidation on biodiesel fuel quality during long-term storage presents a significant concern for biodiesel producers, suppliers, and consumers [11] Antioxidants Assay Studies Assays for antioxidant protection against oxidative damage generally depend on measurements of decreases in a marker of oxidation. Many terms have been used by different researchers to describe antioxidant capacity including total antioxidant capacity, efficiency, power, parameter, potential, potency, and activity. The activity of a chemical would be meaningless without the context of specific reaction conditions such as pressure, temperature, reaction media, co-reactants, and reference points. Because the antioxidant activity measured by an individual assay reflects only the chemical reactivity under the specific conditions applied in that assay, it is inappropriate and misleading to generalize the data as indicators of total antioxidant activity. The other terms listed above are more independent of specific reactions and have similar chemical meanings. To remain consistent, we use capacity to refer to the results obtained by different assays. Oxidant-specific terms such as peroxyl radical scavenging capacity, superoxide scavenging capacity, ferric ion reducing capacity and the like would be more appropriate to describe the results from specific assays than the loosely defined terms total antioxidant capacity and the like [1]. 141

3 On the basis of the chemical reactions involved, major antioxidant capacity assays can be roughly divided into two categories: (1) hydrogen atom transfer (HAT) reaction based assays and (2) single electron transfer (ET) reaction based assays. The ET-based assays involve one redox reaction with the oxidant (also used as the probe for monitoring the reaction) as an indicator of the reaction endpoint. Most HAT-based assays monitor competitive reaction kinetics, and the quantitation is derived from the kinetic curves. HAT-based methods generally are composed of a synthetic free radical generator, an oxidizable molecular probe, and an antioxidant. HAT- and ETbased assays are intended to measure the radical (or oxidant) scavenging capacity, instead of the preventive antioxidant capacity of a sample. Because the relative reaction rates of antioxidants (or substrates) against oxidants, particularly peroxyl radicals, are the key parameters for sacrificial antioxidant capacity, autoxidation and its inhibition kinetics are analyzed before in-depth analysis of the individual assays [12, 13]. Mechanism Assays involving hydrogen atom transfer reactions ROO* + AH ROOH + A* ROO* + LH ROOH + L* Assays by electron-transfer reaction M(n) + e (from AH) AH* + + M(n - 1) Other assays Antioxidant assays ORAC (oxygen radical absorbance capacity) TRAP (total radical trapping antioxidant parameter) ABTS method Crocin bleaching assay IOU (inhibited oxygen uptake) Inhibition of linoleic acid oxidation Inhibition of LDL oxidation FRAP (ferric ion reducing antioxidant parameter) TEAC (Trolox equivalent antioxidant capacity) SOS method DPPH (diphenyl-1-picrylhydrazyl) Copper(II) reduction capacity Total phenols assay by Folin-Ciocalteu reagent TOSC (total oxidant scavenging capacity) Inhibition of Briggs-Rauscher oscillation reaction Chemiluminescence Electrochemiluminescence Table 4.1: Mechanism of antioxidant assays 142

4 Antioxidants with free radical scavenging activities may therefore be relevant in the prevention and therapeutics of diseases where free radicals are implicated. WHO has recommended the use of natural antioxidants that can delay or inhibit the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative chain reactions. Antioxidants are substances that when present at low concentrations, compared to those of the oxidisable substrate significantly delays or inhibits the oxidation of the substrate [9]. An important role of antioxidants is to suppress free radical mediated oxidation by inhibiting the formation of free radicals by scavenging radicals. Radical scavenging action is dependent on both the reactivity and concentration of the antioxidant. Research on the role of antioxidants in biology focused earlier on their use in preventing the oxidation of unsaturated fats, which is the cause of rancidity [14] Biodiesel Storage and oxidation stability studies The parameter of oxidation stability has been fixed at a minimum limit of a 6-hour induction period at 110 C [15]. The method adopted for determination of the oxidation stability is the so called Rancimat method which is commonly used in the vegetable oil sector. Especially high contents of unsaturated fatty acids, which are very sensitive to oxidative degradation, lead to very low values for the induction period. Thus, even the conditions of fuel storage directly affect the quality of the product. Several studies showed that the quality of biodiesel over a longer period of storage strongly depends on the tank material as well as on contact to air or light. Increase in viscosities and acid values and decreases in induction periods have been observed [16] during such storage. Although there are numerous publications on the effect of natural and synthetic antioxidants on the stability of oils and fats used as food and feed, little is available on the effect of antioxidants on the behavior of FAME used as biodiesel. To retard oxidative degradation and to guarantee a specific stability, it becomes necessary to find appropriate additives for biodiesel. Simkovsky et al studied the effect of different antioxidants on the induction period of rapeseed oil methyl esters at different temperatures but did not find significant improvements [17]. Schober et al tested the influence of the antioxidant TBHQ on the peroxide value of soybean oil methyl esters during storage and found good improvement of stability [18]. Canakci et al described the effect of the antioxidants TBHQ and α-tocopherol on fuel properties of methyl soyate and found beneficial effects on retarding oxidative degradation of the sample [19]. Das et al 143

5 described effect of commercial antioxidants used in kharanja biodiesel for storage stability [20]. Most recently Karavalakis et al described the effect of synthetic phenolic antioxidants used for storage stability and oxidative stability. The storage stability of different biodiesel blends with automotive diesel treated with various phenolic antioxidants has also been investigated over a storage time of 10 weeks [21]. In the previous studies, numerous methods for assessing the oxidation status of biodiesel have been investigated, including acid value, density, and kinematic viscosity. The peroxide value may not be suitable because, after an initial increase, it decreases due to secondary oxidation reactions, although the decrease likely affects only samples oxidized beyond what may normally be expected. Thus there is the possibility of the fuel having undergone relatively extensive oxidation but displaying an acceptable peroxide value. The peroxide value is also not included in biodiesel standards. Acid value and kinematic viscosity, however are two facile indicators for rapid assessment of biodiesel fuel quality as they continuously increase with deteriorating fuel quality [22]. In this chapter, oxidative and storage stability of biodiesel was investigated using commercially available antioxidants. The experimental details of antioxidant assay (scavenging activity test) for five commercially available antioxidants such as BHA, BHT, GA, TBHQ and PY are provided. FRAP (ferric reducing ability of plasma) radical scavenging activity, ABTS (2,2,- azinobis(3-ethylbenzoline-6-sulfonic acid)) radical scavenging activity, superoxide anion scavenging activity (SOS), and DCF/AAPH/TRAP assays were carried out. The aim of the present study was to investigate the antioxidant capacity of five commercially available antioxidants such as BHA, BHT, GA, TBHQ and PY, and the oxidative and storage stabilites of Pongamia pinnata (PBD) and Jatropha curcus (JBD) biodiesels. FRAP (ferric reducing ability of plasma) radical scavenging activity, ABTS (2,2,- azinobis(3-ethylbenzoline-6-sulfonic acid)) radical scavenging activity, superoxide anion scavenging activity (SOS), and DCF/AAPH/TRAP assays were carried out to analyze antioxidant activity. The Rancimat procedure and the ASTM procedure were employed to investigate oxidation stability and storage stability, respectively, of the biodiesels. 144

6 Fuel properties such as acid value (AN) and kinematic viscosity (KV) of PBD and JBD were determined at regular periods of time, using the selected antioxidants at different concentrations. 2. Experimental 2.1. Superoxide anion scavenging activity assay The scavenging activity of extract towards superoxide anion radicals was measured by superoxide anion scavenging activity assay [1, 12, 13]. 1 ml of nitro blue tetrazolium solution (156 µm in 100 mm phosphate buffer, ph 7.4), 1 ml nicotine amide adenine dinucleotide solution (468 µm in 100 mm phosphate buffer, ph 7.4) and 0.1 ml of different concentrations of commercially available antioxidants dissolved in water were mixed together. The reaction was initiated by adding 100 µl of phenazine methosulphate (PMS) solution (60 µm) in 100 mm phosphate buffer, (ph 7.4) to the mixture. The reaction mixture was incubated at room temperature for 5 min and the absorbance at 560 nm was measured against reagent blank in spectrophotometer. The superoxide anion scavenging activity was calculated according to the following equation. % Inhibition = control test/control 100, where control was the absorbance of the control and test was the absorbance in the presence of the antioxidant. The experiment was repeated in triplicate ABTS radical scavenging activity assay The ABTS radical scavenging activity of the antioxidant was measured by ABTS radical scavenging activity. Radical cation (ABTS + ) was produced by reacting ABTS solution (7 mm) with 2.45 mm ammonium persulphate and the mixture was allowed to stand in dark at room temperature for hours before use. Different concentrations (10-50 µg/ ml) of antioxidant (0.5 ml) were added to 0.3 ml of ABTS solution and the final volume was made up to 1 ml with solvent. The absorbance was read at 745 nm and the% inhibition was calculated. The experiment was performed in triplicate. The ABTS radical scavenging activity was calculated as per the following equation. % inhibition = control test/control

7 2.3. FRAP radical scavenging activity assay Ferric reducing ability of plasma (FRAP) agent was prepared by mixing 25 ml of acetate buffer (500 mm) with 2.5 ml of tripyridyltriazine (TPTZ) (10 mm) and 2.5 ml of ferric chloride (20 mm) solution. To 900 µl of FRAP reagent, 100 µl antioxidant solution at different concentrations (20, 40, 60, 80 and 100 µg/ ml) were added. The increase in absorbance at 593 nm was measured after 4 min. FeSO 4.7H 2 O was used as a standard. An increase in absorbance indicated enhanced reducing potential of plasma [1, 12, 13] DCF/AAPH assay (TRAP) An azo initiator, AAPH, was used to produce peroxyl radicals, and the scavenging activity of the antioxidant was monitored via the spectrophotometric analysis of 2,7- dichlorofluorescin-diacetate [1, 12, 13]. The activation of DCF was achieved by mixing DCF (3.41 µl of 50 µg/ ml solution) and NaOH (1.75 ml of 0.01 N solution) and allowing the mixture to stand for 20 min before adding ml of sodium phosphate buffer (25 mm, ph 7.2). The reaction mixture contained 170 µl activated DCF solution and 10 µl of antioxidant solution at different concentrations (20, 40, 60, 80 and 100 µg/ ml). The reaction was initiated by the addition of 20 µl of 600 mm AAPH. After 10 min, the absorbance was read at 490 nm using a Spectrophotometer Biodiesel storage condition Normally, 200 ml of PBD and JBD biodiesel samples were stored in open Borosil glass bottles of 250 ml capacity and kept indoors, at a temperature of either 30 C or 42 C. The samples were exposed to air under daylight condition. The storage studies on PBD and JBD using commercially available antioxidants are presented in this section. Experiments were carried out at different storage conditions. The storage conditions employed were: 1) ordinary glass bottle with open space (OGOS), 2) ordinary glass bottle with closed space (OGCS), 3) ordinary glass bottle with closed space containing nitrogen (OGCSN), 4) amber glass bottle with open space (AGOS), 5) amber glass bottle with closed space (AGCS) and 6) amber glass bottle with closed space containing nitrogen (AGCSN). Ambient humidity was between 41% to 72%. The samples were analyzed weekly. 146

8 2.6. Determination of oxidative stability Oxidative stability (OS) of biodiesel sample was studied with a Rancimat 873 instrument (Metrohm, Switzerland), as followed by Tang et al. [9] Das et al. [20] Karavalakis et al. [21] and Knothe [22]. The sample was heated at a constant temperature of 110 C with excess airflow which passed through a conductivity cell filled with distilled water. During this oxidation process volatile acids were formed which were carried along with the airflow resulting in a gradual increase in the conductivity of distilled water present in the conductivity cell. After a period of time, called induction period, the gradual increase leads to a sudden increase in the conductivity of the distilled water. The induction period of PBD and JBD were determined with and without the addition of antioxidants like BHA, BHT, TBHQ, PY and GA, used at different concentrations (100, 200, 500, 1000, 2000, 3000 and 5000 ppm) Evaluation of storage stability To evaluate the storage stability, the ASTM procedures D weeks and D weeks were employed. In the ASTM D weeks procedure, the KV, PV and AN values were determined at 30 C every week over a period of 25 weeks. In the ASTM D procedure, the KV, PV and AN values were monitored at 43 C every week over a period of 12 weeks. The storage container used was 250 ml bottle containing 200 ml of biodiesel and 50 ml of free space. The ambient humidity in the storage room was 41% to 72%. 3. Results and Discussion 3.1. Antioxidant assay Five commercially available antioxidants BHA, BHT, GA, TBHQ and PY were chosen for the assay. Experimental details and results of antioxidant assays (scavenging activity tests) are discussed in this chapter. FRAP (ferric reducing ability of plasma) radical 147

9 scavenging activity, ABTS (2,2,-azinobis (3-ethylbenzoline-6-sulfonic acid)) ) radical scavenging activity, superoxide anion scave enging activity (SOS), DCF/AAPH/TRAP assay were carried out. Chemical structure of the selected antioxidants are given below. These antioxidants may offer resistance to oxidative stress by scavenging free radicals and inhibiting lipid peroxidation. Results of various free radical-scavenging assays have established the antioxidant potential of the five commercially available antioxidants which might be responsible for the observed in vitro free radical-scavenging activity. Figure 4.1: Chemical structure of commercially available antioxidants. The grouped bar chart (Figure 4.2) reveals the ABTS radical cation scavenging potential of each antioxidant to be in the range of µg/ ml. The mean values across the concentration range indicate that pyrogallal is highly potent in neutralising ABTS cation radicals. ABTS assay is based on the inhibition of the absorbance of the radical action ABTS +, which has a characteristic long wavelength absorption spectrum [12]. The results obtained clearly imply that pyrogallol and butylatedhydroxy toluene (BHT) inhibit the radical or scavenge the radical in a concentration dependent manner. All the selected antioxidants exhibited an inhibition ranging from 22% to 78% at a concentration of 148

10 100 µg/ ml. Pyrogallol (commercial antioxidant) showed an inhibition of 78.63% at a concentration of 100 µg/ ml. Figure 4.2: Antioxidants assay results. a) ABTS assay b) SOS assay, c) TRAP assay, d) FRAP assay. Polyphenolic compounds have an important role in stabilising lipid oxidation and are associated with antioxidant activity besides being known for their powerful chain breaking abilities [23]. The scavenging effect of commercially available antioxidants on superoxide anion free radical is shown in Figure 4.2. In the range of µg/ ml, the antioxidants showed a dose-dependent inhibition on the superoxide anion free radical. The superoxide radicals were generated in vitro by the phenazine methosulphate system. The scavenging 2- activity of the extract was determined by the NBT reduction method. In this method, O 2 reduces the yellow dye (NBT 2+ ) to produce the blue formazan, which is measured spectrophotometrically at 560 nm. Antioxidants are able to inhibit the purple NBT formation. The results are expressed as the percentage inhibition of the NBT reduction with respect to the 149

11 reaction mixture without sample. The five commercially available antioxidants exhibited inhibition ranging from 22% to 78% at a concentration of 100 µg/ ml. The antioxidant pyrogallol showed an inhibition of 78.63% at a concentration of 100 µg/ ml and butylated hydroxyl anisole showed an inhibition of 78.78% at a concentration of 100 µg/ ml, while PY and BHA exhibited almost the same results. The FRAP assay gives fast, reproducible results with plasma, with single antioxidants in pure solution and with mixtures of antioxidants in aqueous solution which are added to the plasma. The dose response characteristics of different antioxidants showed different activities, but the dose response of each individual antioxidant tested was linear, showing that activity was not concentration dependent, at least over the concentration ranges tested in this study. The dose response lines at 593 nm for 100 µg/ ml solutions of BHT, BHA, TBHQ, GA and PY are shown in Figure 4.2. This method was initially developed to assay plasma antioxidant capacity, but can be used to measure the antioxidant capacity in a wide range of biological samples and pure compounds like fruits, wines, and animal tissues [24]. The FRAP assay can be readily applied to both water and ethanol extracts of different plants. In this assay, the antioxidant activity is determined on the basis of the ability to reduce ferric (III) iron to ferrous (II) iron. The results are expressed as µmol ferrous iron equivalents per 100 g of dry weight of plant material. The five commercially available antioxidants exhibited inhibitions ranging from 8% to 67.5% at a concentration of 100 µg/ ml while TBHQ showed an inhibition of 67.5% and PY showed an inhibition of 61.9%. Most of the methods applied to biological fluids determine the total reactive antioxidant potential (TRAP) and correspond to the first category. TRAP is an indicator of the free-radical scavenging ability of an antioxidant against peroxyl radical using hydrogen atom transfer. The five selected antioxidants exhibited an inhibition ranging from 33% to 93.95% in the concentration range µg/ ml. BHT exhibited an inhibition of 93.95% and TBHQ showed an inhibition of 86.81%, both at concentrations of 100 µg/ ml. 150

12 3.2. Biodiesel storage studies The study on karanja oil methyl ester was carried out by Das et al., by using three antioxidants namely butylated hydroxy anisole, butylated hydroxy toluene and propyl gallate [20]. They varied the load level of the three antioxidants from 100 to 1000 ppm and noticed decrease in peroxide values as the concentration of antioxidants increased. Peroxide value is not listed as a parameter in the biodiesel fuel specification, though viscosity and acid value were among the specifications listed within PS121 (provisional fuel standard guideline for biodiesel) and are known to be affected by the autoxidation of biodiesel. Changes in viscosity and acid value were therefore monitored in this study, to understand the oxidative stability of Pongamia biodiesel. Dunn examined the effects of oxidation under controlled accelerated conditions on fuel properties of methylsoyate, where the author employed only TBHQ and α- tocopherol as antioxidants which were found to have beneficial effects on retarding oxidative degradation of methylsoyate biodiesel [18]. Karavalakis et al., [21] in their study evaluated the impact of biodiesel concentration in diesel fuel on the stability of the final blend. They have also discussed the effect of sulphur content in the base diesel on the oxidation stability of the blend. Results of storage studies of Pongamia and Jatropha biodiesel using commercially available antioxidants are presented in this chapter. Experiments were carried out at different storage conditions. The storage conditions employed were: 1) ordinary glass bottle with open space (OGOS), 2) ordinary glass bottle with closed space (OGCS), 3) ordinary glass bottle with closed space containing nitrogen (OGCSN), 4) amber glass bottle with open space (AGOS), 5) amber glass bottle with closed space (AGCS) and 6) amber glass bottle with closed space containing nitrogen (AGCSN). The effect of five different antioxidants on the oxidative stability and storage stability of Pongamia and Jatropha biodiesel were evaluated using standard Rancimat procedure (EN 14214) and recommended ASTM procedure D weeks. Storage in amber glass bottle with nitrogen atmosphere was found to be the best storage condition exhibiting minimum oxidation. Figure 4.3 shows the variance of kinematic viscosity, acid value and peroxide value with respect to time in the presence of nitrogen atmosphere. 151

13 Neat 100 ppm 200 ppm 500 ppm 1000 ppm 2000 ppm 3000 ppm 5000 ppm Neat 100 ppm 200 ppm 500 ppm 1000 ppm 2000 ppm 3000 ppm 5000 ppm Neat 100 ppm 200 ppm 500 ppm 1000 ppm 2000 ppm 3000 ppm 5000 ppm Kinamatic Viscosity (mm²/s) Peroxide Value (mg/kg) Acid Value (mgkoh/g) Weeks Weeks Weeks Figure. 4.3: Storage studies using pyrogallol as additive in Pongamia biodiesel 16 Neat 100 ppm 200 ppm 500 ppm 1000 ppm 2000 ppm 3000 ppm 5000 ppm 7 Neat 100 ppm 200 ppm 500 ppm 1000 ppm Neat 100 ppm 200 ppm 500 ppm 1000 ppm 2000 ppm 3000 ppm 5000 ppm Kinematic Viscosity (mm²/s) Weeks Peroxide Value (mg/kg) Weeks Acid Value (mgkoh/g) Weeks Figure. 4.4: Storage studies using pyrogallol as additive in Jatropha biodiesel Studies using various concentrations reveal that 1000 ppm is sufficient for a storage time of 25 weeks. Figure 4.3 and 4.4 shows the variance of kinematic viscosity, acid value and peroxide value with respect to time in the presence of nitrogen atmosphere. The effects of commercial antioxidants on Pongamia and Jatropha biodiesel at different atmospheric conditions and various concentrations of antioxidants were studied and are discussed below Jatropha biodiesel with BHA as additive In the absence of antioxidants (neat) JBD exhibits a KV value of mm 2 /s, PV of 9.3 mg/kg and AV of 2.47 mg KOH/g. The storage studies also reveal that in the absence of antioxidants PBD shows a KV value of 14.7 mm 2 /s, PV of 6.38 mg/kg and AV of 1.68 mg KOH/g. The results presented below are from the storage studies carried out under AGCSN conditions. All other storage conditions showed higher values of KV, PV and AV). 152

14 AO ( ppm) Neat Kinematic viscosity Peroxide value Acid value

15 Figure. 4.5: Storage stability of Jatropha biodiesel with BHA as antioxidant carried out for 25 weeks 154

16 Pongamia biodiesel with BHA as additive AO ( ppm) Neat Kinematic viscosity Peroxide value Acid value

17 Figure 4.6: Storage stability of Pongamia biodiesel with BHA as antioxidant carried out for 25 weeks 156

18 Results of storage studies of Pongamia and Jatropha biodiesel using BHA as the antioxidant are shown in Figures 4.5 and 4.6. Various storage conditions such as: 1) OGOS, 2) OGCS, 3) OGCSN, 4) AGOS, 5) AGCS and 6) AGCSN were tested. Storage in amber glass bottle with nitrogen atmosphere was found to be the best storage condition exhibiting minimum oxidation. Results reveals that JBD with 5000 ppm BHA, under nitrogen atmosphere stored in a amber glass bottle exhibited a KV of 5.21 mm 2 /s, PV of 5.42 mg/kg and AV of 0.44 mg KOH/g. The storage studies also reveal that PBD with 5000 ppm of BHA showed KV of 5.27 mm 2 /s, PV of 6.2 mg/kg and AV of 0.44 mg KOH/g. The storage stability increased when the concentration of antioxidant was increased Jatropha biodiesel with BHT as additive AO ( ppm) Neat Kinematic viscosity Peroxide value Acid value

19

20 5000 Figure.4.7: Storage stability of Jatropha biodiesel with BHT as antioxidant carried out for 25 weeks Pongamia biodiesel with BHT as additive AO ( ppm) Neat Kinematic viscosity Peroxide value Acid value

21

22 5000 Figure.4.8: Storage stability of Pongamia biodiesel with BHT as antioxidant carried out for 25 weeks Results of storage studies of Pongamia and Jatropha biodiesel using BHT as the antioxidant under various storage conditions such as, 1) OGOS, 2) OGCS, 3) OGCSN, 4) AGOS, 5) AGCS and 6) AGCSN are presented above (Figures 4.7 and 4.8). Storage in amber glass bottle with nitrogen atmosphere was found to be the best storage condition exhibiting minimum oxidation. Figure 4.7 and 4.8 show the variance of kinematic viscosity, acid value and peroxide value with respect to time in the presence of nitrogen atmosphere. JBD with 5000 ppm BHT under nitrogen atmosphere stored in a amber glass bottle exhibited a KV of 4.7 mm 2 /s, PV of 5.28 mg/kg and AV of 0.39 mg KOH/g. The study also reveals that PBD with 5000 ppm BHT exhibits KV of 5.1 mm 2 /s, PV of 6.22 mg/kg and AV of 0.41 mg KOH/g. The storage stability increased when the concentration of antioxidants was increased. 161

23 Jatropha biodiesel with GA as the additive AO (ppm) Neat Kinematic viscosity Peroxide value Acid value

24 Figure.4.9: Storage stability of Jatropha biodiesel with GA as antioxidant carried out for 25 weeks 163

25 Pongamia biodiesel with GA as additive AO (ppm) Neat Kinematic viscosity Peroxide value Acid value

26 Figure.4.10: Storage stability of Pongamia biodiesel with GA as antioxidant carried out for 25 weeks Results of storage studies of Pongamia and Jatropha biodiesel using GA as the antioxidant under various storage conditions such as, 1) OGOS, 2) OGCS, 3) OGCSN, 4) AGOS, 5) AGCS and 6) AGCSN are presented above. Storage in amber glass bottle with nitrogen atmosphere was found to be the best storage condition exhibiting minimum oxidation. 165

27 Figure 4.9 and 4.10 show the variance of kinematic viscosity, acid value and peroxide value with respect to time in the presence of nitrogen atmosphere. JBD with 5000 ppm GA, under nitrogen atmosphere stored in a amber glass bottle, exhibited a KV of 5.4 mm 2 /s, PV of 5.33 mg/kg and AV of 0.45 mg KOH/g. The storage studies also reveals that 5000 ppm of GA in PBD shows KV of 5.1 mm 2 /s, PV of 6.22 mg/kg and AV of 0.41 mg KOH/g. The storage stability increased with an increase in the concentration of GA. 166

28 Jatropha biodiesel with TBHQ as additive AO (ppm) Neat Kinematic viscosity Peroxide value Acid value

29 Figure.4.11: Storage stability of Jatropha biodiesel with TBHQ as antioxidant carried out for 25 weeks 168

30 Pongamia biodiesel with TBHQ as additive TBHQ AO (ppm) Kinematic viscosity Peroxide value Acid value Neat

31 Figure.4.12: Storage stability of Pongamia biodiesel with TBHQ as antioxidant carried out for 25 weeks 170

32 Results of storage studies of Pongamia and Jatropha biodiesel using TBHQ as the antioxidant were investigated under different storage conditions such as, 1) OGOS, 2) OGCS, 3) OGCSN, 4) AGOS, 5) AGCS and 6) AGCSN. Storage in amber glass bottle with nitrogen atmosphere was found to be the best storage condition exhibiting minimum oxidation. Figure 4.11 and 4.12 shows that JBD with 5000 ppm TBHQ, under nitrogen atmosphere stored in a amber glass bottle, exhibited a KV of 4.48 mm 2 /s, PV of 5.28 mg/kg and AV of 0.40 mg KOH/g. PBD with 5000 ppm TBHQ under similar conditions shows KV of 4.98 mm 2 /s, PV of 6.12 mg/kg and AV of 0.38 mg KOH/g. The storage stability increased when the concentration of TBHQ was increased. 171

33 Jatropha biodiesel with PY as additive AO (ppm) Kinematic viscosity Peroxide value Acid value Neat

34

35 5000 Figure.4.13: Storage stability of Jatropha biodiesel with PY as antioxidant carried out for 25 weeks Pongamia biodiesel with PY as additive AO (ppm) Kinematic viscosity Peroxide value Acid value Neat

36

37 Figure.4.14: Storage stability of Pongamia biodiesel with PY as antioxidant carried out for 25 weeks Results of storage studies of Pongamia and Jatropha biodiesel using PY as the antioxidant were investigated under different storage conditions such as, 1) OGOS, 2) OGCS, 3) OGCSN, 4) AGOS, 5) AGCS and 6) AGCSN. Storage in amber glass bottle with nitrogen atmosphere was found to be the best storage condition exhibiting minimum oxidation. Figure 4.13 and 4.14 reveals that JBD with 5000 ppm PY, under nitrogen atmosphere stored in a amber glass bottle, exhibited a KV of 4.57 mm 2 /s, PV of 5.23 mg/kg and AV of 0.38 mg KOH/g. The storage studies reveal that PBD with 5000 ppm PY shows KV of 4.75 mm 2 /s, PV of 5.23 mg/kg and AV of 0.38 mg KOH/g. The storage stability increased when the concentration of antioxidants was increased. 176

38 3.3. Oxidative stability studies of Pongamia and Jatropha biodiesel using commercially available antioxidants Rancimat method, the most commonly employed method in the vegetable oil sector, was adopted for the determination of oxidation stability. A high content of unsaturated fatty acids, which is very sensitive to oxidative degradation, leads to decreased induction times. Thus, even the conditions of fuel storage directly affect the quality of product. Several studies showed that the quality of biodiesel over a longer period of storage strongly depends on the tank material as well as on contact to air and light. Increase in viscosity and acid values leads to decrease in induction periods [11, 16]. The oxidative stability of biodiesel was studied by the Rancimat method as per EN14112 methodology using commercially available antioxidants. All the other antioxidants like BHA, BHT, GA, and TBHQ were also used in the studies with concentrations ranging from 100 ppm to 5000 ppm under various atmospheric conditions. The different storage conditions are ordinary glass bottle with open space, ordinary glass bottle with closed space, ordinary glass bottle with nitrogen atmosphere, amber coloured bottle with open space, amber coloured bottle with closed space and amber coloured bottle with closed space containing nitrogen atmosphere. 177

39 178

40 Figure 4.15: Oxidative stability results on Pongamia and Jatropha at different atmospheric conditions and different commercially available antioxidants. When investigating phenolic antioxidants, it was found that their anti-oxidative capabilities bear a relationship to the number of phenol groups occupying 1, 2 or 1, 4 positions in an aromatic ring, as well as to the volume and electronic characteristics of the ring substituent present. Generally, the active hydroxyl group can provide protons that inhibit the formation of free radicals or interrupt the propagation of free radical and thus delay the rate of oxidation. The effectiveness of PY, TBHQ, GA, can be explained based on their molecular structure (Figure 4.15). These additives possess two OH groups attached to the aromatic ring, while both BHT and BHA possess one OH group attached to the aromatic ring. Thus, based on their electro-negativities, TBHQ and PY offer more sites for the formation of a complex between free radical and antioxidant radical for the stabilization of the ester chain. Another contributing factor for the poor antioxidant performance of both BHT and BHA is their relatively low volatility, which under the operating conditions of the Rancimat method will lead to loss of additives during the early part of the test [18]. 179

41 Antioxidant concentration (1000 ppm) Figure. 4.16: Oxidative stability sudies on PBD and JBD at 1000 available antioxidants. ppm commercially Figure 4.16 shows that the pyrogallal was found to be the best antioxidant among those commercially available, with a induction period of 3.51 h at a concentration of 1000 ppm, h at a concentration of 5000 ppm, and h at concentration of 5000 ppm for both Pongamia and Jatropha biodiesel. 180

42 4. Conclusion The commercially available antioxidants which were studied, exhibit good radical scavenging activity (for a concentration of 100 µg/ ml) as shown in Table 4.2. Compound SOS (%) ABTS (%) FRAP (%) TRAP (%) TBHQ PY BHT BHA GA Table 4.2: Results of radical scavenging assay at a concentration of 100 µg/ ml Biodiesel samples stored in amber glass bottles with closed space containing nitrogen gave the best results as compared to the other 5 conditions tested. Pyrogallol was found to be the best commercial antioxidant for both Pongamia and Jatropha biodiesels as it increased the induction time from 0.05 h to 4 h, at 1000 ppm. Kinematic viscosity and acid values are good indicators of storage stability of biodiesels. BHT, TBHQ, PY, GA, and BHA, at concentrations of 1000 ppm, improved the storage stability of biodiesel. 181

43 REFERENCES 1. Oyaizu. Food Res. Int. 2008, 41, Dunn, R. O. J. Am. Oil Chem. Soc. 2005, 82, Lacoste, F.; Lagardere, L. Eur. J. Lipid Sci. Technol. 2003, 105, Schober, S.; Mittelbach, M. Eur. J. Lipid Sci. Technol. 2004, 106, Mittelbach, M.; Schober, S. J. Am. Oil Chem. Soc. 2003, 80, Schober, S.; Mittelbach, M. Eur. J. Lipid Sci. Technol. 2004, 106, Dunn, O. Fuel Process. Technol. 2005, 86, Ryu, K. Biores. Technol. 2010, 101, Tang, H.; Guzman, R. C. D..; Salley, S. O.; Simon, N. G. K. Y. Lipid Technol. 2008, 20, Bouaid, A.; Martinez, M.; Aracil, J. Biores. Technol. 2009, 100, McCormick, R. L.; Westbrook, S. R. Energy Fuels 2010, 24, Evans, R. ABTS free radical scavenging modified method Cui, Y. J. Ethno pharm. 2005, 96, Selvakumar, K.; Madhan, R.; Srinivasan, G.; Baskar, V. As. J. Pharm. Tech. 2011; Vol. 1: Issue 4, Pg Mittelbach, M.; Gangl, S. J. Am. Oil Chem. Soc. 2001, 78, Bondioli, P.; Gasparoli, A.; Lanzani, A.; Fedeli, E.; Veronese, S.; Sala, M. J. Am. Oil Chem. Soc. 1995, 72, Simkovsky, N. M.; Ecker, A. Erdöl Erdgas Kohle 1999; 115: Dunn, R. J. Am. Oil Chem. Soc. 2002, 79, Canakci, M.; Monyem, A.; Gerpen, J. V. Trans. ASAE 1999, 42, Das, L. M.; Bora, D. K.; Pradhan, S.; Naik, M. K.; Naik, S. N. Fuel 2009, 8, Karavalakis, G.; Hilari, D.; Givalou, L.; Karonis, D.; Stournas, S. Energy 2011, 36, Knothe, G. Eur. J. Lipid Sci. Technol. 2006, 108,

44 23. Wanasundara, P. K. J. P. D.; Shahidi, F. Antioxidants: Science, Technology, and Applications. Bailey s Industrial Oil and Fat Products, Sixth Edition; Sixth Volume: Edited by Fereidoon Shahidi. 24. Enayat, S.; Banerjee, S. Food Chemistry, 2009, 116,