Waste Clay for Biodiesel through Base Catalyzed Transesterification of Residual Cotton Seed Oil

After processing of cotton seed oil (CSO) the activated bleaching clay (ABC) is converted to low valued waste bleaching clay (WBC). The chemical composition of ABC from Mirpur, Azad Kashmir region of Pakistan is found out as; SiO2 71.34, Al2O3 15.54, CaO 2.72, MgO 1.48, Na2O 0.51, K2O 0.23, Fe2O3 0.02. The clay has bleachability (74 %) and oil retention (32.70 %) by standard method (ASTM). The cotton seed oil recovered (CSOR) with polar/nonpolar solvents (31.1-36.3 %) have different FFA values (0.2-0.85). However, lower percentage of tri-esters (88 %) was found out in dark coloured CSOR as compare to freshly n-hexane extracted CSO (92.5 %) from edible oil refinery. The lower FFA (0.2) valued CSOR with n-hexane is transesterified at optimized conditions to mono alkyl esters (CSOR-FAME). The reaction was optimised by performing series of experiments to observe molar conc., of methanol-oil (3-18:1), catalysts; NaOH, NaOCH3, KOH, KOCH3oil (0.25-1.5), temperature (20-80°C), reflux time (120 min) and mixing intensity (200-650 rpm). The maximum yield of biodiesel (98.5 %) has been found out by NaOCH3 (1.00 %) as catalyst, methanol-oil (6:1) at temperature (65°C) and stirring intensity (650 rpm). The properties of CSOR-FAME (biodiesel) are also under limits as per standards; ASTM 6751, EN 14214 and WBC/ CSOR-FAME appear to be an acceptable feedstock for fatty acids/biodiesel production as renewable fuel. DOI : COMING SOON Corresponding author: Zeeshan Ali, Complex, Shahrah-EJalal-ud-Din Roomi Road, Lahore-54600, Pakistan., Ph (off): 0092 42 9230688-95 / Ext-323, Email: drzeeshan2009@yahoo.com

The utilization of waste materials and byproducts is very important to fulfill demands of quality products and to substitute depleting resources of the world.The hydrocarbons of fossils being conventional source of fuels are continuously decreasing; the fluctuating mineral oil prices and environmental impacts have intensified the search for alternate fuels.Biodiesel is defined as the mono-alkyl esters of vegetable oils or animal fats [1], biofuels has attracted a great deal of interest during the past decade as a renewable, biodegradable, non-toxic and eco-friendly clean fuel.
Biodiesel is found to be the best substitute of petrodiesel fuel not only for its comparable calorific value but also for its several other advantages such as biodegradability, low toxic emission, higher flash point, excellent lubricity, carbon neutral, & environmentally acceptance fuel [2].The oils/fats are found to be the best candidates as an alternate and ecofriendly energy source.Oils/fats are not only used for edible purposes but varieties of industrial products are also developed from this commodity.However, the use of edible sources comes under heavy criticism since fuel-for food concept, which is regarded as an unethical by many quarters.Alternatively, the use of waste edible oil could be a better solution as it is available in abundant [3] Triglyceride for biodiesel production comes from various sources; edible oil, inedible oil, waste/used oils, animal fats [4] and also from microorganisms [5].The waste bleaching clay (WBC) is used for oil processing is being identified for ecofriendly and renewable fuel; it is an important byproduct of oil processing industries.

The consumption of edible oils/fats only in
Pakistan is 2500 mmt, about 565 mmt or 23 % of oils/ fats requirements is met through oil seed cultivation and animal resources [6].The cotton is a major crop of the country, cottonseed is the by product and cottonseed oil (CSO) is extracted from the seeds of the cotton plant after the removal of cotton lint.The country contributes about 10 % of the total CSO produced all over the world [7].Cotton (Gossypium hirsutum L.), belonging to the Malvaceae family is an important crop that yields the natural fibre used by the textile industry.It is one of the second best potential sources for plant proteins after soybean and ninth best oil-producing crop [8].The locally available bleaching clay is frequently used to process cotton seed oil to meet the standards for edible usage.The activated bleaching clay (ABC) is used to remove colouring matters, soap, gums, metals (iron, nickel), oxidized compounds and polymers.This substance consists primarily of hydrated aluminium silicate, reports indicated that bleaching clays retains 20-40 % of oil/fat and importantly, the adsorbed oil represents the major part of bleaching cost as reported by Ong [9].After processing of CSO, large quantities of bleaching clays are disposed off in landfills, causes pollution hazard and the retained organics are wasted.
The direct use of oils/fats as fuel is also limited due to two main reasons; high viscosity and low volatility [2].Therefore, oils/fats are chemically reacted with alcohols (transesterification reaction) to produce fatty acid alkyl esters/biodiesel [11].The resulting product not only contains alkyl esters but also unreacted starting material, residual alcohol and residual catalyst [2].The optimization of transesterification reaction depends upon; catalyst type & concentration, methanoloil molar ratio, reaction temperature, reaction time, stirring intensity, FFA (free fatty acid value) and moisture contents in oils and fats [12].Mostly, transesterification is being carried out by alkaline catalysts in homogeneous phase i.e., NaOH, KOH, NaOCH 3 and KOCH 3 [13][14].The transesterification is also reported by enzymatic esterification of CSO [15].However, no work has been carried out to study the base catalyzed transesterification of waste cotton seed oil from processed BC.Keeping in view, the consumption of fixed oils/fatty acids for production of edible oils, oleochemicals and allied products, the thorough investigation regarding base catalysed transesterification of cottonseed oil recovered (CSOR) from WBC has been carried out.The work includes the reaction parametric studies and produced fatty acids methyl esters (FAME) "biodiesel" (CSOR-FAME) properties.

Evaluation of Freshly Extracted CSO
The analysis of freshly extracted CSO i.e., FFA value, saponification value, iodine value, peroxide value, unsaponifiables was carried out by official methods of American Oil Chemist's Society (AOCS) [16].The glyceride components & fatty acids analysis were carried out by GC and TLC [17] The gas chromatograph; GC-14A & data processor C-R-4A was used for the identification of methyl esters by using a polar column (2.5 m × 3 mm id), coating material GP-10%-SP-2330 on supporting media 100-120 chromosorb WAW.The FID detector was used with requisite temperature of detector and injector; 250°C and 230°C respectively.It was operated under temperature programming 180-210°C at the rate of 4°C/minute & Nitrogen flow rate of 30 mL/minute.The fatty acid methyl esters were identified by the comparison of their corresponding retention times with standard methyl esters of fatty acids; C 12 -C 24 under the same conditions [18].

Evaluation of ABC
The analysis of ABC ie; compositional analysis, bleachability, moisture contents, oil filtration rate, oil retention and bulk density was carried out by standard methods [19].

Extraction of Oil from WBC & Evaluation of CSOR
The lipids were extracted through cyclic solvent extraction (Soxhlet apparatus) by using solvents; nhexane, methanol, ethanol, petroleum ether & their combinations with repeated extractions.The solvent was removed under vacuum by rotary film evaporator (Hëidolph, Germany).The analysis of CSOR i.e., free fatty acid value (FFA), saponification value, iodine value, per oxide value, unsaponifiables was carried out [16].Analysis of Biodiesel

Results and Discussion
The indigenous BC belonging to Azad Kashmir region of Pakistan is frequently processed to produce ABC.The chemical composition and processing technology relates to bleaching quality of ABC.The ABC is analyzed by standard methods [19] as shown in  The stoichiometrically the transesterification reaction requires 3 mol of alcohol for 1 mol of triglyceride to yield 3 mol of methyl esters & 1 mol of glycerol.The transesterification is a reversible/ equilibrium reaction.Therefore an excess methanol is required for successful completion of reaction.The molar ratios of methanol to oil; 3:1, 6:1, 9:1, 12:1, 15:1 & 18:1 have been employed to determine the effects of excess methanol quantities.The yields of methyl esters with respect to time at different molar ratio of methanol-oil from 3:1 up to the level of 18:1 exhibited encouraging effects on the yield of esters (Figure -4).The higher molar ratio then the stoichiometeric value resulted in a greater ester formation [28] & could ensure complete reaction.The limited effect on the ester yield was found with the increase in molar ratio of methanol to oil.On the other h&, the reaction was also incomplete for a molar ratio less then 6:1.It has been shown that beyond the molar ratio of 6;1, further methanol addition had no considerable effect on ester formation.Excess amount of catalyst also complicate ester recovery and raised process cost [29].In case of  molar ratios greater then 6:1, a dilution effect is the likely cause while for molar ratios less then 6:1, insufficient mixing of the reactants in the biphasic transesterification reaction system is the likely cause.
The current results of optimum yield (98.5 %) of CSOR-FAME with oil/ methanol molar ratio of 1:6 are in agreement with work carried out by Freedman etal and Usta N etal [12,30] which reported optimum conversion of various vegetable oils and tobacoo seed oil in to their corresponding esters with a molar ratio of 1:6.So the unwanted impurities retained by ABC during processing of cotton seed oils have least effects for their conversions to methy esters.
Optimization of Reaction Temperature.
The transesterification of CSOR were studied at different temperatures i.e., 20, 35, 50, 65 & 80°C.The reaction time of 120 min was constant for each experiment.The Sodium methaoxide (1.0 %) as optimized in the previous section, methanol/oil molar ratio (6:1) & rate of stirring 650 rpm were applied for each experiment at different temperatures.The Figure-5 shows that 85 % ester yield was achieved in just 15 min.After 120 min, the reaction was completed & esters yields; 89.0, 95.5, 98.5 & 98.7 % were found out at 20, 35, 50, 65 and 80°C respectively.The lower ester yields can not be up to the standards due to higher contents of glycerides & fatty acids.The temperature optimization 65°C for conversion of recovered cotton seed oil to esters results are in accordance to the work of Mehr etal [31] regarding optimization of alkali-catalyzed transesterification of Pongamia Pinnata seed oil for production of biodiesel.Encinar etal and Karaosmanoglu etal [32][33] findings (65°C) are also same for production of biofuels direct from oils of Cynara Cardunculus L and rape seed respectively.

Optimization of Rate of Stirring
The effect of stirring on CSOR-FAME production was investigated by performing four experiments at different stirring rates (200, 350, 500 & 650 rpm).In all experiments, an oil/methanol molar ratio of 1:6, a reaction temperature of 65° C, & a NaOCH 3 catalyst (1.00 %) were used.The Figure-6 showed direct correlation between the stirring rate & ester yield; i.e., as the rate of agitation was increased, an increase in yield was observed.Accordingly, a mixing rate of 650 rpm afforded the optimum conversion of CSOR to CSOR-FAME (98.5%).The different stirring rates concluded that elevated speeds promoted the homogenization of reactants, leading to higher methyl ester yields.This is in accordance with earlier studies [34].

Quality of CSOR-FAME (Biodiesel)
The nature of fatty acids plays an important role on the qualities of biodiesel, the fatty acids were analyzed by GC.The major fatty acid is linoleic acid (53.8 %), followed by oleic acid (20.6 %) while palmitic acid (19.5 %) is the predominant saturated acid as shown under

Vol- 1
Issue 1 Pg.no.-2 Introduction : Activated bleaching clay (ABC) of Azad Kashmir (Pakistan) region and waste bleaching clay (WBC) are attained from Hamza vegetable oil refineries, Lahore.Cotton seeds (CS) belongs to Punjab region (Pakistan).Solvents/reagents used are of analytical-grade, mostly purchased from Merck-Darmstadt, Germany and Riedelde-Haën, Germany.Silicagel HF 254 , Merck Ref. 7739 was used for TLC and standards are product of BDH, UK.The

CSOR
TLC used to determine the lipid components & extent of transesterification reactions.The thin layer chromatography (TLC-20 x 20 cm) of 0.5 mm thickness were prepared by coating silicagel for the separation/ identification of lipid components.The solvent system used for the fractionation of lipids components was; hexane-diethylether-acetic acid (80/20/2) & for monoalkyl esters; hexane-diethylether (80/20).The locating agent 2,7-dichlorofluorescein was used, which gave purple yellow colored bands under an ultraviolet light at λ ; 366 nm.Experimental Procedure Experiments were carried out to ascertain the methanoloil molar ratio, catalyst type & concentration, reaction temperature & agitation intensity on transesterification reaction.The reaction time (120 min) was kept constant throughout experimental studies.The molar ratio of methanol-oil was varied as 3:1, 6:1, 9:1, 12:1, 15:1 & 18:1.The catalysts; NaOH, NaOCH 3 , KOH, KOCH 3 were used.NaOCH 3 was found the most efficient catalyst so its concentrations were varied; 0.00, 0.25, 0.50, 0.75, 1.00, 1.25 & 1.50 % to found optimum concentration.The employed temperatures were; 20, 35, 50, 65 & 80°C .Agitation rates were; 200, 350, 500 & 650 rpm.Ttransesterification of CSORThe chemical reaction was carried out using a 500 mL round-bottomed flask, equipped with thermostat, sampling outlet, mechanical stirrer (Eyela Tokyo, Japan) with tachometer reading 15 × 10 rpm DCM & reflux condensation systems.The CSO (250 g) was preheated to the set temperatures; 20, 35, 50, 65 & 80°C.The fixed amounts of freshly prepared methanolic solutions of catalysts; sodium hydroxide, potassium hydroxide, sodium methaoxide & potassium methaoxide were mixed with oil & considered the time; 0.00 mint.After consistent intervals 2 mL of sample was withdrawn for chromatographic analysis.All the experiments were conducted for maximum of 120 min in order to ensure the complete esterification of CSOR into methyl esters ) and TLC were frequently used for the analysis of CSOR-FAME.The GC details & conditions have already mentioned in section 2.2.However, for TLC, the chromatograms (20×20 cm) of thickness (0.50 mm) & adsorbent (Silicagel HF 254 ) were prepared by the use of Quickfit TLC applicator.The eluting solvent mix; n -hexane-diethylether (10:90) was used to fractionate & identify methyl esters/ glycerides in order to ensure the complete conversion of the CSOR into methyl esters (CSOR-FAME).The non-destructive locating reagent 2,7dichlorofluorescein used for coloured spots of esters & glycerides under ultra violet light; λ 366 nm.Production, Separation & Purification of CSOR-FAME (biodiesel) After optimization of reaction parameters, the experiment was carried out to produce biodiesel by using NaOCH 3 (1 % w/w), methanol-oil (6:1 w/w), temperature (65°C) & stirring intensity (650 rpm) for maximum yield of biodiesel.On achieving the maximum yield of alkyl esters, the reaction stopped & the excess methanol was recovered with rotary film evaporator (Hëidolph, Germany) at 50°C under vacuum.The residue transferred to separating funnel & washed with water (50-55°C) & n-hexane.After, some time the mixture is cooled down & two phases are separated.The upper phase consisted of CSOR-FAME while the lower phase contained the glycerol with other materials (methanol, catalyst, soaps, and some entrained methyl esters and partial glycerides).After separation of the two layers, the upper methyl esters layer was purified by removing residual methanol at 50°C by evaporator under vacuum.The remaining catalyst, methanol and glycerol were removed by successive rinses with distilled water.The residual water was removed by drying with anhydrous Na 2 SO 4 and filtered.The lower glycerol containing phase was acidified with a calculated amount of sulphuric acid, to neutralize any unreacted sodium methoxide and to decompose soaps formed during transesterification reaction.The mixture obtained was subjected to distillation at 65°C under a moderate vacuum to recover the excess methanol.This assisted the separation of glycerol from entrained methyl esters and soaps.The biodiesel yield was determined as: CSOR -FAME (%) = FAME (g)/CSOR (g) × 100

Figure- 3 .
Figure-3.The best yields were offered at concentration of 1.00 %.The lowest concentration of sodium methaoxide i.e., 0.25 % was not effective to catalyze the reaction for maximum yield of products.

Table 1 ,
the prominent chemical components of clay structure, mesh size, physical parameters, activation process of clay and nature of oil.The bleaching process, impurities and retention time leads to the quantity and quality of recovered cotton seed oil.The cotton seed oil CSO are recovered from WBC with polar and non polar solvents for maximum oil yield as shown in Table-2, the accumulated oil yields (37.2-39.8%) with combination of solvents are higher as compare to usuall n-hexane extractions at refineries due to different conditions of process in the oil refineries.The colour of polar extractions was also darker (brown) than that of the non -polar extractions.The polar solvents yielded more oil as compare to non-polar due to higher solubility of impurities and unwanted materials.The oil yields extracted with different solvents with respect to solvent cycles and FFA of CSOR are summarized in Figure-1.proceeding work.The CS yield oil near to WBE (35 %) as shown in Table 1, there is also a little difference in bulk densities of cotton seeds and WBE.So, WBE have almost comparable potential of oil with cotton seeds.The WBE is ignored due to more impurities, dark in colour and limited/nonfesible technology for the recovery of edible oil.But it is equally important as a source of fatty acids required for biodiesel production.The physicochemical values and glycerides components determined by TLC are also comparable as shown in Table-3 and Figure-2, except difference in

Table - 2: Extraction of residual cottonseed oil from WBC with different solvents/combinations *
percentage based on clay weights and all the extracts were collected after 24 cycles.clay from 1 st extraction is subjected to 2 nd extraction with the listed solvents b deoiled, dried