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 Riedel-de-Haën, Germany. Silicagel HF₂₅₄, Merck Ref. 7739 was used for TLC and standards are product of BDH, UK. The BF₃-methanol complex (Merck-Schuchardt, Germany) was used for fatty acids analysis through GC. The fatty acids’ methyl esters (C_12-C₂₄) are attained from Supelco^®,USA for GC analysis. The colouring reagent; 2,7-dichlorofluorescein (Merck, Germany) used to identify components under UV light; λ 366 nm.

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; C₁₂-C₂₄ under the same conditions 18.

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.

The lipids were extracted through cyclic solvent extraction (Soxhlet apparatus) by using solvents; n-hexane, 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. 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.

Experiments were carried out to ascertain the methanol-oil 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, NaOCH₃, KOH, KOCH₃ were used. NaOCH₃ 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.

The 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 CSOR-FAME.

The chromatographic techniques; gas chromatography (GC) 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 HF₂₅₄) 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 2,7-dichlorofluorescein used for coloured spots of esters & glycerides under ultra violet light; λ 366 nm.

After optimization of reaction parameters, the experiment was carried out to produce biodiesel by using NaOCH₃ (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 Na₂SO₄ 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

The fuel properties of CSOR-FAME ie; acid value, density, kinematic viscosity at 40°C, oxidative stability, lubricity, cloud point, pour point, cold filter plugging point, flash point, ash content, copper strip corrosion test, ester contents, free glycerine, total glycerine, mono, di and tri glyceride contents were determined according to American Standards for Testing Materials (ASTM) and European Standard (EN). Each experiment was conducted in triplicate and data are reported as mean ± standard deviation (SD).

The efficiency of different basic catalysts is illustrated in the Figure 2, KOH showed least amount of CSOR-FAME yield, while highest yield of esters is attained by using CH₃ONa. Sodium & Potassium methaoxides exhibited higher yields as compare to hydroxides as shown in Figure 3, the lower yield of esters by using hydroxide catalysts is may be due to formation of water which leads to hydrolysis of esters & formation of soaps 21

Six experiments were conducted by varying the sodium methaoxide concentrations; 0.25, 0.50, 0.75, 1.00, 1.25 & 1.50 % (oil weight basis). The effect of NaOCH₃ concentration on ester formation is shown in 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. The conversion did not increase by increasing the catalyst amount beyond 1.00 % as well to obtain a fuel meeting biodiesel standards. It was observed that with the increase in catalyst concentration above 1.00 %, the yields were lower. The overload of sodium methaoxide emulsified the product. With the increase in the concentration of catalyst above 1.00 % there was decrease in the yield of methyl esters. This decline in ester yield might be attributed to the increased formation of glycerol & soap. The current work agree with the results that the formation of soap in the presence of higher amount of catalysts increases the viscosity of the reactants thus lowers the ester yield 2223. The addition of an excessive amount of alkaline catalyst causes formation of emulsions, increasing viscosity & making recovery of the methyl esters difficult 2425. An optimal catalyst concentration is required for successful transesterification, in the present case it is determined to be 1.00 % sodium methaoxide. However, it is found that potassium hydroxide (1.0 %) offered the best yield during the methanolysis of Pongamiapinnata oil 26 & used frying oil 27.

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 1230which 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.

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 3233 findings (65°C) are also same for production of biofuels direct from oils of Cynara Cardunculus L and rape seed respectively.

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 NaOCH₃ catalyst (1.00 %) were used. The Figure 6showed 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.

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 Table 4. The recovered oil contains saturated fatty acids (23.8 %), monounsaturated fatty acids (21.2 %) and polyunsaturated fatty acids (54.9 %). The CSOR-FAME were purified with solvent extraction. The impurities had deleterious effects on the fuel properties, especially with regard to diminished temperature performance, manifested by increased cloud, pour, & cold filter plugging points. The important fuel properties; acid value, density, kinematic viscosity (40°C), oxidative stability, lubricity, cloud point, pour point, cold filter plugging point, flash point, ash content, copper strip corrosion of CSOR-FAME as measured according to accepted ASTM methods are depicted in Table 5. In addition to mentioned properties the ester contents, free glycerine, total glycerine, mono, di & tri glyceride contents were also determined. Although CSO contains higher contents of triglycerides (92.5%) as compare to triglycerides of CSOR (88.3%) but lower contents of mono & diglycerides are present in CSO as shown in table 3. Therefore, higher yield of esters have been found out by the optimised transesterification of CSOR as shown in table 5. The properties of biodiesel depend heavily on its raw materials 35, however, properties of produced CSOR-FAME investigated in this study also satisfied nearly all prescribed ASTM D 6751 & EN 14214 specifications. Therefore the indigenous ABC, abundantly used for processing of CSO have appreciable contents of fatty acids, after certain optimized processing the product is economical & ecofriendly source of energy.

ABC:

Activated bleaching clay

AOCS:

American Oil Chemists’ Society, Official methods

ASTM:

American standard for testing materials

CSO:

Cottonseed oil

CSOR:

Cotton seed oil recovered

CSOR-FAME:

Cotton seed recovered oil-fatty acid methyl esters (biodiesel)

FFA:

Free fatty acid value

GC:

Gas chromatography

SD:

Standard deviation

TLC:

Thin layer chromatography

WBC:

Waste bleaching clay