**2. Materials and methods**

#### **2.1 Sample preparation**

230 Biodiesel – Feedstocks and Processing Technologies

JCL oil is obtained only after going through the following steps: collection of fruit from the trees, separation of seed from the hull, seed drying (Chew, 2009), oil pressing and filtration. Pressing oil from the kernel yields kernel cake (40-50%) and crude oil (50-60%). At present, in the majority of cases oil is generally pressed directly from the seed without separating the kernel and shell. This method produces seed cake (70-75%) and crude oil (25-30%) (Chew, 2009). Much of the un-extractable oil still remains in the seed cake; hence better ways of extracting the oils are needed. Among the extraction techniques reported in the literature include the use of Soxhlet extraction method (Castro and Ayuso, 1998; Ayuso and Castro, 1999; Szentmihalyi et al., 2002; Darcia and Castro, 2004), aqueous enzymatic oil extraction (Rosenthal et al., 1996; Sharma and Gupta, 2006; Jiang et al., 2010) and enzyme assisted three phase partitioning (Shah et al., 2004; Gaur et al., 2007). Some of these extraction methods, however, required a longer extraction time (Chew, 2009). Nowadays, many researchers (Papamichail et al., 2000; King et al., 2001; Cao and Ito 2003; Machmudah et al., 2008) turns to supercritical extraction techniques which is relatively rapid because of the low viscosities

Transesterification is the general term used to describe the important class of organic reactions where an ester is transformed into another ester through interchange of the alkoxy moiety. Several aspects, including the type of catalyst (alkaline, acid or enzyme), alcohol/vegetable oil molar ratio, temperature, purity of the reactants (mainly water content) and free fatty acid content have an influence on the course of the transesterification. In the conventional transesterification of fats and vegetable oils for biodiesel production, free fatty acid and water always produce negative effects, since the presence of free fatty acids and water causes soap formation, consumes catalyst and reduces catalyst effectiveness, all of which result in a low conversion (Demirbas, 2007). In addition to that, more catalyst is required to neutralize free fatty acids of oil with higher free fatty acids content (Kusdiana and Saka, 2004). Thus, the catalytic processes have a high production cost and are energy intensive. One primary problem is due to the vigorous stirring required for the mixing of the two-phase mixture of oil and alcohol. Another problem is the separation of catalyst after the reaction (Madras et al.,

Supercritical fluid extraction using polar solvent such as methanol as an extraction solvent is highly potential extraction technique to be used whereby high yield of oil can be achieved within a shorter time (Hawash et al., 2009). Further, at supercritical state, the solvent solubility increased dramatically, and the extracted oil is relatively low in impurities (Tan et al., 2009). However, there is no details on the maximum crude biodiesel yield can be obtained related to

In situ transesterification differs from the conventional reaction in the sense that the oilbearing material contacts acidified alcohol directly instead of reacting with purified oil and alcohol. That is, extraction and transesterification of the seed powder proceed within the same process, with alcohol acts as an extracting solvent as well as esterification reagent (Fukuda et. al., 2001). In situ transesterification (Harrington and Evans, 1985; Marinkovic and Tomasevic, 1998; Kildiran et al., 1996; Hass et al., 2004), a biodiesel production method that utilizes the original agricultural products instead of purified oil as the source of triglycerides for direct transesterification, eliminates the costly hexane extraction process and works with virtually any lipid-bearing material. It could reduce the long production system associated with pre-extracted oil and maximize alkyl ester yield. The use of reagents and solvents is reduced, and the concern about waste disposal is avoided. This process reduces the cost of final product as this process has less number of unit operations. It is the best non-renewable source of energy with good environmental impact and easy recovery.

and high diffusivities associated with supercritical fluids.

2004). Therefore, non-catalytic transesterification has been investigated.

the in-situ supercritical methanol transesterification direct from the seeds.

The *Jatropha curcas* L. (JCL) fruits were obtained with cooperation from the Plantation Unit of Universiti Teknologi MARA Perlis, Malaysia. JCL fruits were cleaned and de-hulled to separate the hull from the seeds. The seeds were then dried in an oven at 105 °C for 35 min (Akbar et al., 2009). The JCL seeds were ground using grinder and sieved through progressively finer screen to obtain particle sizes (*dp*) of < 1 mm (Augustus et al., 2002). Sieving was accomplished by shaking the JCL powder in a Endecotts Shaker Model EFL2 for about 30 min and finally stored in a tightly-capped plastic container. The seeds need to be dried and ground in order to remove surface moisture content to obtain constant weight and weaken or rupture the cell walls to release oil for extraction, respectively (Akpan, 2006).

#### **2.2 In-situ supercritical methanol transesterification**

A batch type reactor at supercritical methanol was used for in-situ supercritical methanol transesterification of biodiesel from JCL seeds. The in-situ transesterification was carried out at temperatures and pressures ranging from 180 – 300 °C and 6 – 18 MPa, respectively. After a leak-check test, the reactor was pressurized with nitrogen to the desired pressure and heated to reaction temperature at a rate of 5 °C/min. After reaching desired temperature, the reaction was held for periods of 5 – 35 min. A JCL seeds-to-methanol ratio (1:15, 1:20, 1:30, 1:40 and 1:45 w/v) was also investigated. After each reaction, the vessel was removed from the heater and placed into a cold water bath to quench the reaction and depressurized to ambient pressure. The extracted product was discharged from the reactor and was vacuum-filtered on a Buchner funnel and the filter cake was washed with methanol. The extracted products from the in-situ transesterification were allowed to settle and separated into two phases in 500 ml separating funnel. It took about 30 min to separate into two phases, i.e., the top phase consists of the biodiesel (fatty acid methyl ester) and the lower phase consists of the glycerol and other minor components. The schematic diagram of the experimental apparatus of the batch-wise extraction system is shown in Fig. 1.

Fig. 1. Schematic diagram of batch-wise extraction system.

Production of Biodiesel Via In-Situ Supercritical Methanol Transesterification 233

**180 200 240 280 300**

**Temperature (°C) Total FAMEs Methyl palmitate Methyl oleate Methyl linoleate Methyl stearate Others** Fig. 2. In-situ supercritical methanol transesterification results from JCL seeds at various

From Table 1, the results indicate that the percent of FAMEs yields obtained at temperatures of 180 to 300 °C were in the range of 63.9 – 97.9%. The saturated FAMEs content of the seed samples are low, which is between 10.3 – 18.1% for methyl palmitate and 3.6 – 7.1% for methyl stearate. Meanwhile, the content of unsaturated FAMEs, methyl oleate and methyl linoleate are considerably higher at 27.9 – 39.5% and 22.1 – 33.2%, respectively. It should be noted that the critical temperature of methanol is at 240 °C and therefore, the conditions at 180 – 200 °C, 240 and >240 – 300 °C represent subcritical, supercritical and postcritical states

At 180 °C, which is the lowest temperature of investigation, low yields of FAMEs (63.9%) were obtained. This observation might be due to the subcritical state of methanol or the instability of the supercritical state of methanol. It was observed that by increasing the reaction temperature to supercritical conditions had a favorable influence on the yield of ester conversion (Demirbas, 2008). Similar results have been reported by Cao et al., (2005), Madras et al., (2004)

Apparently, by increasing the reaction temperature from 200 to 280 °C, the conversion increases significantly with FAMEs yields increased from 76.0 – 97.9%. The higher conversions observed in the supercritical state can be attributed to the formation of a single phase between alcohol and oil (Madras et al., 2004). Under supercritical conditions, the solubility parameter of alcohol reduces and was close to the solubility parameter of oil (Han et al., 2005). According to Petchmala et al., (2008), the increase in temperature causes the polarity of methanol to decrease, as a result of the breakdown of the hydrogen bonding of methanol, leading to an increased in the solubility of fatty acids in methanol. The complete solubility occurs as the temperature approaches the mixture critical temperature, at which point the reaction mixture became homogeneous and reaction took place rapidly. In addition, higher temperature contributed to the decomposition of cell walls, and as a result

At 300 °C, the percent of FAMEs (90.9%) yields were slightly decreased. This observation was due to the decomposition of polyunsaturated methyl esters and unreacted triglycerides in postcritical methanol at severe high temperature (Tan et al., 2009). This finding was further supported by Xin et al., (2008) who suggested that the favorable reaction temperature adopted

and Bunyakiat et al., (2006) on soybean oil, sunflower oil and coconut oil, respectively.

of the medium, respectively.

temperatures on percent of FAMEs yield and its contents.

crude biodiesel yield was increased (Machmudah et al., 2007).

**Yield (%)**

#### **2.3 FAMEs analysis**

The FAMEs analysis was quantified by Agilent Technologies 6890N with HP-5 5% Phenyl Methyl Siloxane capillary column (30 m by 320 µm by 0.25 mm) and a flame ionization detector. Methyl heptadecanoate (10.0 mg; internal standard) was dissolved in 1 ml hexane to prepare the standard solution. Approximately 100 mg crude methyl ester was dissolved in 1 ml standard solution for GC analysis (Hong, 2009). Approximately 1 µl sample was injected into the GC at an oven temperature of 210 C with Helium as the carrier gas. The GC oven was programmed at 210 C, isothermally for 15 min. the FAMEs content was calculated by use of the Equation 1:

$$\text{IC} = \frac{\sum A - A\_{IS}}{A\_{IS}} \quad \times \quad \frac{C\_{IS} \times V\_{IS}}{m} \quad \times \quad 100\% \tag{1}$$

#### **Where:**

*∑A* = total peak area of methyl ester *AIS* = peak area of internal standard (methyl heptadecanoate) *CIS* = concentration of the internal standard solution, in mg/ml *VIS* = volume of the internal standard solution used, ml *m* = mass of the sample, in mg
