**3.2 Fourier-transform infrared spectroscopy (FT***-***IR)**

FT-IR spectroscopy data of the mid-infrared region of biodiesel samples to recognize functional groups and the bands analogous to various stretching and bending vibrations is highlighted in **Table 2**.

## **3.3 Nuclear magnetic resonance (NMR)**

The FAMEs NMR spectrum was acquired by (Bruker Avance III 400 NMR Spectrometer, Karlsruhe, Germany) at 400 MHz (1 H-NMR) or 100 MHz (13C-NMR). Denatured chloroform was used as solvent and tetramethylsilane as the internal standard. The biodiesel <sup>1</sup> H NMR (300 MHz) spectrum was noted with a cycle delay of 1.0 s, and eight times scans with a pulse duration of 30°, (**Table 3**). A carbon 13C NMR (75 MHz) spectrum was recorded with pulse duration of 30°and a cycle delay of 1.89 s, followed by scanning for 160 times (**Table 4**).

**Figure 5.** *(A) Instrument for Soxhlet extraction (chemical extraction); (B) instrument for mechanical oil extraction.*


**Table 1.**

*The oil content (%) of 4 plants, using mechanical oil extraction and Soxhlet extraction methods.*


#### **Table 2.**

*FT-IR data presenting various functional groups in FAMEs.*


#### **Table 3.**

*1 H NMR spectroscopic data depicting chemical composition of various methyl esters in biodiesel (FAMES) samples.*

*Optimization and Characterization of Novel and Non-Edible Seed Oil Sources for Biodiesel… DOI: http://dx.doi.org/10.5772/intechopen.97496*


**Table 4.**

*13C NMR spectroscopic data depicting the chemical shift values matching to various structural features in FAMEs.*

## **3.4 GC–MS procedure**

The outcome of biodiesel in our studies was evaluated by GCMS (QP2010SE, Shimadzu, Japan), furnished with a capillary column: PEG-20 M (30 m × 0.32 mm × 1 μm film thickness). Helium gas flow rate 1.2 mL/min; split ratio 40:1; the injector temperature and injection volume were 220°C and 1 uL; Furnace heat up mode was 100°C for 1 min, then from 100°C rises to 210°C at the increase rate of 10°C/min. Sensor heat mode was 210°C, and then for 20 min, the temperature was continuing at 210°C; ion source temperature of 200°C; for electron impact 70 eV ionization mode used; mass range of 35–500 m/z. The FAMEs of all plant sources were identified with the mass spectrometry fragmentation design provided by the GCMS system software, as matched with those stored in the mass spectrometry library NIST14, and their fatty acid identity was further verified by matching with known standards and values [39–42].

The comparative GC based identified FAMEs major compositions (%) of prepared biodiesel from four non-edible plant sources is given in **Table 5**.

#### **3.5 ICP-OES procedure for elemental analysis in biodiesel**

Inductively Coupled Plasma Spectrometer (Spectro-blue, Germany) and Elemental Analyzer (Vario EL CUBE, Germany) were used for the presence of metals in the biodiesel. For the ICP-OES test, 1 g of oil sample was taken for incinerating. The ashing process involved an increase in the oven temperature to 200°C in one hour; then the heat levels were mainatained upto 500 °C for 2 h, and finally


**Table 5.** *GC based identified FAMEs major compositions (%) of prepared biodiesel.*

#### *Botany - Recent Advances and Applications*

to 800°C for 5 h. The ash was dissolved in 10 mL of 2% HNO3. The prepared sample was used for elements finding and concentration test of the biodiesel.

The account of ICP-OES comparative element concentrations of 4 non edible oil plant species is given in **Table 6**.

#### **3.6 Elemental analyzer (EA) procedure for elemental analysis**

The element analyzer (Vario EL CUBE, Germany) was used to detect the H, N, C and O concentrations of biodiesel obtained from plant sources [39–42]. About 0.5 mL of biodiesel, 3 mL of concentrated HCl and 1 mL of nitric acid were taken in a tube and kept them at rest for 10–15min, to dissolve the oil in the solution. Fresh reagents can be used for sample preparation. The aqua regia amount was twice than the sample. About 1 mL of prepared solution was taken in a new tube and added deionized water making it up to 5 mL. The technique was repeated for 2–3 times until the sample appeared as clear and vivid and ready for evaluation of C, H, N, and O concentrations.

The comparative account of elemental analysis of biodiesel obtained from 4 non edible oil plant species is given in **Table 7**.

### **3.7 Physiochemical properties of biodiesel seed oil from four non edible oil plant species**


The comparative account of physiochemical properties of biodiesel seed oil obtained from four non edible oil plant species is given in **Table 8**.

*Abbreviations: Koelreuteria paniculata Biodiesel Oil (KPOB); Rhus typhina Biodiesel Oil (RTOB); Acacia farnesiana Biodiesel Oil (AFOB); Albizzia julibrissin Biodiesel Oil (AJOB).*

#### **Table 6.**

*ICP-OES element concentrations of 4 non edible oil plant species.*

*Optimization and Characterization of Novel and Non-Edible Seed Oil Sources for Biodiesel… DOI: http://dx.doi.org/10.5772/intechopen.97496*


*Abbreviations: Koelreuteria paniculata Biodiesel Oil (KPOB); Rhus typhina Biodiesel Oil (RTOB); Acacia farnesiana Biodiesel Oil (AFOB); Albizzia julibrissin Biodiesel Oil (AJOB).*

#### **Table 7.**

*Elemental analysis of biodiesel from 4 non edible oil plant species.*


*Abbreviations: Koelreuteria paniculata Biodiesel Oil (KPOB); Rhus typhina Biodiesel Oil (RTOB); Acacia farnesiana Biodiesel Oil (AFOB); Albizzia julibrissin Biodiesel Oil (AJOB).*

#### **Table 8.**

*Physiochemical properties of biodiesel (FAMEs) samples.*
