*2.9. Characterization of Biodiesel*

Biodiesel produced by Fe3O4\_PDA\_Lipase catalyzed transesterification was monitored by Fourier transfer infrared (FTIR) spectroscopy. The FTIR spectra of feedstock and synthesized biodiesel were taken over the scanning range 400–4000 cm<sup>−</sup><sup>1</sup> using Cary 630 Agilent FTIR spectrometer. Oil FTIR spectra was compared to the FTIR of synthesized biodiesel to confirm the formation of fatty acid methyl esters. The GC/MS scan of biodiesel was taken to reveal compositional profile of synthesized biodiesel. GCMS QP 2010 system with a dB 5 column and a diameter of 0.15 mm was used. One μL sample size and 1:100 split ratio was selected. Helium as the carrier gas at 1.2 mL/min rate was used to elute the sample. The column temperature was set in the range 150–250 ◦C, at rate of 4 ◦C/min. GCMS mass scanning range was 30 to 550 m/z. Detection of FAMEs was done using NIST MS library of GCMS. The compatibility of WCO based biodiesel being used as fuel was confirmed by the estimation of its fuel properties, which were investigated according to the ASTM D methods.

#### *2.10. Selection of Suitable Models for Optimization*

Based on the experimental outputs, the best fitted model out of linear, 2Fi, cubical and quadratic models were selected for optimization purpose. The fitness of selected models was further ascertained through summery statistics such as R2, adjusted R2, model significance and lack-of-fit test. Moreover, normality and predicted values versus actual values plots were also employed for the above said purpose. The impact of selected reaction variables on the response i.e., biodiesel yield was revealed by 3D response surface plots.

## **3. Results and Discussion**

#### *3.1. Synthesis and Evaluation of Nano-Biocatalyst E*ff*ectiveness*
