The biogenic synthesis of Fe3O4 nanoparticles was boosted by the use of a recently found organic acid produced from Citrus sinensis. Fe3O4 nanoparticles were synthesized at 350 °C and 450 °C, and their physical and chemical properties were studied using a number of different characterisation techniques. Crystalline structure and phase were determined by X-ray diffraction (XRD) analysis of as-synthesized and annealed Fe3O4 nanoparticles. This finding demonstrates that the Fe3O4 nanoparticles have a unique, cubic spinel structure with only one phase. With the use of well-established formulae, we were able to calculate the crystallite size and lattice parameter of the synthesized Fe3O4 nanoparticles, and found that their size increased as a function of the annealing temperature, from 11 nm to 34 nm. The FTIR method was used to analyse the as-synthesized and annealed Fe3O4 nanoparticles for functional groups, molecular structure, and chemical bonding. Metallic components at 465, 551 and 1628 cm−1 were found after analysing the three samples. Field Emission Scanning Electron Microscopy (FESEM) was used to analyse the surface morphology and grain size fluctuations of Fe3O4 nanoparticles, while Energy Dispersive X-ray Spectroscopy (EDX) was used to study the elemental composition. Fe3O4 nanoparticles' thermal characteristics change depending on the annealing temperature. The antibacterial efficacy of Fe3O4 nanoparticles, manufactured using green methods, was assessed against two different types of bacteria: Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative).
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