Effect of Calcination Temperature and Time on the Synthesis of Iron Oxide Nanoparticles: Green vs. Chemical Method

Citation: Abdullah, J.A.A.; Jiménez-Rosado, M.; Guerrero, A.; Romero, A. Effect of Calcination Temperature and Time on the Synthesis of Iron Oxide Nanoparticles: Green vs. Chemical Method. Materials 2023,16, 1798. https://doi.org/10.3390/ ma16051798 Academic Editor: Federico Cesano Received: 13 January 2023 Revised: 19 February 2023 Accepted: 20 February 2023 Published: 22 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). materials Article Effect of Calcination Temperature and Time on the Synthesis of Iron Oxide Nanoparticles: Green vs. Chemical Method Johar Amin Ahmed Abdullah 1,* , Mercedes Jiménez-Rosado 1, Antonio Guerrero 1and Alberto Romero 2,* 1Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 41011 Sevilla, Spain 2Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012 Sevilla, Spain *Correspondence: [email protected] (J.A.A.A.); [email protected] (A.R.); Tel.: +34-95-455-7179 (J.A.A.A. & A.R.) Abstract: Nowadays, antioxidants and antibacterial activity play an increasingly vital role in biosystems due to the biochemical and biological reactions that involve free radicals and pathogen growth, which occur in many systems. For this purpose, continuous efforts are being made to minimize these reactions, including the use of nanomaterials as antioxidants and bactericidal agents. Despite such advances, iron oxide nanoparticles still lack knowledge regarding their antioxidant and bactericidal capacities. This includes the investigation of biochemical reactions and their effects on nanoparticle functionality. In green synthesis, active phytochemicals give nanoparticles their maximum functional capacity and should not be destroyed during synthesis. Therefore, research is required to establish a correlation between the synthesis process and the nanoparticle properties. In this sense, the main objective of this work was to evaluate the most influential process stage: calcination. Thus, different calcination temperatures (200, 300, and 500 ◦ C) and times (2, 4, and 5 h) were studied in the synthesis of iron oxide nanoparticles using either Phoenix dactylifera L. (PDL) extract (green method) or sodium hydroxide (chemical method) as the reducing agent. The results show that calcination temperatures and times had a significant influence on the degradation of the active substance (polyphenols) and the final structure of iron oxide nanoparticles. It was found that, at low calcination temperatures and times, the nanoparticles exhibited small sizes, fewer polycrystalline structures, and better antioxidant activities. In conclusion, this work highlights the importance of green synthesis of iron oxide nanoparticles due to their excellent antioxidant and antimicrobial activities. Keywords: Phoenix dactylifera L.; nanoparticles; green synthesis; chemical synthesis; iron oxide; calcination temperature 1. Introduction The eco-friendly green synthesis of iron oxide has increased in popularity in recent years due to its effectiveness, low cost, non-toxicity, and environmentally friendly nature. Furthermore, these methods can produce nanoparticles with high crystallinity in a variety of sizes, morphologies, and high magnetic properties [ 1 – 8 ]. These approaches are based on an active substance (phenolic compounds) present in plants, which acts as a reducing agent on metal salts to obtain metal oxide nanoparticles [ 9 – 11 ]. This led to the improved production of nanoparticles with unique, specific, and magnetic properties [ 12 – 15 ]. Nevertheless, nanoparticles based on phenolic compounds need further investigation to evaluate factors such as extraction methods, reactant concentrations, pH solution, metal salt, and reaction and calcination temperature and time [ 16 , 17 ]. In this way, the control of nanoparticle size and functionality is still a challenge in green methods. During synthesis, the mixing of reducing agents with precursors results in rapid nucleation of the nanoparticles, followed by their aggregation. Therefore, it is evident that the rapid nucleation at the nanoparticle formation results in larger nanoparticle aggregates, heterogeneous crystal phases, and lower efficiency. This effect could be resolved by applying a post-processing technique, such as thermal treatment [ 18 , 19 ]. The thermal treatment Materials 2023,16, 1798. https://doi.org/10.3390/ma16051798 https://www.mdpi.com/journal/materials Materials 2023,16, 1798 2 of 18 can eliminate impurities, control the chemical phase of the capping agent, and determine the final phase, purity, and functionality of the final product. In this sense, homogenous or heterogeneous crystal phases could be modified by the final calcination treatment [ 18 – 20 ]. Nevertheless, this treatment has different effects on green and chemical synthesis. Thus, it can degrade the functional groups provided by green synthesis, reducing nanoparticle functionality [ 20 ]. Conversely, the chemical methods require a high calcination temperature to remove any excessive or secondary agents formed during the synthesis [ 18 ]. Thus, it is necessary to specify the calcination temperature and time to achieve the desired properties. Natural and synthetic iron oxide nanoparticles are available in various forms and quantities, including iron oxides, hydroxides, and/or oxide-hydroxides complexes. Their atomic composition contains the same elements (Fe, O, and OH), although their crystal structures and iron valence may be different. However, the most important iron oxide nanoparticles (Fe x O y -NPs) are based on magnetite (Fe 3 O 4 -NPs), hematite (Fe 2 O 3 -NPs), and maghemite ( γ -Fe 2 O 3 -NPs) [ 21 – 29 ], since they have a significant impact on the physicochemical and functional properties [ 30 ]. Iron oxide nanoparticles range in diameter from 1–100 nm and display a high surface-area-to-volume ratio, providing them with a significantly enhanced binding capacity and high dispersibility in solutions. Moreover, iron oxide nanoparticles < 20 nm in size display superparamagnetic properties [ 31 ]. These properties, in addition to their biocompatibility and nontoxicity, favor their potential application in biomedicine, environmental remediation, defense and aerospace, electronics, construction, healthcare, automotive textiles, agriculture, and the food industry [32,33]. Smaller iron oxide nanoparticles exhibit excellent antioxidant and antibacterial activities. Thus, they have a high inhibitory ability against foodborne pathogen growth, including Escherichia coli and Staphylococcus aureus [ 34 ]. This capacity to kill bacteria is due to the production of reactive oxygen species ( • OH, • O 2− ), which damage DNA and the bacteria protein, causing mitochondrial dysfunction [ 31 , 35 , 36 ]. Magnetic iron oxide nanoparticles have also been used to isolate and identify the DNA of pathogens in dairy products [37]. In biosystems, antioxidants and antibacterial activity play a crucial role. The biochemical and biological reactions inside living cells generate lethal free radicals, which are found in a variety of biological systems. However, little information is available regarding the study of magnetic iron oxide nanoparticles as antioxidants and antibacterial agents. Therefore, the effects of iron oxide nanoparticles on biochemical reactions should be investigated, as it is necessary to understand their antioxidant and antibacterial activity. They have been reported in previous works, concluding that several factors (e.g., particle size and magnetic behavior) could alter these properties [ 38 ]. Nevertheless, no study has extensively examined the effect of photochemical degradation on the efficiency and functionality of nanoparticles, especially those prepared from Phoenix dactylifera L. extract. The active photochemical responsible for donating the nanoparticle to its maximum functional capacity should not be degraded or destroyed during the synthesis. Therefore, the main objective of this work was to provide a novel comparison between the green and chemical synthesis of iron oxide nanoparticles and to demonstrate the impact of the calcination stage on their properties and functionality. In this way, Phoenix dactylifera L. (PDL) was used for its polyphenol content, as reported in previous works [ 39 , 40 ]. Factors like calcination temperatures and times, which may influence iron oxide nanoparticles’ properties, were investigated. Thus, iron oxide nanoparticles were synthesized by colloidal precipitation using two reducing agents: polyphenols extracted from PDL (green method, GS-NPs) and sodium hydroxide (chemical method, CS-NPs). Nanoparticles of both approaches were investigated under different calcination temperatures (200, 300, 500 ◦ C) and times (2, 3, 5 h) and were further characterized by XRD, FTIR, SEM, and TEM. Furthermore, the obtained iron oxide nanoparticles were tested for their antioxidant activity towards DPPH and their antibacterial activity towards Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Materials 2023,16, 1798 3 of 18 2. Materials and Methodology 2.1. Materials In this study, the following chemicals: iron chloride hexahydrate (FeCl 3· 6H 2 O 98%), sodium hydroxide (NaOH), ethanol (CH 3 CH 2 OH 99.9%), methanol (CH 3 OH 99.9%), gallic acid (C 7 H 6 O 5 ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and dimethyl sulfoxide anhydrous (DMSO) (C 2 H 6 SO 99.9%) were all supplied by Sigma Aldrich (Darmstadt, Germany) and used. The experiments were performed employing only analytical-grade chemicals and reagents. Polyphenols were extracted from PDL, which was collected from Seville (Spain), following a previous protocol [ 41 , 42 ]. Briefly, leaves were dried for 44 days at 22 ±3◦C and 35% RH. Polyphenols were extracted by the Soxhlet method with distilled water (water: leaf powder ratio 10:1) for 8 h. The extract had a total polyphenol content (TPC) of 39 ±2 mg GAE/g extract. 2.2. Nanoparticle Synthesis Iron oxide nanoparticles were produced in two ways: greenly (GS-NPs) by adding 20 mL of PDL extract dropwise into 20 mL of iron chloride (FeCl 3· 6H 2 O) 1 M solution, and chemically (CS-NPs) by adding 20 mL of NaOH into 20 mL of FeCl 3· 6H 2 O 1 M solution. NaOH 5 M was added to adjust the pH of the reaction to 7.5. The resulting solutions were heated on a hot plate at 50 ◦ C under stirring for 2 h. Thereafter, the mixture was filtrated using Whatman n º 1 filter paper, and then, a series of washings with distilled water was conducted to eliminate contaminants and foreign particles. The precipitates were subjected to pre-treatment at 100 ◦ C for 8 h in an oven. Then, they were calcined. This last step was evaluated using different calcination temperatures (200, 300, and 500 ◦ C) for different times (2, 4, and 5 h). A more detailed description of this synthesis is provided in previous works [41–43]. 2.3. Nanoparticle Characterization 2.3.1. X-ray Diffraction (XRD) The XRD patterns were acquired using a Brand diffractometer (Bruker model D8 advance A25 diffractometer with Cu anode, Bruker) for the confirmation of the crystalline phase. The nanoparticle diffractograms were performed between 2 θ◦ = 15 and 70 ◦ . The X-ray with a beam power of 40 kV and a current of 30 mA made contact with the samples at 0.015 ◦ (pitch angle) and 0.1 s (passing time), while the samples were rotating at 30 rpm [ 44 ]. As described in previous studies [ 41 , 42 ], the size and crystallinity of the magnetic iron oxide nanoparticles were calculated using the Debye-Scherrer formula. 2.3.2. Fourier Transform Infrared Spectroscopy (FTIR) A DTGS-KBr sensor was used in transmittance mode to perform FTIR spectroscopy on a Hyperion 100 Spectrometer (Bruker, Billerica, MA, USA). The iron oxide nanoparticles were characterized from 4000 to 400 cm −1 to gather their structural information. Fe-O bonds in FexOycan be identified in the fingerprint region (800–400 cm−1) [41,42]. 2.3.3. Scanning Electron Microscopy (SEM) An SEM study was performed for the purpose of gathering information on the iron oxide nanoparticles’ morphology and size. It was carried out using a scanning electron microscope (Zeiss EVO, Dublin, CA, USA) at 10 kV. As part of this comparison, the samples were photographed at different magnifications and evaluated using the freeware Image-J v1.53q (NIH, Bethesda, MD, USA) [42]. 2.3.4. Transmission Electron Microscopy (TEM) A TEM study was conducted to examine the crystalline characteristics of the size of nanoparticles. The observations were made at 200 kV through a TEM (Talos S200 Materials 2023,16, 1798 4 of 18 microscope, FEI, Hillsboro, OR, USA). Images of GS-NPs and CS-NPs were evaluated through Image-J 1.53q free software [42]. 2.4. Functional Properties 2.4.1. Antioxidant Activity Iron oxide nanoparticles in this study were tested for their antioxidant activity using the DPPH method (free radical DPPH). This method involves the scavenging of free radicals through DPPH. Thus, the DPPH test can determine the number of free radicals trapped by an antioxidant. It was conducted following the protocol described in previous work [ 42 ]. Briefly, 2 mL of nanoparticle dispersion (4, 2, 1, 0.5, 0.25, 0.125 mg/mL in DMSO) was mix-agitated with 2 mL of DPPH. Then, their absorbances were recorded at 517 nm after incubation for 0.5 h. An equal volume of DPPH solution and DMSO was used as a control solution. The necessary anti-radical that could cause 50% inhibition of DPPH (IC 50 ) was determined by measuring the inhibition percentage (IC) for each concentration using GraphPad Prism 9 software (GraphPad Prism for Windows version 9.0.0, San Diego, CA, USA). 2.4.2. Antimicrobial Activity The GS-NPs were tested for their antimicrobial activity on agar plates according to Behera et al. (2012) with a few modifications [ 45 ]. The antibacterial properties of GS- NPs were tested against two pathogen types: Staphylococcus aureus (S. aureus gram+) and Escherichia coli (E. coli gram − ) inoculated on agar gels. To this end, 20 µ L of dispersion ( 50 mg/mL ) of each selected GS-NPs (the nanoparticles were dispersed in distilled water by ultrasound for 1 / 3 h) was dropped in holes of 7 mm diameter, which were made using a cone-shaped pipette as a perforator. A standard was prepared by filling wells of 2 mm in diameter with 10 µL of Gentamicin antibiotic (50 µg/mL). Diameters of inhibition zones surrounding the GS-NPs solutions were measured after 24–48 h of incubation time at 37 ◦ C by Image-J 1.53q (Free software) to assess the antibacterial activity of the different samples [43]. 2.5. Statistical Analysis At least three replicates were performed to achieve a reliable comparison. Standard deviations and significant differences were calculated for the statistical analysis using GraphPad Prism9 and IBM SPSS Statistics (version 26.0.0 software). One-way ANOVA was used to signify the differences between observations. These significances were obtained through HSD Tukey statistical analysis, with p< 0.05 (95% confidence interval). 3. Results and Discussion 3.1. Nanoparticle Characterization 3.1.1. XRD The X-ray diffractograms are shown in Figure 1(2 θ and planes in red indicate the magnetite phase and 2 θ and planes in black indicate the hematite phase). Further investigations were conducted on the effects of calcination time and temperature on crystallinity, crystallite size, and phase composition. Regarding the green synthesis (Figure 1a), the diffraction peaks of the GS-NPs calcined at 200 ◦ C for 2 h could be attributed to 98.3% of polycrystalline structures of magnetite (36.2% monoclinic, 32.6% trigonal with hexagonal axis, and 29.3% cubic structures) and 1.7% of cubic hematite (Tables 1and 2). These proportions were extracted from (JCPDS n º . 00-153-2800, 00-152-8611, 00-900-7706, 00-900-2673, and 00-210-1535 standard iron oxide powder diffraction pattern) [46–50]. At 4 h of calcination, the diffraction peaks were attributed to 95.3% of polycrystalline magnetite (9.2% monoclinic, 50.0% trigonal with hexagonal axis, and 36.1% cubic structure) and 4.7% of cubic hematite (Tables 1and 2) (JCPDS n º . 00-153-2800, 00-152-8611, 00-152- 6955 and 00-900-9768 standard iron oxide powder diffraction pattern) [ 46 , 50 – 52 ]. A higher increase in calcination time (5 h) resulted in the formation of 100% magnetite with crystal Materials 2023,16, 1798 5 of 18 systems (44.9% monoclinic and 55.1% trigonal with hexagonal axis) (Tables 1and 2) (JCPDS nº. 00-153-2800, and 00-152-6955 standard iron oxide powder diffraction pattern) [46,51]. Materials 2023, 16, x FOR PEER REVIEW 5 of 21 trigonal with hexagonal axis, and 29.3% cubic structures) and 1.7% of cubic hematite (Tables 1 and 2). These proportions were extracted from (JCPDS nº. 00-153-2800, 00-152- 8611, 00-900-7706, 00-900-2673, and 00-210-1535 standard iron oxide powder diffraction pattern) [46–50]. Figure 1. XRD patterns of the nanoparticles (JCPDS standard) calcined at different temperatures (200, 300, and 500 ◦ C) and times (2, 4, and 5 h): ( a ) GS-NPs profile using aqueous extracts of PDL and ( b ) CS-NPs profile using NaOH. Materials 2023,16, 1798 6 of 18 Table 1. Results obtained for different parameters of GS-NPs using PDL extract and CS-NPs using the chemical method at different temperatures (200, 300, and 500 ◦C ) and times (2, 4, and 5 h): average size in nm (from XRD measurements and SEM images), crystallinity percentage (Crl, %), composition (proportion of Fe 2 O 3 and Fe3O4), and antioxidant activity (DPPH IC50 in µg/mL). GS-NPs T◦C 200 ◦C 300 ◦C 500 ◦C Time XRD (nm) SEM (nm) Crl (%) Fe2O3 (%) Fe3O4 (%) IC50 (µg/mL) XRD (nm) SEM (nm) Crl (%) Fe2O3 (%) Fe3O4 (%) IC50 (µg/mL) XRD (nm) SEM (nm) Crl (%) Fe2O3 (%) Fe3O4 (%) IC50 (µg/mL) 2 h 10.2 ±0.4 f11.7 ±0.2 f98.5 a1.7 98.3 86 ±6e7.6 ±0.5 f5.8 ±0.1 e98.4 a55.0 45.0 8±2f22.0 ±0.3 e21.8 ±0.6 c96.6 b25.4 74.6 1488 ±449 f 4 h 11.8 ±0.6 e14.2 ±0.3 e96.7 b4.7 95.3 55 ±2f16.3 ±0.2 e19.3 ±0.2 d98.6 a57.1 42.9 39 ±3e24.6 ±0.8 d26.0 ±1.7 b97.0 a91.3 8.7 4586 ±343 c 5 h 18.5 ±1.2 d16.8 ±3.6 d87.6 f- 100 403 ±41 d27.7 ±0.3 d23.5 ±1.8 c94.6 c86.6 13.4 109 ±3d23.5 ±0.6 d20.8 ±0.1 d91.1 d62.4 37.6 2100 ±10 e CS-NPs 2 h 29.8 ±0.8 b24.2 ±0.4 b93.4 c91.1 8.9 4213 ±990 c32.1 ±1.1 b25.3 ±0.9 b93.6 d88.6 11.4 4810 ±165 c32.3 ±0.6 a36.0 ±1.0 a91.7 d96.9 3.1 97,778 ±2320 a 4 h 31.9 ±0.3 a26.3 ±0.4 a90.5 e92.9 7.7 12,602 ±779 b39.0 ±1.7 a31.8 ±0.9 a95.0 b93.5 6.5 19,280 ±1249 a31.3 ±0.1 b37.0 ±1.0 a94.9 c100.0 - 3735 ±1650 d 5 h 22.2 ±0.7 c19.2 ±0.1 c92.4 d95.5 4.5 23,442 ±1362 a29.5 ±0.9 c23.0 ±1.0 c91.0 e98.1 1.9 16,550 ±1208 b29.6 ±0.9 c27.5 ±0.5 b85.6 e98.7 1.3 7100 ±400 b Note: Different superscript letters (a–f) within a row indicate significant differences among mean observations (p< 0.05). Table 2. Results obtained for different parameters of GS-NPs using PDL extract and the CS-NPs using the chemical method at different temperatures (200, 300, and 500 ◦ C) and times (2, 4, and 5 h): Crystal systems attributed to hematite (Fe 2 O 3 ) and magnetite (Fe 3 O 4 ) proportions; Mo: Monoclinic; Tri (hex): Trigonal (hexagonal axis); Tri (rohm): trigonal (rhombohedral axis); Cu: Cubic; Te: tetrahedral. GS-NPs T◦C 300 ◦C 300 ◦C 500 ◦C Time Phase MO Tri (Hex) Tri (rohm) Cu Te Total MO Tri (Hex) Tri (rohm) Cu Te Total MO Tri (Hex) Tri (rohm) Cu Te Total 2 h Fe2O31.7 1.7 0.7 0.6 47.9 5.8 55.0 25.4 25.4 Fe3O436.2 32.6 29.4 98.3 35.3 6.6 3.1 45.0 74.6 74.6 4 h Fe2O34.7 4.7 7.2 49.9 57.1 91.3 91.3 Fe3O49.2 50.0 36.1 95.3 42.9 42.9 1.8 1.4 5.5 8.7 5 h Fe2O30.5 86.1 86.6 62.4 62.4 Fe3O444.9 55.1 100 7.9 2.5 3.0 13.4 19.0 1.0 17 0.6 37.6 CS-NPs 2 h Fe2O31.5 89.6 91.1 88.6 88.6 3.4 46.7 46.8 96.9 Fe3O46.3 2.6 8.9 11.4 11.4 2.1 1.0 3.1 4 h Fe2O32.9 2.5 87.5 92.9 0.7 92.8 93.5 2.2 2.8 95.0 100 Fe3O42.9 4.2 7.1 5.7 0.8 6.5 5 h Fe2O36.1 89.4 95.5 1.5 96.6 98.1 60.3 36.1 2.3 98.7 Fe3O44.5 4.5 1.1 0.8 1.9 1.3 1.3 Materials 2023,16, 1798 7 of 18 On the other hand, the increase in calcination temperature caused a pronounced change in the Fe x O y -NP type, which resulted in a greater proportion of hematite. For example, the diffraction peaks of GS-NPs calcined at 300 ◦ C for 2, 4, and 5 h were attributed to 55.0, 57.1, and 86.6% of polycrystalline hematite, respectively (Table 2) [46,47,50,51,53,54] , while the diffraction peaks of the GS-NPs calcined at 500 ◦ C for 2, 4 and 5 h were assigned to 25.4, 91.3, and 62.4% of monoclinic, cubic, and trigonal with hexagonal axis hematite [46,47,49–52,55–60]. The chemically synthesized nanoparticles were mostly hematite (Tables 1and 2). The diffraction peaks of the CS-NPs calcined at 200 ◦ C for 2, 4, and 5 h were attributed to 91.1, 92.9, and 95.5% of the polycrystalline structure of hematite, respectively (Figure 1b and Table 2) [ 46 , 56 , 61 – 63 ]. A temperature increment to 300 ◦ C generated a change in nanoparticles, finding 88.6, 93.5, and 98.1% of the polycrystalline structure of hematite for the calcination times 2, 4, and 5 h, respectively (Figure 1b and Table 2) [ 46 , 51 , 56 , 61 , 64 ]. The further calcination at higher temperatures (500 ◦ C) resulted in nearly pure hematite. Thus, the diffraction peaks were attributed to 96.9, 100, and 98.7% of the polycrystalline structure of hematite for the calcination times 2, 4, and 5 h, respectively (Figure 1b and Table 2) [46,51,56,62,64]. These results are in good accordance with those that have been documented in the literature. Among them, Jafari et al. noted a transition phase of iron oxide nanoparticles from Fe 3 O 4 to Fe 2 O 3 when the particles were annealed in the range of 250–550 ◦ C [ 65 ]. Shangguan et al. observed an increase in Fe 2 O 3 proportion by increasing the calcination time (0.5–2.5 h) and calcination temperature in the range of 450–500 ◦ C [ 66 ]. Rusul et al. also obtained polycrystalline α -Fe 2 O 3 nanoparticles at elevated annealing temperatures (500 and 700 ◦ C) [ 67 ]. In this way, it seems that the mechanism for the production of iron oxide nanoparticles is due to the reduction of iron ions by phenolic compounds (green) or OH (chemical). Thereafter, two oxidation-reduction reactions were suggested: 1-Reduction and precipitation of iron ions Fe +3 of iron chloride (FeCl 3· 6H 2 O) in the presence of phenolic compound (-OH), where the reduction of iron ions to magnetite will normally take place at low calcination temperature and time, according to Equation (1) [65]: 3Fe+3(aq) + 8OH−→Fe3O4↓+ 4H2O (1) Additionally, and/or the reduction of Fe 2 O 3 nanoparticles with hydrogen to precipitate Fe3O4nanoparticles, according to Equation (2) [68]: 3Fe2O3+ H22Fe3O4↓+ H2O (2) 2-Oxidation of iron germs growing in an aqueous solution (resulted in Fe 2 O 3 ) through replacement and armoring process with the increase in calcination temperature and time in the presence of oxygen atoms, according to Equation (3) [65]: 4Fe3O4+ O2→6Fe2O3(3) In this way, magnetite fractions, Fe 3 O 4 -NPs, were more abundant in the GS-NPs, due to the great capacity of phenolic compounds to reduce more Fe +3 ions to Fe +2 (Fe 3 O 4 -NPs). The absence of phenolic compounds (CS-NPs) generated the oxidation of Fe +2 (Fe 3 O 4 ) ions into Fe+3 (Fe2O3) [69]. Several studies have reported similar results [70,71]. Regarding the size and crystallinity of GS-NPs and CS-NPs, a summary of these parameters, as well as the proportions of Fe x O y -NPs, is presented in Table 1. It was observed that the increase in calcination temperature and time increased the size of the nanoparticles (case GS-NPs). The crystallinity degree was higher when the nanoparticles’ size was smaller (Table 1) [ 72 , 73 ]. The decrease in nanoparticle size at low calcination temperature was attributed to oxygen vacancies created on the surface. On the other hand, the increase in calcination temperature up to 300 ◦ C led to an increase in the particle size due to the Ostwald Ripening mechanism (i.e., the particles with larger sizes grow at the Materials 2023,16, 1798 8 of 18 expense of the smaller ones) [ 74 , 75 ]. The formation of relatively smaller nanoparticles happens during the calcination rather than the loss of water in the particles [76]. A decrease in size was also reported by Mornani et al., who investigated the effect of calcination temperatures on the magnetic iron oxide nanoparticles using a chemical method in the range of 400–650 ◦ C [ 18 ]. An increase in nanoparticle sizes was also observed by Koupaei et al. when the calcination time increased [77–79]. Nevertheless, the decrease in size after increasing the calcination time to 5 h at 500 ◦ C (GS-NPs) and at 200, 300, and 500 ◦ C (CS-NPs) (Table 1) is probably due to the oxidation of magnetite at the grain surface to form smaller hematite (Fe2O3) [79,80]. 3.1.2. FTIR Figure 2a,b shows the general FTIR spectra of GS-NPs and CS-NPs, respectively. The bonds between 3500 and 3420 cm −1 are a result of the vibrational stretching of the OH-phenolic compound present in the extract (GS-NPs) and OH of NaOH (CS-NPs) [81]. Materials 2023, 16, x FOR PEER REVIEW 10 of 21 observed that the increase in calcination temperature and time increased the size of the nanoparticles (case GS-NPs). The crystallinity degree was higher when the nanoparticles’ size was smaller (Table 1) [72,73]. The decrease in nanoparticle size at low calcination temperature was attributed to oxygen vacancies created on the surface. On the other hand, the increase in calcination temperature up to 300 °C led to an increase in the particle size due to the Ostwald Ripening mechanism (i.e., the particles with larger sizes grow at the expense of the smaller ones) [74,75]. The formation of relatively smaller nanoparticles happens during the calcination rather than the loss of water in the particles [76]. A decrease in size was also reported by Mornani et al., who investigated the effect of calcination temperatures on the magnetic iron oxide nanoparticles using a chemical method in the range of 400–650 °C [18]. An increase in nanoparticle sizes was also observed by Koupaei et al. when the calcination time increased [77–79]. Nevertheless, the decrease in size after increasing the calcination time to 5 h at 500 °C (GS-NPs) and at 200, 300, and 500 °C (CS-NPs) (Table 1) is probably due to the oxidation of magnetite at the grain surface to form smaller hematite (Fe 2 O 3 ) [79,80]. 3.1.2. FTIR Figure 2a,b shows the general FTIR spectra of GS-NPs and CS-NPs, respectively. The bonds between 3500 and 3420 cm −1 are a result of the vibrational stretching of the OH- phenolic compound present in the extract (GS-NPs) and OH of NaOH (CS-NPs) [81]. Figure 2. General FTIR spectra (4000–400 cm −1 ) of the nanoparticles calcined at different temperatures (200, 300, and 500 ◦ C) and times (2, 4, and 5 h): ( a ) GS-NPs profile using aqueous extracts of PDL and (b) CS-NPs profile using NaOH. Materials 2023,16, 1798 9 of 18 The sharp peaks at 2912–2838 cm −1 are associated with the C-H extension. The band around 1630 cm −1 indicates C=C stretching vibration or aromatic ring deformation. Around 1733 cm −1 , the bands could be related to the C=O of ketones, aldehydes, and esters. Ester groups show also one band around 1380 cm −1 [ 69 , 82 ]. The asymmetric stretching vibrations of C-O-C and C-O, corresponding to polyphenolic compounds, could be observed between 1038 and 1071 cm −1 and between 1201 and 1248 cm −1 , respectively [ 69 ]. Furthermore, the bonds between 1159 and 1104 cm −1 belong to C–O–H in polyphenolic compounds [ 69 ]. The reduction of the precursor (FeCl 3· 6H 2 O) by phenolic groups (–OH) results in the segmentation of the band around 1642 cm −1 into three peaks around 1652, 1632, and 1622 cm −1 . Figure 3a,b shows especially the fingerprint of FTIR spectra of GS-NPs and CS-NPs, respectively. Materials 2023, 16, x FOR PEER REVIEW 11 of 21 Figure 2. General FTIR spectra (4000–400 cm −1 ) of the nanoparticles calcined at different temperatures (200, 300, and 500 °C) and times (2, 3, and 5 h): (a) GS-NPs profile using aqueous extracts of PDL and (b) CS-NPs profile using NaOH. The sharp peaks at 2912–2838 cm −1 are associated with the C-H extension. The band around 1630 cm −1 indicates C=C stretching vibration or aromatic ring deformation. Around 1733 cm −1 , the bands could be related to the C=O of ketones, aldehydes, and esters. Ester groups show also one band around 1380 cm −1 [69,82]. The asymmetric stretching vibrations of C-O-C and C-O, corresponding to polyphenolic compounds, could be observed between 1038 and 1071 cm −1 and between 1201 and 1248 cm −1 , respectively [69]. Furthermore, the bonds between 1159 and 1104 cm −1 belong to C–O–H in polyphenolic compounds [69]. The reduction of the precursor (FeCl 3 ·6H 2 O) by phenolic groups (–OH) results in the segmentation of the band around 1642 cm −1 into three peaks around 1652, 1632, and 1622 cm −1 . Figure 3a,b shows especially the fingerprint of FTIR spectra of GS- NPs and CS-NPs, respectively. Figure 3. Fingerprint region of FTIR spectra in the (900–400 cm −1 ) of the nanoparticles calcinated at different temperatures (200, 300, and 500 °C) and times (2, 3, and 5 h): (a) GS-NPs profile using aqueous extracts of PDL and (b) CS-NPs profile using NaOH. Figure 3. Fingerprint region of FTIR spectra in the (900–400 cm −1 ) of the nanoparticles calcinated at different temperatures (200, 300, and 500 ◦ C) and times (2, 4, and 5 h): ( a ) GS-NPs profile using aqueous extracts of PDL and (b) CS-NPs profile using NaOH. Materials 2023,16, 1798 16 of 18 37. Paul, S.; Saikia, J.P.; Samdarshi, S.K.; Konwar, B.K. 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