Optimization of reaction parameters for the green synthesis of zero valent iron nanoparticles using pine tree needles

2.1 Materials

Pinus eldarica needle leaves were collected from garden campus of the Fasa university of Medical sciences (Fasa, Fars province, Iran). Ferric chloride (FeCl₃·6H₂O) was purchased from Merck Chemicals (Darmstadt, Hessen, Germany). Millipore water (Millipore Corp., Bedford, MA, USA, conductivity range of 0.055-0.294 μS/cm) was used for all the experiments.

2.2 Leaf extract preparation

Needle leaves were rinsed with deionized water in order to remove possible dusts and mud particles. The needles were dried at room temperature under dust free condition for about two weeks and then powdered with household miller and passed through a particular mesh sieve. Five gram of leaves powder was mixed with 100 mL deionized water in a 250 mL round bottom flask [36,37]. The mixture was heated up to boiling by using a heater mantel under reflux. After 15 min boiling resultant extract was cooled to room temperature and filtered through a Whatman filter paper (Reeve angel, Grade 201) to remove the sludge. The filtrate was centrifuged (5000 rpm, 12 min) to remove fine leaf particles. A clear supernatant was obtained and used for further experiments.

2.3 Experimental design

The MODDE software version 9 (Umetrics, Sweden) as a tool for statistical design of experiment was employed with two predominant stages in order to optimize the factors involved in synthesis process for maximizing the production of INPs. First stage was carried out to specify the important factors with significant effects on the amount of synthesized nanoparticles. A fractional factorial design was utilized to screen the effect of four variables namely plant extract quantity, iron precursor concentration, reaction temperature, and reaction time.

Second stage was carried out to achieve an empirical model to specify the optimum concentrations of selected efficient factors. In this step, central composite face (CCF) design and RSM were used to optimize the effective factors selected from screening stage. The variables and their range of values are depicted in Table 1.

2.4 Synthesis of INPs

For INPs fabrication, leaf extract (9 mL) and deionized water (750 μL) were added to a 50 mL round bottom flask and the mixture was stirred vigorously at room temperature. Consequently, 250 μL iron precursor (FeCl₃·6H₂O, 1 M) was added to the flask and reaction was followed for 30 min at room temperature. The reaction product was centrifuged and resulting black precipitate was washed with deionized water for three times to remove any unreacted solutes and phytochemicals. Finally, the black pellet was oven dried at 50°C for 48 h.

2.5 Characterization of INPs

Physicochemical properties of the synthesized nanoparticles through optimum experiment were evaluated by material analysis techniques. Transmission electron microscope (TEM, Zeiss, EM900, HT-100 KV) studies were carried out to identify the morphology and size of the prepared particles [38, 39, 40]. Analyses were done without any sample preparation. A drop of INPs suspension was dripped on a cupper grid and dried at room temperature. Particle size analysis was conducted by using an image analysis software (ImageJ version 1.47v, developed by NIH, http://imagejnihgov/ij/) Fourier-transform infrared (FTIR) spectroscopy (Perkin Elmer Spectrum One) analysis was done using KBr pellets for characterizing the synthesized INPs and also for understanding the existence of surface functional groups on the nanoparticles. The IR absorption analysis was done from 4500 cm^-1 to 400 cm^-1 [13,41]. The crystallographic analysis of INPs was performed by X-ray powder diffractometer (Siemens D5000). Resulting XRD pattern was evaluated by X’Pert High Score version 1.0d (PAN analytical B.V., Almelo, the Netherlands) [42,43]. The magnetic properties of nanoparticles and values of magnetic parameters such as saturation magnetization (M_s), coercive force (H_c) and magnetic remanence (M_r) were characterized using

vibrating sample magnetometer (VSM) (American-Lake Shore Cryotronics company, 7407 Model) with increasing magnetic field up to 19 kOe and field sweeping from −19 to +19 kOe [44,45]. Thermo gravimetric analysis (TGA) and derivative thermo gravimetric (DTG) analyses were done by a TGA, 209 F3 Tarsus. This technique was done to determine the thermal stability, presence, and the quantification of organic compounds from Pinus eldarica leaf extract in the final INPs product. TGA thermo grams were recorded for 10.8 mg of powder sample at a heating rate of 10°C/min in the temperature range of 30 to 600°C under air atmosphere. Energy-dispersive X-ray spectrometer (EDS) (Rontec analyser, Germany) was employed to determine the elemental composition of nanoparticles.

3.1 Effective parameters in the synthesis reaction

Reaction time, iron precursor concentration, leaf extract quantity, and reaction temperature were selected to investigate their effect on INPs production. DoE was carried out based on these four parameters and the amount of prepared nanoparticles was chosen as the response [35]. The results of initial screening for effective parameters in the synthesis reaction are depicted in Table 1. Table 2 illustrates the statistical analysis and variables coefficient in order to evaluate the main effects of single parameters and their interactions on the weight

of produced nanoparticles [34]. These results indicate that two factors namely iron precursor concentration and amount of leaf extract had significant positive effect on nanoparticles production (P-value less than 0.05 indicates that the factor is significant). These findings are in close agreement with previous report for the synthesis of iron nanoparticles using green tea extract. Where, FeCl₃-to-green tea extract ratio found to be the effective parameter in the amount of nanoparticles formation [46].

3.2 Optimization of nanoparticles production

Determination of optimum amount for iron precursor and leaf extract to enhance the INPs production was carried out by using the central composite face (CCF) design as shown in Table 3. The ANOVA test (Table 4) indicates a good fitness of the model, because of the high F value of 160.421 and a very low probability value. The linear regression coefficient R² = 0.994 and the adjusted

determination coefficient R² of 0.988 for the model demonstrate the accuracy of the model.

The response surface plots are shown in Figure 1. Analysis of the plots demonstrates that the maximum INPs is achievable when the final concentration of iron precursor and leaf extract are set as 25-45 mM and 8.5-9 mL, respectively. As can be seen in Figure 1, formation of nanoparticles decreases when the values of ferric chloride and leaf extract are set to be more than 50 mM and less than 8.5 mL, respectively. The highest productivity was predicted by the model to be 0.015 g per reaction (1.5 mg per mL of the reaction mixture) with optimum values of ferric chloride (25 mM) and leaf extract (9 mL). Consequently, the validation experiment was carried out under the optimized condition which resulted in the production of 0.015 g mL^-1 INPs.

Figure 1

Response surface plot (a) and response counter plot (b) for the fabrication of INPs showing the effects of iron precursor concentration and amount of leaf extract in a green synthesis reaction, all reactions were done in 10 mL final volume.

Although vast investigations have been done in regards to green synthesis of nanoparticles, there are very rare investigations for the optimization of nanoparticles fabrication [46]. Therefore, in the present study, the effects of reaction parameters on the fabrication of INPs were investigated and the reaction condition was optimized to achieve highest amount of nanoparticles in a constant reaction volume. Reaction condition has an immense influence on the physicochemical characteristics of the green synthesized nanoparticles [17,22,23]. So, investigations must be done to determine the impacts of synthesis conditions and involved factors on the reactivity, morphology, and other properties of the synthesized nanoparticles. For instance, the influences of various parameters such as the ratio of iron precursor (Fe²⁺) and leaf extract (tea extract), reaction temperature, and reaction pH were systematically investigated and have been reported by Lanlan Huang [47]. It was revealed that the reactivity of the prepared nanoparticles was highly depended to the synthesis conditions and the reduction rate is kinetically dominated by these factors.

3.3 Characterization of INPs

Physicochemical properties of the prepared INPs under optimal reaction condition were evaluated. Figure 2a is illustrating TEM micrograph of the produced nanoparticles and corresponding size distribution histogram is depicted in Figure 2b The obtained particles were dominantly spherical in shape with 8-34 nm in diameter with the mean particle size of 18 nm. Green synthesized INPs with similar particle size distribution were also reported by using a red wine, pomegranate, mulberry, and cherry leaf extract [12,48]. Based on the TEM micrographs, INPs were heavily surrounded by biological components from leaf extract (Figure S1), therefore, the resulting structure is a microstructure which composed of INPs and phytochemical compounds. Similar phenomenon is reported for the green synthesis of metal nanoparticles using a variety of plant extracts [16,23,49,50]. In some cases it has been confirmed that this biologic matrix is mainly composed of carbohydrates from leaf extract [23,49].

Figure 2

TEM micrograph of the biosynthesized INPs (a), which indicates that nanoparticles are spherical and surrounded by a biologic matrix from leaf extract, and corresponding particle size distribution histogram (b), more TEM image is provided in supplementary materials as Figure S1.

FTIR analysis was conducted to determine the chemical components which were responsible for stabilizing and capping of INPs. FTIR spectrum of the prepared INPs is presented in Figure 3. The OH groups induce a broad indicative peak which appeared at 3420.97 cm⁻¹ [41]. The carbonyl group stretching vibration can be seen at 1616.05 cm⁻¹ and the peak at 1069.05 cm^–1 is due to C–O bonds [41]. The peaks of aliphatic C–H appeared around 2918 cm^–1 [38, 39, 40]. The FTIR results indicate that prepared INPs are capped with biologic compounds which were derived from Pinus eldarica leaf extract [13,22,51]. These finding are in agreement with TEM micrograph which shows that INPs are surrounded by a biologic matrix. The Fe-O characteristic peaks of iron oxide nanoparticles commonly appear at about 640 cm⁻¹ and 450 cm^–1 [42,43, 4452,53]. These bonds were not observed in the FTIR spectrum of the prepared particles, which is

Figure 3

Fourier transform infrared (FTIR) spectra of the prepared INPs, indicating presence of organic compounds from Pinus eldarica.

indicative of zero-valent INPs. Similar spectra were also reported for chemically or plant mediated synthesized zero-valent INPs [16,54]. Oxidation of the exposed iron atom to iron oxide/hydroxide results in a core-shell structure that Fe⁰ forming the core which is surrounded by an iron oxide shell [11,55,56]. The thin iron oxide shell lead to the appearance of Fe-O absorption bonds in the FTIR spectra, but in lower intensity than that usually observed for iron oxide nanoparticles [44]. Absence of these peaks in FTIR spectra of the prepared particles indicated the absence of iron oxide shell. Organic capping from leaf extract seems to protect the surface of zero-valent iron atoms from oxidation.

The XRD pattern of the INPs is presented in Figure 4. XRD analysis confirmed that no distinctive peaks were present on the spectra. This indicated that the INPs were not crystalline in nature and were amorphous structures. Fabrication of amorphous zero-valent INPs was also reported by using leaf extract of different plants such as eucalyptus, mulberry, pomegranate, and cherry [57]. The broad shoulder peak from 20° to 30° of 2θ values was proposed to be due to the presence of organic component from leaf extract which are responsible for capping and stabilizing nanoparticles [26]. Some researchers also considered a tinny peak appearing at around 2θ of 44-45° as indicative peak for zero-valent INPs [11,58,59]. Production of other amorphous nanostructures was also reported for the green synthesized INPs. For instance, green synthesis of INPs by using extracts of Eucalyptus tereticornis, Melaleuca nesophila, and Rosemarinus officinalis was reported to result in amorphous iron-polyphenol nanoparticles [4,60].

Figure 4

X-ray powder diffraction (XRD) pattern of the synthesized INPs; no characteristic peak was observed which indicates amorphous structure of the nanoparticles.

TGA and DTG curves of the produced INPs are provided in Figure 5, demonstrating a significant weight loss processes. The initial weight loss below 263.7°C should be attributed to the evaporation of residual and adsorbed water. The weight loss above 263.7°C corresponds to the decomposition of the capping materials [16,61]. Decomposition of organic materials makes the main peaks at 373.9°C and 415.9°C in the DTG curve [16,62]. There was no weight loss at above 440°C. TGA analysis showed that the weight percentage of biologic coating in the product is about 74.43% [15]. Since now, the weight percentage of organic compounds in the green synthesized nanoparticles was reported to be up to 60% [63]. So, pine tree needles can be introduced as a biologic source for reducing and capping agents to provide nanoparticles with heavy coating. This unique feature can be a great advantage for future applications.

Figure 5

TGA and DTG curves of the synthesized INPs; the weight loss below 263.7°C was due to water evaporation and above 263.7°C was due to decomposition of biologic coating.

The magnetization curve as a function of the applied magnetic field at room temperature is shown in Figure 6. The sample showed no hysteresis and the magnetization curve was completely reversible that exhibits a superparamagnetic behavior of nanoparticles. The saturation magnetization value of the synthesized particles was found to be 33 memu/g. The very low saturation value is possibly due to the diamagnetic properties of biologic capping material and amorphous state of the prepared INPs [15]. Previous investigations have indicated that increase in the intensity of biological coating resulted in significant reduction of saturation magnetization [52,53]. Also, Santra et al., reported that the magnetization of the uncoated magnetic nanoparticles (1.3 emu/g) is higher than coated nanoparticles (0.5 emu/g) [64]. In the other experiment, the saturation magnetization values of the iron nanoparticles which were synthesized by using Plectranthus amboinicus leaf extract was reported to be 1.25 emu/g [65]. Low saturation magnetization value of the prepared nanoparticles in contrast to previous report can be in agreement with TGA results which indicate a heavy biologic coating.

Figure 6

Magnetization curve of the prepared INPs, magnetization value of the synthesized INPs was measured to be 33 memu/g.

Localized elemental information of the iron nanoparticles was determined by EDS and results are depicted as Figure 7. There are intense peaks of C, O, Ca, and Fe in the spectrum and quantitative data indicated that the atomic percentages of the prepared material were 53.08% C, 44.76% O, 0.30% Ca, and 1.87% Fe. The high percentage of carbon and oxygen elements originated mainly from the polyphenol groups and other C, O-containing compounds in Pinus eldarica extract. Also, Ca element is attributed to the plant extract. As can be observed from the TEM graph, the produced iron nanoparticles were heavily surrounded by biologic compounds from leaf extract, which is accountable for the low iron content in the sample. These results are in agreement with previous findings for the green synthesized INPs [57,58,66]. But, in comparison, the low Fe weight percent is due to heavy biological coating that is provided by Pinus eldarica extract [58]. This value (1.87%) for iron content is one of the least atomic percentage that already were reported.

Figure 7

EDS spectrum of the synthesized INPs.