A Comprehensive Study of Synthesis and Analysis of Anisotropic Iron Oxide and Oxyhydroxide Nanoparticles

One-dimensional anisotropic nanoparticles are of great research interest across a wide range of biomedical applications due to their specific physicochemical and magnetic properties in comparison with isotropic magnetic nanoparticles. In this work, the formation of iron oxides and oxyhydroxide anisotropic nanoparticles (ANPs) obtained by the co-precipitation method in the presence of urea was studied. Reaction pathways of iron oxide and oxyhydroxide ANPs formation are described based on of X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), and pulse magnetometry studies. It is shown that a nonmonotonic change in the Fe3O4 content occurs during synthesis. The maximum content of the Fe3O4 phase of 47.4% was obtained at 12 h of the synthesis. At the same time, the reaction products contain ANPs of α-FeOOH and submicron isotropic particles of Fe3O4, the latter formation can occur due to the oxidation of Fe2+ ions by air-oxygen and Ostwald ripening processes. A subsequent increase in the synthesis time leads to the predominant formation of an α-FeOOH phase due to the oxidation of Fe3O4. As a result of the work, a methodological scheme for the analysis of iron oxide and oxyhydroxide ANPs was developed.


Introduction
Recently, one-dimensional (1D) nanosized structures (nanorods, nanowires, nanoellipsoids, nanoneedles) including iron oxides and oxyhydroxides have been in the focus of scientific research due to their fundamental importance and practical significance for materials science and medicine [1][2][3][4]. For example, magnetite (Fe 3 O 4 ) nanorods have found many applications in industry as magnetic storage devices, catalysts, cooling devices, gas sensors, electrodes in lithium-ion batteries, as well as in various medical diagnostics contexts [5,6]. Anisotropic nanoparticles (ANPs) of FeOOH can be used as selective sorption materials [7], catalysts [8], and templates for the synthesis of other iron oxides compounds [9]. The transition from traditional isotropic to ANPs is accompanied by a change in their optical (spectral), biological, magnetic properties, chemical reactivity, and catalytic activity [10]. This is demonstrated not only for iron-based nanoparticles, but also for other valuable nanoobjects [11,12]. Anisotropic gold nanoparticles of different aspect ratio exhibit a shift of the surface plasmon-absorption spectra further into the near-infrared region that improve in vivo tissue penetration in comparison with isotropic nanoparticles [13]. ANPs offer

Synthesis of Anisotropic Nanoparticles
In order to synthesize ANPs, we used a method adapted from Lian et al. [27]. The calculated amount of FeCl 3 ·6H 2 O (6.756 g, 0.250 M), FeSO 4 ·7H 2 O (3.426 g, 0.123 M), urea (12.0 g, 2.0 M), and 100 mL of deionized water were placed into a two-necked round flask equipped with a reverse condenser ( Figure 1). The obtained solution was stirred by magnetic stirrer at 90-95 • C in an oil bath (300 rpm). After 3,6,9,12,18, and 24 h of the synthesis, a 15 mL probe sample was taken for further study. The resulting particles were separated by the magnet and washed several times with deionized water to a neutral pH. oxyhydroxide nanoparticles and their detailed analysis in order to determine a set of analytical methods for studying the structure, composition, and physicochemical properties of individual iron oxides (oxyhydroxide) and their mixtures.

Synthesis of Anisotropic Nanoparticles
In order to synthesize ANPs, we used a method adapted from Lian et al. [27]. The calculated amount of FeCl3•6H2O (6.756 g, 0.250 M), FeSO4•7H2O (3.426 g, 0.123 M), urea (12.0 g, 2.0 M), and 100 mL of deionized water were placed into a two-necked round flask equipped with a reverse condenser (Figure 1). The obtained solution was stirred by magnetic stirrer at 90-95 °C in an oil bath (300 rpm). After 3,6,9,12,18, and 24 h of the synthesis, a 15 mL probe sample was taken for further study. The resulting particles were separated by the magnet and washed several times with deionized water to a neutral pH.

Characterization of the Samples
The phase composition of ANPs was analyzed by XRD on a Shimadzu XRD 6000 (Shimadzu Corporation, Kyoto, Japan) diffractometer (CuKα radiation) at a range of 10 to 70° (scan rate 1°/min). The XRD data were analyzed by the Rietveld method using the Match! Software (v. 3.13, Bonn, Germany) to assess the phase composition of the samples.
Raman spectra were recorded on an NT-MDT system (NT-MDT Spectrum Instruments, Zelenograd, Russia) equipped with a 100× objective. Excitation was performed Lian et al. suggest that the formation of Fe 3 O 4 occurs according to the following chemical reactions [25]:

Characterization of the Samples
The phase composition of ANPs was analyzed by XRD on a Shimadzu XRD 6000 (Shimadzu Corporation, Kyoto, Japan) diffractometer (CuKα radiation) at a range of 10 to 70 • (scan rate 1 • /min). The XRD data were analyzed by the Rietveld method using the Match! Software (v. 3.13, Bonn, Germany) to assess the phase composition of the samples.
Raman spectra were recorded on an NT-MDT system (NT-MDT Spectrum Instruments, Zelenograd, Russia) equipped with a 100× objective. Excitation was performed with a laser at wavelengths of 633 nm with a maximum power of 60 mW. To prevent heating and oxidation of magnetite, no more than 1% of the laser power was used.
To characterize the surface of ANPs, X-ray photoelectron spectroscopy (XPS) was performed using a Thermo Fisher Scientific XPS NEXSA spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with a monochromated Al Kα Alpha X-ray source working at 1486.6 eV. XPS survey spectra were acquired at a pass energy of 200 (eV) and energy resolution of 1 eV from the surface area of 200 µm 2 . The high-resolution spectra were Nanomaterials 2022, 12, 4321 4 of 18 acquired at a pass energy of 50 eV and energy resolution 0.1 eV. A flood gun was used to compensate for the charge.
Prior to the study of the morphology, a conductive Au coating was deposited on the samples. SEM (Quanta 200 3D electron microscope (FEI Company, Hillsboro, OR, USA)) equipped with the energy dispersive spectroscopic analysis (EDS) (FEI Company, Hillsboro, OR, USA) was performed to evaluate the changes in the morphology and elemental composition of the samples.
The structure of the samples was studied using HRTEM (ThemisZ electron microscope, Thermo Fisher Scientific, Waltham, MA, USA) with an accelerating voltage of 200 kV and a limiting resolution of 0.07 nm. The images were recorded using a Ceta 16 CCD sensor (Thermo Fisher Scientific, Waltham, MA, USA). The device is equipped with a SuperX (Thermo Fisher Scientific, USA) energy-dispersive characteristic X-ray spectrometer (EDX) with a semiconductor Si detector with an energy resolution of 128 eV.
The magnetic properties of ANPs were investigated at a temperature of 295 K with an external pulsed magnetic field up to 6.5 kOe using a pulsed magnetometer (Tomsk State University, Tomsk, Russia).

Results and Discussion
To study the formation of ANPs in detail and determine occurring reactions and processes, an evaluation was carried out at different synthesis times. The structure, phase and chemical composition of ANPs were studied by XRD, Raman spectroscopy, and XPS. Figure 2a shows the XRD pattern of the samples at different synthesis times. After 3 h of synthesis, XRD patterns corresponded to akaganeite (β-FeOOH) ( [38]. After 12 h of synthesis, the intensity of magnetite peaks decreases and the intensity of goethite peaks increases. The quantitative phase composition of the samples is presented in Table 1. The results of Raman spectroscopy ( Figure 2b) are in agreement with XRD data. The spectrum of the sample after 3 h of synthesis contains the characteristic peak of β-FeOOH at 310, 419, and 725 cm −1 . The Raman spectra of the samples after 6,9,12,18, and 24 h of synthesis include two peaks of α-FeOOH located at 299 and 386 cm −1 , in addition to other less intense peaks at 245, 299, 386, 481, and 552 cm −1 . An increase in the synthesis time up to 12 h leads to an intensification of the characteristic peak of Fe 3 O 4 at 670 cm −1 . A further increase of the time of the synthesis is accompanied by a gradual decrease of the intensity of the 670 cm −1 peak. A broadening of the magnetite peak is observed due to the formation of maghemite (γ-Fe 2 O 3 ), which generally occurs due to the presence of Fe 2+ in the Fe 3 O 4 structure as a result of oxidation both by air oxygen during the synthesis and by laser beam in the process of Raman spectra registration.
The SEM images of the obtained samples presented in Figure 3 reveal details of their morphology. After 3 h of synthesis, β-FeOOH needle-like structures began to form. Increase of the synthesis time should lead to transformation of β-FeOOH needles to α-FeOOH nanorods, which interact with Fe(OH) 2 during increasing pH of the solution (formation of ammonia via urea decomposition) according to the mechanism proposed by Lian et al. [27] to eventually form Fe 3 O 4 nanorods. However, SEM images in combination with XRD and Raman results do not reliably confirm the formation of magnetite nanorods. The SEM images of the obtained samples presented in Figure 3 reveal details of their morphology. After 3 h of synthesis, β-FeOOH needle-like structures began to form. Increase of the synthesis time should lead to transformation of β-FeOOH needles to α-FeOOH nanorods, which interact with Fe(OH)2 during increasing pH of the solution (formation of ammonia via urea decomposition) according to the mechanism proposed by Lian et al. [27] to eventually form Fe3O4 nanorods. However, SEM images in combination with XRD and Raman results do not reliably confirm the formation of magnetite nanorods.    The SEM images of the obtained samples presented in Figure 3 reveal details of their morphology. After 3 h of synthesis, β-FeOOH needle-like structures began to form. Increase of the synthesis time should lead to transformation of β-FeOOH needles to α-FeOOH nanorods, which interact with Fe(OH)2 during increasing pH of the solution (formation of ammonia via urea decomposition) according to the mechanism proposed by Lian et al. [27] to eventually form Fe3O4 nanorods. However, SEM images in combination with XRD and Raman results do not reliably confirm the formation of magnetite nanorods.  XPS analysis was used to more precisely reveal the chemical, phase, and molecular composition of the synthesized ANPs ( Figure 4). Survey spectra demonstrate predominant peaks of Fe and O. There are also peaks of C as an adventitious carbon and peaks of S and Cl of initial iron salt at the earliest stage of the synthesis. High-resolution Fe 2p and O 1s XPS spectra in the case of all time intervals of the synthesis have similar peaks because of the close position of peaks energy of α-FeOOH and Fe 3 O 4 as the main phases [39]. Fe 2p spectra include Fe p 3/2 and Fe 2p 1/2 peaks at 711 and 724 eV, respectively, which are characteristic of iron oxide and oxyhydroxide derivatives ( XPS analysis was used to more precisely reveal the chemical, phase, and molecular composition of the synthesized ANPs ( Figure 4). Survey spectra demonstrate predominant peaks of Fe and O. There are also peaks of C as an adventitious carbon and peaks of S and Cl of initial iron salt at the earliest stage of the synthesis. High-resolution Fe 2p and O 1s XPS spectra in the case of all time intervals of the synthesis have similar peaks because of the close position of peaks energy of α-FeOOH and Fe3O4 as the main phases [39]. Fe 2p spectra include Fe p3/2 and Fe 2p1/2 peaks at 711 and 724 eV, respectively, which are characteristic of iron oxide and oxyhydroxide derivatives (Table 2) [40]. Deconvoluted spectra contain peaks of Fe 3+ and Fe 2+ presented in the surface of all studied samples. XPS O 1s spectra demonstrate 4 predominant peaks at 528.7, 530.3, 531.6, and 532.3 eV corresponding to surface OH, lattice Fe-O, lattice Fe-OH, and adsorbed H2O, respectively [41]. An increase in the Fe/O ratio during synthesis (until 12 h) is connected with decreased oxygen content due to the formation of Fe3O4. After 12 h of synthesis, the Fe/O ratio starts to decrease. A slight decrease in the Fe/O ratio after 12 h may be due to the formation of OH groups on the surface of the samples.  For a detailed study of the morphology and fine structure of ANPs, TEM images were recorded. Figure 5 shows TEM and HRTEM images of the sample obtained during 12 h.  For a detailed study of the morphology and fine structure of ANPs, TEM images were recorded. Figure 5 shows TEM and HRTEM images of the sample obtained during 12 h.
Analysis of microphotographs indicates that the sample contains mainly ANPs of various sizes (Figure 5a,b). The length and diameter distributions of the ANPs are shown in Figure 6. The sample also contains submicron particles of about 200-600 nm in size, which are agglomerates of two or more particles (Figure 5a).
In addition to the XRD and Raman spectroscopy results, the phase composition of the ANPs is described by the detailed study of HRTEM images (Figure 5d,e) using interplanar spacing (FFT analysis). Since the complexity of the analysis of iron oxides and oxyhydroxides ANPs lies in their close interplanar spacings (Table 3), the FFT analysis should simultaneously take into account several values of interplanar spacings. According to FFT analysis of submicron isotropic particles (Figure 5d), these relate to Fe 3 O 4 to confirm interplanar spacings of 2.4, 2.5, 2.1, 4.8, and 2.9 Å corresponding to (222), (311), (400), (111), and (220) crystallographic planes, respectively. Conversely, ANPs correspond to the α-FeOOH phase that supports by the characteristic interplanar spacings of 2.5, 4.9, and 2.5 Å corresponding to (040), (020), and (101) crystallographic planes, respectively (Figure 5e). The investigated interplanar spacings in the HRTEM images are in agreement with the XRD data (Table 3). In addition, measured angles between the planes equal 55.7 • and 63.9 • also confirm that these particles belong to the Fe 3 O 4 ( Figure 5d) and α-FeOOH (Figure 5e) phases, respectively. Analysis of microphotographs indicates that the sample contains mainly ANPs of various sizes (Figure 5a,b). The length and diameter distributions of the ANPs are shown in Figure 6. The sample also contains submicron particles of about 200-600 nm in size, which are agglomerates of two or more particles (Figure 5a). In addition to the XRD and Raman spectroscopy results, the phase composition of the ANPs is described by the detailed study of HRTEM images (Figure 5d,e) using interplanar spacing (FFT analysis). Since the complexity of the analysis of iron oxides and oxyhydroxides ANPs lies in their close interplanar spacings (Table 3), the FFT analysis should simultaneously take into account several values of interplanar spacings. According to FFT analysis of submicron isotropic particles (Figure 5d), these relate to Fe3O4 to confirm interplanar spacings of 2.4, 2.5, 2.1, 4.8, and 2.9 Å corresponding to (222), (311), Analysis of microphotographs indicates that the sample contains mainly AN various sizes (Figure 5a,b). The length and diameter distributions of the ANPs are s in Figure 6. The sample also contains submicron particles of about 200-600 nm in which are agglomerates of two or more particles (Figure 5a). In addition to the XRD and Raman spectroscopy results, the phase composit the ANPs is described by the detailed study of HRTEM images (Figure 5d,e) usi terplanar spacing (FFT analysis). Since the complexity of the analysis of iron oxide oxyhydroxides ANPs lies in their close interplanar spacings (Table 3), the FFT an should simultaneously take into account several values of interplanar spacings. A  In some cases of ANPs morphology micro pores were observed (Figure 5c). They can be formed during the dehydroxylation of α-FeOOH in the process of TEM study under the influence ultra-high vacuum and the energy of the beam of the microscope [45]. This is consistent with the XRD results, since the nonuniform broadening of the diffraction peaks in the samples can be explained by the formation of pores [46]. At the same time, a layer of hematite (α-Fe 2 O 3 ) of about 13.5 nm with interplanar spacing of 3.7 Å is observed on the surface of the goethite ANPs [47], which is formed as a result of its dehydroxylation and transformation to a more stable α-Fe 2 O 3 phase (Figure 5d) [19]. These disordered crystallites have sizes within the range of 1-3 nm. Thus, the obtained after 12 h reaction mixture includes both isotropic Fe 3 O 4 nanoparticles and ANPs of α-FeOOH.
It is known that the magnetic properties of magnetite particles can be affected by various factors, of which the most significant are the size and shape of crystallites, the presence of weakly magnetic impurities and crystallinity. The value of the Ms of magnetite decreases with a low crystallite size and with the presence of weakly magnetic impurities in the material [48,49]. The hysteresis loops of the synthesized nanoparticles Fe 3 O 4 and their magnetic characteristics are shown in Figure 7 and in Table 4, respectively. Based on obtained results and the mechanism suggested by Lian et al. [27], the possible pathway of reaction during ANPs formation was proposed. While the observed chemical processes during ANP synthesis are in general agreement with the reactions described in the Materials and Methods section, some differences are observed. A scheme  Synthesis for 3 h did not lead to the formation of the magnetite phase. The sample consisted of akageneite β-FeOOH, which, although paramagnetic at a temperature of 300 K, did not exhibit significant magnetic properties. An increase in the synthesis time leads to the formation of magnetite nanoparticles, which is confirmed by XRD analysis (Figure 2a). An increase in the content of the Fe 3 O 4 phase leads to a growth in M s values up to 46.83 ± 0.29 emu/g (synthesis time 12 h). This is confirmed by the data on the phase composition, which are presented in Table 1. The saturation magnetization is much lower than the M s value of a magnetite polycrystal, which is 92 emu/g [50]. This is due to the low content of the magnetic phase and the low-dimensional state of Fe 3 O 4 crystallites ( Figure 5). Another reason for the decrease in magnetic properties both at the beginning and at the end of magnetite synthesis is that the α-FeOOH particles surrounding the Fe 3 O 4 particles reduce the magnetic dipole interactions between neighboring magnetic particles.
A further increase in the synthesis time of magnetite particles leads to a decrease in the M s value to 1.78 ± 0.04 emu/g (the synthesis time is 24 h). This is explained by the transformation of Fe 3 O 4 into the antiferromagnet α-FeOOH at a Curie temperature of 393 K. Despite α-FeOOH being antiferromagnetic, it has a nonzero magnetic moment due to incomplete compensation of the magnetic moments of the sublattices when in the form of nanoparticles.
While hysteresis losses are practically absent, the coercive force increases with the duration of the synthesis. The higher H c values as compared to pure magnetite [51] can be explained in terms of the presence of the α-FeOOH phase in the samples. Due to the canting of the moments in the magnetic structure of goethite, four sublattices can be distinguished instead of two. As a result, the coercivity of pure goethite can reach high values, which is possibly attributable to permanent magnetism [52]. It should be mentioned that M s values in the case of ANPs of Fe 3 O 4 obtained by the other research groups differ from both the analogical isotropic nanoparticles and the bulk material [53]. For instance, M s of Fe 3 O 4 ANPs is 84 [54], 54 [32], 28 [35], and 17 [9] emu/g (more values presented in Table 5). Such an inhomogeneity in the values of the M s is associated with high shape anisotropy of ANPs which prevents them from magnetization in directions other than along their easy magnetic axes [29], with the increase in surface spin canting effect depending on the particle size [53], and with a presence of nonmagnetic side phases [23]. The latter factor has a greater effect on the M s in the case of the synthesis of ANPs obtained by the co-precipitation method. Thus, to reliably confirm the structure and phase composition in order to explain the magnetization characteristics of ANPs, it is necessary to use a combination of XRD, Raman, HRTEM, and pulse magnetometry or vibrating-sample magnetometry (VSM).
Based on obtained results and the mechanism suggested by Lian et al. [27], the possible pathway of reaction during ANPs formation was proposed. While the observed chemical processes during ANP synthesis are in general agreement with the reactions described in the Materials and Methods section, some differences are observed. A scheme of the proceeding stages is presented in Figure 8. Since magnetite ANPs is obtained by the co-precipitation of iron salt in two oxidation states, the solubility of formed hydroxides (oxyhydroxides) should be taken into account for the determination of reaction routes. Since the solubility product of Fe(OH) 3 (K sp (Fe(OH) 3 ) = 2.79·10 −39 ) is much smaller than that of Fe(OH) 2 (K sp (Fe(OH) 2 ) = 4.87·10 −17 ) [55], Fe(OH) 3 is the first to be precipitated during the reaction. As one of the condensed aqua-hydroxo complexes, Fe(OH) 3 is known not to exist in solution in such a form [56].  (Figure 8b). While the adsorption of Fe 2+ ions onto the α-FeOOH (Fe 3+ source) surface at the next stage proceeds according to a mechanism suggested by Lian et al. [27], this does not occur in our synthesis. To explain this regularity, we should consider two possible reaction pathways in which Fe 2+ ions can participate. On the one hand, Fe 2+ ions can adsorb on the surface of α-FeOOH ANPs and then transform to Fe3O4 ANPs during further precipitation reaction (Figure 8). On the other hand, Fe 2+ ions can be oxidized by air oxygen (the reaction system is not isolated) and form isotropic submicron nanoparticles of Fe3O4 (Figure 8b-d). However, the latter does not lead to the formation of anisotropic morphology in the case of Fe3O4 due to its characteristic cubic crystal structure and different rate of Fe 2+ and Fe 3+ deposition. The probability of one of the occurring processes will be determined by their thermodynamic and kinetic regularities. The kinetics of Fe 2+ oxidation was demonstrated by Morgan and Lahav [59]. They noticed a high rate of the oxidation process, which can be thermodynamically and kinetically enhanced by surface hydroxyl groups of α-FeOOH. Later, Chen and Thompson found that the rate of Fe 2+ oxidation increases significantly in the presence of α-FeOOH (4 h vs. a few minutes), which catalyzes the process [60]. These statements, At first, hydrolysis of FeCl 3 is accompanied by the formation of β-FeOOH nanoellipsoids (including dehydration of iron hydroxide) that act as a template for the further synthesis of ANPs (Figures 2a and 3a). Thus, this process can be considered as a shapedetermining stage. Predominant formation of β-FeOOH at the earliest stages is due to the stabilization of its structure by Cl − ions incorporating into the tunnels located in it structure (Figure 8a) [57]. The initial concentration of FeCl 3 affects the size of β-FeOOH nanoellipsoids [58]. For this reason, it should be taken into account when synthesizing ANPs of a specified shape and size.
During synthesis at 95 • C, a gradual decomposition of urea occurs to generate OH − ions. With increasing synthesis time and pH of the solution, Cl − ions are replaced by OH − ions to transform β-FeOOH into α-FeOOH (Figure 8b). While the adsorption of Fe 2+ ions onto the α-FeOOH (Fe 3+ source) surface at the next stage proceeds according to a mechanism suggested by Lian et al. [27], this does not occur in our synthesis. To explain this regularity, we should consider two possible reaction pathways in which Fe 2+ ions can participate. On the one hand, Fe 2+ ions can adsorb on the surface of α-FeOOH ANPs and then transform to Fe 3 O 4 ANPs during further precipitation reaction (Figure 8). On the other hand, Fe 2+ ions can be oxidized by air oxygen (the reaction system is not isolated) and form isotropic submicron nanoparticles of Fe 3 O 4 (Figure 8b-d). However, the latter does not lead to the formation of anisotropic morphology in the case of Fe 3 O 4 due to its characteristic cubic crystal structure and different rate of Fe 2+ and Fe 3+ deposition. The probability of one of the occurring processes will be determined by their thermodynamic and kinetic regularities. The kinetics of Fe 2+ oxidation was demonstrated by Morgan and Lahav [59]. They noticed a high rate of the oxidation process, which can be thermodynamically and kinetically enhanced by surface hydroxyl groups of α-FeOOH. Later, Chen and Thompson found that the rate of Fe 2+ oxidation increases significantly in the presence of α-FeOOH (4 h vs. a few minutes), which catalyzes the process [60]. These statements, which are in good agreement with our results, explain the formation of isotropic submicron Fe 3 O 4 particles. It is important to note that, although γ-Fe 2 O 3 can also form, its presence is difficult to determine by ex situ methods [19]. The formation of α-Fe 2 O 3 can be observed due to its greater thermodynamic stability as demonstrated by the HRTEM method. A further increase in the synthesis time (up to 12 h) is accompanied by the oxidation of Fe 3 O 4 to α-FeOOH ANPs at high pH due to the presence of air oxygen (Figure 8b-d) [61]. After 24 h of synthesis, almost all of the Fe 3 O 4 converts to α-FeOOH (Figure 8e) as confirmed by XRD and Raman spectroscopy results. The dependence of M s on synthesis time is also in agreement with proposed reaction pathway. After 12 h of synthesis, M s decreases to an almost zero value to support a very low quantity of Fe 3 O 4 due to its transformation to α-FeOOH. Thus, while the co-precipitation method is not suitable for the synthesis of pure Fe 3 O 4 ANPs, it can be used as a method of synthesis of αand β-FeOOH with given morphology.
Along with the features of the reactions occurring during the synthesis of ANPs of iron derivatives, the set of research methods and the sequence of their use are of great importance. The known sets of analytical methods used in the study of ANPs are summarized in Table 5. Although the XPS method cannot be used to distinguish mixture of iron phases due to the close values of binding energies, it is the most informative approach for the analysis of nanoparticles with a modified surface. For this reason, XPS is not included in Table 5. A review of the methods used for the study of iron oxides and oxyhydroxides ANPs (Table 5) shows that the most commonly used research methods are XRD, HRTEM, and VSM, which are used to characterize phase composition, morphology (crystal structure), and magnetic properties, respectively. From the point of view of phase composition, determination of the maghemite (γ-Fe 2 O 3 ) phase by XRD is difficult due to the similarity of its reflection (and crystal structure) to Fe 3 O 4 . Thus, another method or combination of methods is required. For instance, Raman spectroscopy, which represents a powerful method for determining a γ-Fe 2 O 3 phase which demonstrates a characteristic band at 700 cm −1 of Raman shift [62]. Here, it is necessary to avoid high laser power that can affects the real phase composition [63]. Although infrared spectroscopy is a less informative method for iron oxygen-containing compounds, it can be useful for analyzing modified iron oxides (hydroxides) [64] and their active sites by adsorption of specific probe molecules in the case of catalytic application [65]. The morphology and shape of iron-based ANPs are studied by SEM and TEM (HRTEM). While analysis of SEM and HRTEM images can be used to demonstrate the shape and size of synthesized ANPs or isotropic nanoparticles, a comprehensive analysis of ANPs should include evaluation of interplanar spacings; however, for iron-and oxygen-containing compounds, these may have similar values. Thus, it becomes necessary to measure several values of interplanar spacings and angles between definite crystallographic planes [66]. In the case of magnetic iron oxide ANPs, M s is a basic parameter that should be measured to determine their magnetic characteristics. Saturation magnetization mainly depends on phase composition, particle size, and the presence of organic or polymer surface modifiers [63]. However, M s is usually measured both to confirm the purity of the magnetic phase and to evaluate its potential as a magnetic component of magnetoactive materials for different application areas.
A methodological scheme for the analysis of iron oxide and oxyhydroxide ANPs based on our results and those of other studies is presented in Figure 9. The first group of methods is aimed at establishing the structure, phase, and functional composition of studied nanoparticles. First, it is necessary to confirm by XRD that the studied sample is a monophasic compound containing exactly the target phase. In addition to data on the phase composition, Raman and Infrared spectra should be registered to identify the presence of all iron oxides and oxyhydroxides ANPs that cannot be reliably determined by XRD (as an example, γ-Fe 2 O 3 ). Moreover, IR spectroscopy and XPS are useful for analysis of the modified surface of ANPs. The second group of methods, including SEM and TEM (HRTEM), are used to determine the morphology of the studied iron-based nanoparticles. As well as being used to confirm the anisotropic shape and form of the synthesized nanoparticles, these methods are used to calculate the size distribution and evaluate the tendency to agglomeration. The third group of methods is aimed at confirming the phase composition and crystal structure of ANPs, as well as studying their magnetic properties. The use of these methods, along with traditional methods for studying the structure, phase, and functional composition (methods of group 1), is due to the rather similar properties of various iron oxides and oxyhydroxides, which complicates the reliable assessment of the properties of studied iron based ANPs. Assignment of ANPs to a certain phase should be carried out based on detailed FFT analysis of several interplanar spacings corresponding to a definite set of crystallographic planes and the angles between them. In the case of magnetic iron oxide ANPs, their magnetic properties should be determined, mainly in terms of M s . In general, the analysis of hysteresis curves is used to estimate the value of the M s , coercivity, residual magnetization, as well as to determine the magnetic behavior of nanoparticles (e.g., paramagnetic, ferromagnetic, ferrimagnetic). Thus, a proposed methodological scheme is aimed at the standardization of research methods for obtaining the most reliable data on the structure, phase composition, and other important properties of iron oxides and oxyhydroxides ANPs. This methodological scheme will be also useful not only for iron-based ANPs, but also for isotropic ones, as obtained by different synthesis procedures. rather similar properties of various iron oxides and oxyhydroxides, which complicates the reliable assessment of the properties of studied iron based ANPs. Assignment of ANPs to a certain phase should be carried out based on detailed FFT analysis of several interplanar spacings corresponding to a definite set of crystallographic planes and the angles between them. In the case of magnetic iron oxide ANPs, their magnetic properties should be determined, mainly in terms of Ms. In general, the analysis of hysteresis curves is used to estimate the value of the Ms, coercivity, residual magnetization, as well as to determine the magnetic behavior of nanoparticles (e.g., paramagnetic, ferromagnetic, ferrimagnetic). Thus, a proposed methodological scheme is aimed at the standardization of research methods for obtaining the most reliable data on the structure, phase composition, and other important properties of iron oxides and oxyhydroxides ANPs. This methodological scheme will be also useful not only for iron-based ANPs, but also for isotropic ones, as obtained by different synthesis procedures.      8 Oe] (hollow ellipsoids) [72] a Data of the corresponding method presented in square brackets; b D-diameter of ANPs; c L-length of ANPs; d d-interplanar spacing; e AR-aspect ratio; f IR spectra also include bands of Fe 3 O 4 modifiers; g Phase composition depends on initial Fe(NO 3 ) 3 concentration.

Conclusions
The synthesis of iron oxide and oxyhydroxide ANPs was carried out by co-precipitation of Fe 2+ and Fe 3+ salt in the presence of urea. By varying the synthesis time, changes of their morphology, phase, and chemical composition, as well as their magnetic properties, were studied by SEM, XRD, Raman spectroscopy, XPS, HRTEM, and pulse magnetometry. XRD and Raman spectroscopy results demonstrate that β-FeOOH is formed at the early stages of the synthesis. The formation of the Fe 3 O 4 phase occurs until 12 h of the synthesis and reaches a maximum Fe 3 O 4 phase content of 47.4%. The subsequent increase of time leads to predominant formation of α-FeOOH phase due to oxidation of Fe 3 O 4 by air oxygen. Such observations are in good agreement with M s values which reach a maximum (46.83 emu/g) after 12 h of the synthesis. The presence of both isotropic and anisotropic particles after 12 h is confirmed by SEM and HRTEM. The latter in combination with FFT analysis allows determining phase composition via measurements of interplanar spacings and angles between specific crystallographic planes.
Based on the obtained experimental data, refined reaction pathways of the formation of iron oxides and oxyhydroxides ANPs are proposed. The hydrolysis of FeCl 3 occurring at the beginning of the process (3 h) leads to the formation of β-FeOOH nanoellipsoids due to the stabilization of its structure by Cl − ions. This stage can be considered as shape-determining due to the formation of rod-shaped structures depending on the initial concentration of FeCl 3 . Then, β-FeOOH to α-FeOOH transformation takes place because of the exchange of Cl − by OH − ions forming via urea decomposition. At increased synthesis time and OH − ion concentration, along with rod-shaped α-FeOOH nanoparticles, isotropic Fe 3 O 4 submicron particles are formed during Fe 2+ oxidation and Ostwald ripening process. Compared to the adsorption of Fe 2+ ions on the surface of rod-shaped α-FeOOH, which should lead to the formation of Fe 3 O 4 nanorods, these tend to favor kinetically rapid oxidation accompanied by the formation of submicron isotropic Fe 3 O 4 particles. The isotropic shape of Fe 3 O 4 particles connected with their cubic crystal structure and the difference in Fe 2+ and Fe 3+ deposition are factors that hinder the formation of ANPs. Along with a description of the chemistry of the process, a methodology for analyzing ANPs based on iron oxide or oxyhydroxide is proposed. Such a methodological scheme will be useful for carrying out a detailed analysis of the chemistry, phase composition, and structure of iron oxides and oxyhydroxides ANPs.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.