Formation of Fe(0)-Nanoparticles via Reduction of Fe(II) Compounds by Amino Acids and Their Subsequent Oxidation to Iron Oxides

1 Department of Nutrition and Food Assessment, Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia 2 FLOP Inc., Mandľová 37, 851 10 Bratislava, Slovakia Department of Physical Chemistry, Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia Department of Inorganic Chemistry, Institute of Inorganic Chemistry, Technology and Materials, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia Department of Nuclear Physics and Technology, Institute of Nuclear and Physical Engineering, Faculty of Electrical Engineering and Information Technology, Slovak University of Technology, Ilkovičova 3, 812 19 Bratislava, Slovakia 6 Food Research Institute, Priemyselná 4, P.O. Box 25, 824 75 Bratislava, Slovakia 7 Institute of Materials Science, Faculty of Materials Science and Technology, Slovak University of Technology, Paulínska 16, 917 24 Trnava, Slovakia


Introduction
e standard redox potential of the system Fe(II) → Fe(0) is −0.41 V and Fe(III) → Fe(0) is −0.04 V. Consequently, iron cannot be easily obtained by the reduction of its compounds [1].Attention is paid to the preparation of micro-and nanoparticles of elementary iron.A well-known method for the preparation of iron microparticles is by thermal decomposition of iron pentacarbonyl, Fe(CO) 5 , yielding particles of sizes within 500-700 nm [2].e elementary iron prepared in this way is widely used in electronics (recording media, ferrites) and in pharmacy as an iron dietary supplement.It is also possible to prepare Fe(0) nanoparticles by the reduction of ferrous compounds with agents such as NaBH 4 , N 2 H 4 , or NaH 2 PO 2 in the presence of a protective colloid [3]; in that paper, the iron particles were reoxidized giving -Fe 2 O 3 with a particle size of about 4 nm.An analogous method, without the protective colloid, led to Fe nanoparticles of the size 50-80 nm [4,5].Fe nanoparticles can also be prepared by the reduction of Fe(II) compounds by hydrogen at 700 ∘ C [6].An efficient method for the preparation is the electrochemical reduction providing Fe nanoparticles with a diameter of F 1: UV/VIS absorption spectra of Fe nanoparticles dispersed in water before (solid, black) and aer opening to air (dashed, red).
nanoparticles about 39 nm [7,8].Biological reductions of Fe(II) to Fe(0) employing microorganisms have also attracted much attention [9][10][11][12][13].Another interesting way of preparing Fe(0) nanoparticles is by the reduction of Fe(II) in transferrin by ascorbic acid [14].Fe nanoparticles can also be prepared by the pyrolysis of organic Fe(II) salts, such as oxalates [15][16][17].In this way it is possible to prepare a pyrophoric material applicable, inter alia, for military purposes [18].In biological systems, reduction of Fe(II) to Fe(0) occurs very likely much easier.As an example, ferritins can be mentioned [19][20][21].Samples of brain taken post mortem contain minerals, mainly goethite in protein nanotube.is mineral obviously cannot be physiologically active since it is insoluble, and it should not be encountered in living systems.Hence, it can be supposed that reduction of Fe(II) to Fe(0) takes place under anaerobic conditions.Subsequently, Fe(0) nanoparticles are spontaneously oxidized in the presence of oxygen so yielding iron oxides.e aim of this paper is to prove that the formation of Fe nanoparticles by the reduction of Fe(II) to Fe(0) with amino acid ligands in the coordination sphere of the central Fe(II) atom in living systems is feasible.

2.2.
Apparatus.e products were analyzed by UV/VIS, transmission electron microscopy (TEM), RTG diffractometry, Mössbauer spectroscopy, magnetometry, TG/DTA, and GC/MS.UV/VIS absorption spectra of iron nanoparticles were recorded employing a double-beam Shimadzu 3600 spectrometer.1 mm quartz cuvette �ow cell was used enabling the �lling of the sample under an inert atmosphere.
e TEM investigations were performed on Philips CM 300 LaB 6 microscope.An accelerating voltage of 300 kV was used.A holey carbon �lm/copper net served as support of the nanoparticles.
Mössbauer spectroscopy was performed in transmission geometry at room temperature (300 K).A conventional constant acceleration spectrometer working with a 57 Co/Rh source of radiation was employed.e experimentally measured spectra were �tted by the CO�FIT evaluation soware [22].
e magnetometry data were scanned by a SQUID magnetometer (MPMS-XL7, Quantum Design) in the DC detection mode.e measurements were conducted in two ways: (i) temperature dependence of the sample magnetic moment at temperatures from 1.9 to 300 K at constant magnetic �eld    T� (ii) �eld dependence of the sample magnetic moment within the range of  from 0 to 7 T at constant temperature   6 K. ermogravimetry (TG) measurements were performed using DTG-60 (Shimadzu).e temperature scale was calibrated to the fusion points of In, Sn, and Zn.e measurements were realized under oxygen and in an inert atmosphere of nitrogen at the heating rate of 10 K/min.
Gas chromatography (GC) was carried out using an Agilent Technologies 6890 gas chromatograph equipped with an Agilent Technologies 5973 inert mass selective spectrometer and with chromatographic column model no.J&W 122-503 E DB-5, 30 m × 0.25 mm × 0.5 m.

Synthesis.
A solution of 0.01 mol Fe 2 SO  ⋅7H 2 O in 300 mL of water (pH 7 ± 2) was placed in a three-necked �ask with a magnetic stirrer, thermometer, tube for inert gas inlet and dropping funnel, and with external heating.e solution was purged with an inert gas (nitrogen or argon) and well stirred.When the solution in the �ask was deaerated, the deareated solution of 0.02 mol of amino acid sodium salt in 50 mL of water was added in one portion.Within a few seconds, the complex of the amino acid with the Fe(II) cation was formed as a greyish-blue green dispersion.e dispersion in water was then very slowly heated.When the temperature reached 40 ∘ C, 0.04 mol of deareated solution of the sodium salt of the same amino acid in 100 mL water was added to the dispersion in one portion, and the pH was adjusted to 9.5-9.7 with the same amino acid.In the case of aliphatic amino acids, the reaction started immediately at this temperature.e reduction of aromatic amino acids needed a higher temperature, between 45 and 52 ∘ C. e metallic iron was quickly formed either as a metal mirror or as a yellow colloid.e colloid was transferred in an inert atmosphere from the �ask to a cuvette for UV/VIS measurement.en, the input of inert gas was stopped.e Fe(0) particles oxidized spontaneously with air or with oxygen to iron oxides.e oxides also form of a colloid in water.e UV/VIS spectra of the colloidal iron oxides were also recorded.en, the orange-brownish precipitate was �ltered using a ��chner funnel, well washed with demiwater and freely dried on air.e drying of the raw mixture of the iron oxides and organic by-products was puri�ed by re�ux, �rstly with toluene and secondly with methylalcohol, again �ltered with the ��chner funnel, washed with approx.�0 m� of diethylether and dried in air.

Results and Discussion
e reduction of Fe(II) coordination compounds by the anions of amino acids in an inert atmosphere is described here.In our paper [23], the amino acid degradation in the coordination sphere of Fe(II) complexes has been studied.As mentioned previously, a small increase of temperature leads to reduction of the cation of the central atom to metal iron.In [23] we described a conceivable reaction mechanism of the amino acid degradation catalyzed with Fe(II) complexes where the products formed are carbonyl compounds or carboxylic acids.In the case of the reduction of the central Fe(II) atom, the amino acids yield different products.If phenylalanine was used as a reducing agent, the dominant products identi�ed were amides of carboxylic acids, mainly phenylacetamide.
A spontaneous reduction of ferrous ions takes place in the range of pH 9.5-9.7.For pH higher than 9.7, a greater content of HO − ions is present in the solution.e HO − ion is a stronger electron-donor than the amino group so that it preferentially occupies the axial sites on the central Fe(II) ion where the reaction takes place [23].A similar situation occurs if the amino acid in the form of a zwitterion is added to the water suspension of the complex; then, the carboxylate anion O=C-O − is the electron donor.At pH between 9.5 and 9.7, the -NH 2 group of the amino acid preferentially coordinates with the central Fe(II) ion [23].e reactivity of the amino acid coordinated via the amino group obviously increases, so does the reactivity of the central Fe(II) ion.At lower temperature the degradation of amino acid takes place [23] while the reduction of the central ion to metallic iron occurs at increased temperature.
UV�VIS spectroscopy was employed to con�rm the existence of the iron nanoparticles both in pristine and oxidized form.Figure 1 shows the UV-VIS absorption spectra of the Fe nanoparticles dispersed in water before and aer opening to air.e optical spectra measured immediately aer �lling the sample under an inert atmosphere show a very similar pattern as found for pure Fe nanoparticles dispersed in water prepared by the electroexplosion of wires [24].ere is a broad peak at 352 nm and a peak at 262 nm.e peak at 352 nm is believed to be due to the remnants of collective oscillation of the surface plasmons.On keeping the sample under air, new absorption bands appeared due to oxidation with several maxima indicating various iron oxide nanoparticles.
Composition of the mixtures of iron oxides obtained by the oxidation of Fe(0)-nanoparticles, their size, and properties were studied by several methods.XRPD spectra of iron oxide nanoparticles are shown in Figure 2. e size of the iron oxide nanoparticles, calculated from the data of Figure 2 according to [25,26], is listed in Table 1.As can be seen, the size of the nanoparticles is about 10 nm.is fact is also con�rmed by TEM investigations (Figure 3).
Iron oxides present in nanoparticles were identi�ed using Mössbauer spectroscopy.In addition to the degree of oxidation, structural states of iron ions were also determined.e Mössbauer spectra of the samples prepared with various amino acid anions as reducing agents are shown in Figure 4; the corresponding spectral parameters are listed in Table 2. �t is noteworthy that a signi�cant relative fraction of goethite (iron oxyhydroxide) was revealed in all measured samples.e nanoparticles were further subjected to magnetic measurements.According to the recording functions (Figure 5), the samples can be divided into two groups.e �rst group includes the samples 1-3 with higher magnetoactivity which is manifested by the value of the mass susceptibility around   = 400 m 3 kg −1 and mass magnetization in saturation (at  = 7 T) over   = 60 A m 2 kg −1 .Magnetization measurements were done in the mode of the �eld decreasing.e value of remnant magnetization was   = 20 A m 2 kg −1 (at  = 46 K) which is roughly one-third of the saturation value and can be deducted from the graphs.e samples show the magnetic hysteresis with the value of the coercive �eld   = 003 T. e second group includes the samples 4-5 with lower magnetoactivity which is manifested by the and mass magnetization at saturation (at  =  T) over   = 30 A m 2 kg −1 .ese values are roughly half with regard to the �rst group.e value of remnant magnetization is   = 10 A m 2 kg −1 (at  =  K), and it is one-third of the saturation value.e samples show the magnetic hysteresis with the value of the coercive �eld   = 002 T. On the basis of these facts, it can be summarized that the magnetoactivity of the second group of samples is about a half compared with the �rst group.e detection of magnetic hysteresis in both groups of samples at the experimental temperature is signi�cant information.Increasing the temperature to room value, the pro�le of magnetic hysteresis is reduced (in the case of the complex 5, from the second group, hysteresis expires) (Figure 6).e differences of magnetic moments depending on the amino acid anion are quite interesting.If the central Fe(II) ion is reduced by aliphatic amino acids, the magnetic moment of nanoparticles is higher than in the case of the reduction by aromatic amino acids.is difference can be brought T 2: Spectral parameters derived from Mössbauer spectra of the investigated samples: -spectral area, -isomer shi,  hf -hyper�ne �eld (only for sextets).about by occlusion of degradation products of amino acids in agglomerates of iron oxide nanoparticles; the agglomerates are formed readily due to the magnetic properties of the nanoparticles.Preliminary thermogravimetry measurements revealed that the content of organic matter in the iron oxide nanoparticles was quite high.erefore, the nanoparticles were puri�ed by extraction with toluene and methanol.e aliphatic amino acids used for the reduction of Fe(II) oxidize yielding low-molecular liquid products which can be extracted by the organic solvents.e aromatic amino acids oxidize giving crystalline products with high temperatures of decomposition; these compounds cannot be extracted fully by the solvents used.is can be clearly seen comparing the TG records of iron oxide nanoparticles (Figure 7) where sodium alaninate and sodium phenylalaninate were used as the reducing agents.In the case of sodium alaninate, the mass loss of nanoparticles is about 3% up to 450 ∘ C; this mass loss corresponds obviously to the loss of occluded solvents (up to 150 ∘ C) and the loss of water by dehydroxylation at higher temperatures.In case of sodium phenylalaninate, the mass loss occurs in two stages where the total mass loss is 21% up to 450 ∘ C. e �rst stage corresponds to the decomposition of benzamide, the other one to the decomposition of phenylacetamide.�oth compounds were identi�ed by GC/MS.TG studies thus con�rm that aliphatic amino acids provide purer nanoparticles.
A presumptive mechanism of the reduction is shown in Scheme 1.At low temperature, only one ligand binds to the axial site of the complex as an anion of the amino acid [23].Contrary to the destruction of amino acids by Fe(II) complexes, at higher temperatures two anions of the amino acid are bound to the two axial sites.Such a complex compound may be unstable, and a concerted redox reaction takes place both at the central cation Fe(II) and the ligands bound at the axial sites.As seen from Scheme 1, the amino acid is oxidized at the -carbon atom simultaneously splitting off formiate anion.us, an amide of the acid, shorter by one carbon atom than the original amino acid, is formed.Since the reaction occurs simultaneously at both anions of the amino acid bound at the axial sites, two formiate anions may reduce Fe(II) to Fe(0) by the single-electron transfer under the liberation of CO 2 .e protons formed are neutralized with OH − anions which leads to a slight decrease of pH which was observed.e proposed mechanism elucidates the formation of Fe nanoparticles as well as of amides of carboxylic acids.
We also tried to reduce other transition metal cations as central atoms in coordination compounds with the ligands mentioned here.Co(II), Cu(II) and Mn(II) are not reduced under the conditions described.Exclusively, the reduction of Fe(II) complexes to Fe(0) with amino acids takes place upon the formation of nanoparticles.

Conclusions
A method for the formation of iron and iron oxide nanoparticles by the reduction of the central Fe(II) ion in the coordination compounds with amino acid ligands is described.e anion of the amino acid used as a ligand acts as the reducing agent.Conditions for the reduction are very mild; the temperature does not exceed 52 ∘ C, and the optimum pH is between 9.5 and 9.7.e process is very rapid; under the  conditions mentioned, the reduction is �nished within a few seconds.A mechanism of the reduction is suggested.
e metal iron precipitates as a mirror on the �ask or as a colloid in water.It is impossible to isolate the metal iron prepared in this way due to the very small size of the particles.For this reason the identi�cation of the product was carried out by measuring UV/VIS spectra of the iron nanoparticles in water.e iron nanoparticles were oxidized by oxygen yielding a mixture of iron oxides.Oxidation of Fe(0) to Fe(II) is also very rapid; it takes several seconds under air.e size and properties of iron oxides were studied by UV/VIS, TEM investigation, RTG diffractometry, Mössbauer spectroscopy, magnetometry, thermogravimetry, and GC/MS.
Reduction of Fe(II) by aliphatic amino acid anions runs slightly above the physiological temperature so that a similar reduction in nonheme types of metalloenzymes is likely.is could account for the course of some feverish diseases or could lead to in situ synthesis of iron oxide nanoparticles for diagnostic and treatment.

F 3 :
TEM photographies of iron oxide nanoparticles prepared by the reduction of Fe(II) with alanine and phenylalanine.edegradation of amino acids took place between 20 and 25 ∘ C. Contrary to paper[23], a slight temperature increase leads to the reduction of the central Fe(II) atom to elemental iron instead of the expected degradation of amino acids since the reduction of Fe(II) to Fe(0) occurs between 40 and 52 ∘ C. Such a sharp temperature boundary is very atypical for the reduction of Fe(II).An exception is the reduction of ferrous cation to elemental iron by strong reducing agents, such as complex hydrides, hydrazine, or hypophosphites[4][5][6].

F 7 :
TG records of iron oxide nanoparticles prepared by the reduction of Fe(II) with alanine and phenylalanine, nitrogen atmosphere, heating rate 10 K/min.

Fe II S 1 :
Suggested mechanism of the reduction of Fe(II) to Fe(0) with amino acid ligands in the coordination sphere of central Fe(II) atom.