Mössbauer spectroscopy: epoch-making biological and chemical applications

Chemistry

As part of the chemistry these complexes, a short review on the synthesis of bis-(arene) iron complexes and reactions associated with direct and indirect replacement of arene ligands in these complexes is presented. These reactions lead to a broad variety of novel iron sandwich compounds that now play an important role in the development of specific areas of metaloorganic and metallacarborane chemistry of iron. The review comprises recent synthetic routes and approaches to iron sandwich complexes [1], of which the most significant have been arene and cyclohexadienyl ferradicarba- and ferratricarba-boranes, together with new types of piano-stool structured compounds. Ferrocene chemistry is followed by developments in the area of η⁶-arene iron chemistry, in which the title [FeAr₂]²⁺ [dication complexes (where Ar=η⁶-arene)] play an important role in the synthesis of other iron containing compounds. The most efficient preparative methods affording [FeAr₂]²⁺ complexes described in [1], [12], along with typical reactions of preparation that lead directly or indirectly to the replacement of one or both arene ligands by organic or carborane bases.

Biology

The iron in the body is indispensable; its concentration in biological tissues must be strictly controlled, because iron deficiency leads to cognitive and behavioral changes in humans. Free iron is toxic to cells as it acts as a catalyst in the formation of free radicals from reactive oxygen species via the Fenton Reaction [13]. Hence vertebrates use an elaborate set of protective mechanisms to bind iron in various tissue compartments. Within cells, iron is stored in a protein complex as ferritin or hemosiderin. Apoferritin binds to free ferrous iron and stores it in the ferric state. As ferritin accumulates within cells of the reticuloendothelial system, protein aggregates are formed as hemosiderin. Ferritin is a globular protein complex consisting of 24 protein subunits and is the primary intracellular iron-storage protein in both prokaryotes and eukaryotes, keeping iron in a soluble and non-toxic form. Ferritin that is not combined with iron is called apoferritin. Ferritin is a ubiquitous intracellular protein that stores iron and releases it in a controlled fashion. The protein is produced by almost all living organisms, including algae, bacteria, higher plants, and animals. In humans, it acts as a buffer against iron deficiency and iron overload [14]. Ferritin is found in most tissues as a cytosolic protein, but small amounts are secreted into the serum where it functions as an iron carrier. Plasma ferritin is also an indirect marker of the total amount of iron stored in the body; hence serum ferritin is used as a diagnostic test for iron deficiency anemia [15]. Inside the ferritin shell, iron ions form crystallites together with phosphate and hydroxide ions. The resulting particle is similar to the mineral ferrihydrite. Each ferritin complex can store about 4500 iron (Fe³⁺) ions [16], [17]. The heavy chain of ferritin also possesses ferroxidase activity; this involves the conversion of iron from the ferrous (Fe²⁺) to ferric (Fe³⁺) forms. This limits the deleterious reaction which occurs between ferrous iron and hydrogen peroxide known as the Fenton reaction which produces the highly damaging hydroxyl radical. Ferritin concentrations increase drastically in the presence of an infection or cancer. Endotoxin is an up-regulator of the gene coding for ferritin, thus causing the concentration of ferritin to rise. By contrast, organisms such as Pseudomonas, although possessing endotoxin, cause serum ferritin levels to drop significantly within the first 48 h of infection. Therefore, the iron stores of the infected body are denied to the infective agent, impeding its metabolism [18]. The concentration of ferritin has been shown to increase in response to stresses such as anoxia [19]; this implies that it is an acute phase protein [20]. A normal ferritin blood level, referred to as the reference interval is determined by many testing laboratories. The ranges for ferritin can vary between laboratories and shown in Table 1.

Art

Vivianite is a phosphate mineral (Fe₃(PO₄)₂·8H₂O) with monoclinic crystal structure. In nature, it is found either in the form of crystals or various types of aggregates. The color of the crystals is often very dark with strong pleochroism, earthy vivianite ranges from light grayish green to saturated blue, frequently being light blue [21], [22], [23], [24]. The final blue precipitate of vivianite was left to dry on air under laboratory conditions. The resulting color of the paint with vivianite was usually blue (commonly in mixture with lead white, sometimes also with other blue pigments like azurite, smalt or ultramarine), more rarely green (e.g. in complex mixtures of pigments including yellow lake and yellow earth) [22]. The temperature-related oxidation of vivianite can be study by Mössbauer spectroscopy. One of the studies regarding vivianite as a pigment describes the structural, color and oxidation changes at the temperatures of 200, 300 and 800°C. A more profound Mössbauer study of vivianite’s oxidation by increased temperatures was performed by Hanzel [25], [26]. However, it is not clearly stated at what temperature vivianite starts to oxidize by temperature influence, a graph suggests value around 105°C. To clarify the temperature-related behavior of vivianite especially during the initial state of the changes, a high-temperature X-ray diffraction study was performed; the full range of the temperature stability of the vivianite’s structure is published for the first time. For this purpose, a synthetic vivianite was prepared by an adapted procedure. Subsequently, the damaging effect on vivianite of relatively low temperatures has been monitored by Mössbauer spectroscopy. Afterwards, experiments with vivianite model paint layer samples were carried out in order to study the dependence of changes of color on structural changes that take place in the temperature interval up to 200°C. To test and simulate the actual conditions that may cause vivianite’s temperature degradation in works of art, oil-on-canvas mock-ups were prepared, and subsequently, relined in a traditional way. The changes were monitored by analysis of image histograms and micro-Raman spectroscopy [8].

Preparation of samples

The materials for chemistry, biology, and art were prepared by three different types of preparation with respect to their applications.

Chemistry

The [FeAr₂]²⁺ dications were isolated for the first time by E. O. Fischer and R. Bőtcher in 1956, via treatment of arenes with FeBr₂ in the presence of AlCl₃ [26]. The method was later improved by Helling and Braitsch, who reported syntheses by the reaction between mesitylene (mes) and anhydrous FeCl₂ in the presence of AlCl₃ in boiling cyclohexane [27]. At the room temperature reaction between polymethylated arenes C₆H_6-nMe_n (Me=CH₃) and FeCl₂ is shown in [1], [28]. The presence of AlCl₃ in heptane for 24–36 h proceeded with the formation of the samples. The details of the formation of series of the [Fe(ɳ⁶-C₆H_6-nMe_n)₂]²⁺ dications were described in [28], [29].

Biology

The samples from a human brain, human and horse spleen were extracted post mortem at the Department of Pathological Anatomy, Comenius University in Bratislava, in accordance with the Helsinki Declaration. The samples of human brain were extracted from the region Globus Pallidus which is a part of Basal Ganglia. Fresh, soft tissues were dried in a vacuum (lyophilized) and the resulting samples were obtained in a form of powder.

Art

The precipitation reaction was carried out according to a general principle:

3 Fe 2 + + 3 HPO 4 2 − → Fe 3 ( PO 4 ) 2 + 3 H + + PO 4 3 −

An appropriate amount of FeSO₄·7H₂O p.a. was dissolved in 5 wt.% solution of H₃PO₄ with a final molar concentration of Fe being 0.28 mol/L. The details of preparation of resulting product are described on [8].

Methods of characterization

Mössbauer spectroscopy has been chosen as a main tool of structural characterization of all type of the samples. Mössbauer spectra of the sample were acquired in transmission mode using a conventional constant acceleration-type spectrometer equipped with a ⁵⁷Co(Rh) source embedded in a rhodium matrix at room temperature from all investigated samples exhibit dublet-like features. At room temperature, the samples were measured first in range ±12 mm/s. Mössbauer spectra measured also in a narrow velocity range ±4 mm/s to allow better resolution of the spectral lines. For selected biological samples we measured the temperature dependencies using liquid helium bath cryostat. To characterize the thermal behavior and compositional/structural evolution at thermal treatments of vivianite we used the Mössbauer spectroscopy from room temperature to 140°C. Calibration of the velocity scale was performed with a thin (12.5 μm) α-Fe foil and isomer shifts are given with respect to its room temperature Mössbauer spectrum. All spectra were evaluated using the program CONFIT [30].

We used X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques for structural characterization of the samples. The structure and phase composition of the powders were inspected using the PANanalytical X’pert XRD diffractometer at CoK_α radiation with a small addition of CoK_β. The values of the lattice constants were deduced using an internal standard. SEM/EDX measurements were performed with TESCAN FERA 3 scanning electron microscope equipped with EDAX Octane 60 mm² detector.

Chemistry

Detailed description and characterisation of the [Fe(ɳ⁶-C₆H_6-nMe_n)₂]²⁺ dications complexes is described in [1], [29], [31].

Hyperfine parameters including isomer shift, quadrupole splitting, and relative areas revealed differences in the samples with varying amount of methyl groups. At the room temperature Mössbauer spectra were fitted by several doublets which corresponded to Fe²⁺ and to Fe³⁺. The Fig. 1 shows the evolution of the Mössbauer spectra of the sample from without methyl groups (n=0) to the sample with six methyl groups (n=6). The typical separation of Mössbauer spectrum is describe in the Fig. 2 for the sample with six methyl groups (n=6). This spectrum obtain several doublets corresponded to Fe²⁺ and to Fe³⁺. The Mössbauer parameters of the doublets which corresponded to Fe²⁺ and to Fe³⁺ are listed in Table 2. The dependencies of quadrupole splitting of the doublets to number of methyl group show in Fig. 3. It is readily seen that for the doublet ascribed to Fe³⁺ the isomer shift, equal to ~0.5 mm/s, does not change while the quadrupole splitting varies with the number of arene methyl groups (n). It can be concluded that the effects of the Me-substituents (Fe) on the arene ligands are transmitted via the arene ring onto the iron centre. The effect can be employed for useful inter-comparisons among iron environments that differ in the number of substituents. This dependence is in good agreement with the results of ¹H and ¹¹B NMR spectroscopic measurements and mass spectrometry [32].

Fig. 1:

The evolution of th Mössbauer spectra of the sample from without methyl groups (n=0) to the sample with six methyl groups (n=6).

Fig. 2:

The separation of Mössbauer spectrum with six methyl groups (n=6).

Fig. 3:

The dependence of quadrupole splitting of dublets described to Fe³⁺ and Fe²⁺ to number of methyl groups.

It can be concluded that the effects of the Me-substituents (Fe) on the arene ligands are transmitted via the arene ring onto the iron centre. The effect can be employed for useful inter-comparisons among iron environments that differ in the number of substituents.

Biology

The Mössbauer parameters of room-temperature doublets of the biological tissues are listed in Table 3 and indicate presence of very small particles which exhibit superparamagnetic behavior. They can be assigned to ferritin-like components. No traces of sextets were revealed. While at room temperature of the biological tissues the obtained Mössbauer spectra show exclusively doublet-like behavior they are split into sextets after lowering the temperature of measurement to 4.2 K. The former line shape indicates that the resonant iron nuclei belong to structures that are non-magnetic presumably due to their small dimensions. Taking into consideration the obtained spectral parameters, we can assign them to FeIII atoms located in a ferritin. It is a very good agreement with results described in [4], [5], [6], [7], [33]. According to the hyperfine parameters obtained from low temperature measurements that are listed in Table 3 we can conclude that they are similar to those for hematite, ferrihydrite or possibly magnetite. The Fig. 4 shows the temperature dependencies of brain and spleen tissues. Morphology of samples was revealed by SEM. The scanning electron microscopes Tescan with back scattering electron (BSE) detector were employed to visualize contrast between areas with different chemical compositions. Typical SEM image and SEM picture from BSE describe in Fig. 5. White parts on Fig. 5b corresponds to the iron atoms.

Fig. 4:

The temperature dependencies of Mössbauer spectra of the brain sample (a) and spleen sample (b).

Fig. 5:

SEM images of powder brain sample (a) and the SEM picture from BSE detector of powder brain sample (b).

Art

The first technique applied on synthetic vivianite in order to study its temperature-related behavior was high temperature-X-ray diffraction. The most intense diffraction line at 60°C of vivianite subtly decreases. A more pronounced decrease can be clearly seen at 70°C; this process gradually continues up to 160°C, when vivianite loses its structure and becomes completely amorphous, which showed that it is phase pure vivianite with 15% of Fe³⁺. The whitish by-product was found to be amorphous phosphate with 94% of Fe³⁺. Formula of pure vivianite (Fe²⁺Fe²⁺₂(PO₄)₂·8H₂O) contained only iron Fe²⁺ but the formula of the metavivianite comprised both of iron [(Fe²⁺_3−xFe³⁺_x)(PO₄)₂(OH)_x·(8−x)H₂O] [21].

The grains of vivianite dispersed in oil are completely degraded, while the “lump” still contains bluish grains. Optical and studies of vivianite samples on glass heated to different temperatures, the Fourier-transform Infrared Spectroscopy (FTIR) measurements in transmission mode, the micro-Raman spectroscopy and X-ray analysis are described on [8].

Figure 6 shows the evolution of Mössbauer spectra with increasing temperature, and Table 4 summarizes the amounts of Fe²⁺ and Fe³⁺ after each heating step from room temperature to 140°C. Mössbauer spectra check in the temperature-induced oxidation of vivianite which starts at 90°C and corresponds with the increase of the content of metavivianite. This is caused by transformation of the divalent iron (Fe²⁺) to trivalent iron (Fe³⁺). Bivalent iron content gradually decreases and conversely increases the content of trivalent iron. The ratio of Fe²⁺ to Fe³⁺ is equal to 50:50 at the temperature around 100°C. Increasing temperature leads to an almost complete transformation of divalent iron to trivalent, and above a temperature of 130°C, this process is terminated. Spectra already show no change in the parameters and contents of individual subspectra as shown in Table 4. The heating was performed on air in order to enable the diffusion of oxygen into the vivianite lattice from the surrounding atmosphere. A complex study of pure synthetic (as well as partly oxidized) vivianite covering the whole extent of the temperature-related stability of its structure is being published for the first time in [8].

Fig. 6:

Mössbauer spectra of ground natural vivianite crystals used for the model samples after exposition to a series of heating steps.