**3.3 Mössbauer spectroscopy (MS)**

Mössbauer spectroscopy (MS) is a sensitive technique to the iron ionic state and environments. MS can distinguish the ferric (Fe3+) and ferrous (Fe2+) ions because of their different isomer shifts. Thus, magnetite, maghemite, and hematite, for example, can be detected in a magnetic sample. Since magnetite mainly presents potential biotechnological and biomedical applications, it is very important to know the main iron oxide phases present in the sample. Therefore, the MS technique is very useful for monitoring the processes of preparation and modification of the iron oxides at different sizes. However, small magnetic nanoparticles (below than 10 nm) must be analyzed at low temperatures in order to block the superparamagnetic relaxation of them [35].

Cabrera et al. [36] proposed two magnetic composites as a matrix to immobilize invertase via covalent bonding. For this, clay minerals such as montmorillonite (MMT) and diatomaceous earth (DE) were used as a nonmagnetic component. Using MS technique, it was possible to evaluate the main iron oxide phases present in the magnetic composites (mMMT and mDE). Mössbauer results revealed a mixture of magnetite and maghemite in equal proportion for the mMMT particles, while a pure magnetite phase was observed in the mDE particles.

Storage conditions can also lead to changes in magnetic property due to phase transformation of iron oxides. Rümenapp et al. [37] described strong oxidation of the bare magnetic nanoparticles on the fourth day of preparation. The complete oxidation to maghemite was observed in the fourth week. In order to avoid the oxidation process, the authors suggested a coating process on the magnetic surface. Iron oxide nanoparticles without and with polyaniline (PANI) coating were assessed by Maciel et al. [17] using MS analysis. The authors described the presence of maghemite in the two samples. In addition, the coating with PANI did not change the chemical nature of the magnetic sample. Similarly, Cabrera et al. [2] functionalized with PANI a magnetic composite from diatomaceous earth and proposed it as promising support to enzyme immobilization. Due to the great catalytic performance of the magnetic bio-derivatives, the authors evaluated the magnetic behavior by MS technique. It is not common to analyze the magnetic sample containing the biomolecule may be due to the small amount of biomolecule immobilized in most of the time. MS results showed the magnetite as the major iron oxide phase in the magnetic composites (mDE and mDE@PANI). Moreover, the hematite was not detected in the samples.
