**3. The chemistry of materials**

In this section, a brief presentation of differential physicochemical techniques used to characterize the support and/or to demonstrate the efficiency of the immobilization methodology will be described. The description of theoretical basis, equipment, and conditions of analysis of the techniques are not the purpose of this material. The main information obtained from each analysis method as well as some examples will be presented below.

#### **3.1 Magnetization measurements**

In general, a material with magnetic property is analyzed by the magnetization measurement technique in order to quantify this property. The saturation magnetization (*M*s), remanent magnetization (*M*r), and coercivity (*H*c) are among the main data obtained by this technique. Additionally, the presence of a hysteresis loop, as well as the *M*r and Hc parameters, could help to assess the magnetic behavior of a material. For instance, a superparamagnetic material presents *M*r and *H*c values near to zero.

The inclusion of materials into the magnetic particles decreases their magnetization power although they still can be attracted by an external magnet. The

**35**

preserved.

*Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology*

saturation magnetization for the magnetic composite prepared from levan polymer and iron oxide particles was reduced tenfold. The happening can be attributed to the difficult alignment of magnetic dominions in the composite material due to the coating process. Furthermore, the addition of the levan increased the particles sizes varying from 20 to 60 μm for magnetite only and 100–200 μm for magnetic levan composite. The authors used this magnetic composite to immobilize trypsin by

Gregorio-Jauregui et al. [32] also showed a decrease in the saturation magnetization values as the amount of polymer (chitosan) in the particles was increased. For instance, bare magnetic nanoparticles (without chitosan—0 w/v%) and magnetic nanoparticles coated with chitosan (0.5 w/v%) presented a *M*s near to 70 and

Surface modification processes, including immobilization of enzyme, were evaluated by Defaei et al. [33]. The authors synthesized magnetic nanoparticles coated with silica and functionalized with naringin (MNP@SiO2/NA). This material was employed as support to immobilize α-amylase (MNP@SiO2/NA/AA). After each modification process a decrease on saturation magnetization values was observed due to the increase of thickness of the shell layer on the magnetic nanoparticles. So,

X-ray diffraction (XRD) is an important method used for analyzing the intermolecular structure of ordered materials. However, this technique is not appropriate for quantifying the degree of order. Magnetic materials such as bare iron oxide particles as well as magnetic composites can be characterized by XRD analysis in order to evaluate the presence of different components in the sample. XRD can also be used to estimate the particle size by using the Scherrer equation,

By using XRD technique, to differentiate between magnetite and maghemite is not possible since the iron oxides present similar standard XRD patterns. According to the International Center of Diffraction Data (reference code: ICDD 019-0629), the crystal planes at (111), (220), (311), (400), (422), (511), (440), (620), and (533) corresponding to the 2θ peaks at 18.44, 30.30, 35.67, 43.37, 53.80, 57.35, 62.97, 71.43, and 74.48° are attributed to both magnetite and maghemite [17]. For instance, Gregorio-Jauregui et al. [32] could not differentiate by XRD technique the iron oxides present in magnetic nanoparticles coated with chitosan. The authors attributed the presence of magnetite due to the black color of the magnetic composite. Furthermore, the coating with chitosan did not affect the crystalline structure

Cabrera et al. [2] assessed by XRD the chemical composition (qualitative data) as well as the crystalline structure of magnetic diatomaceous earth coated with polyaniline (mDE@PANI) nanoparticles. The XRD pattern of the mDE@PANI sample displayed characteristic peaks for crystalline and amorphous silica along with albite, polyaniline, and magnetite. The iron oxide was the predominant crystalline phase. Additionally, the authors reported that the coating process with PANI did not affect the crystallinity degree of the magnetic sample since the narrow peaks were

for MNP@SiO2,

polymer (poor crystallinity) leads to a decrease of magnetic response.

the saturation magnetization values were 38, 27, and 22 emu g<sup>−</sup><sup>1</sup>

MNP@SiO2/NA, and MNP@SiO2/NA/AA, respectively.

, respectively. Furthermore, the authors suggested that these findings could be associated with the direct relation between crystallinity and magnetization in magnetic particles. That is, magnetic materials with a good degree of crystallinity will present a large saturation magnetization. However, the addition of chitosan

*DOI: http://dx.doi.org/10.5772/intechopen.85748*

covalent binding [19].

**3.2 X-ray diffraction (XRD)**

of the magnetic nanoparticles.

for example.

45 emu g<sup>−</sup><sup>1</sup>

#### *Magnetic Bio-Derivatives: Preparation and Their Uses in Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.85748*

saturation magnetization for the magnetic composite prepared from levan polymer and iron oxide particles was reduced tenfold. The happening can be attributed to the difficult alignment of magnetic dominions in the composite material due to the coating process. Furthermore, the addition of the levan increased the particles sizes varying from 20 to 60 μm for magnetite only and 100–200 μm for magnetic levan composite. The authors used this magnetic composite to immobilize trypsin by covalent binding [19].

Gregorio-Jauregui et al. [32] also showed a decrease in the saturation magnetization values as the amount of polymer (chitosan) in the particles was increased. For instance, bare magnetic nanoparticles (without chitosan—0 w/v%) and magnetic nanoparticles coated with chitosan (0.5 w/v%) presented a *M*s near to 70 and 45 emu g<sup>−</sup><sup>1</sup> , respectively. Furthermore, the authors suggested that these findings could be associated with the direct relation between crystallinity and magnetization in magnetic particles. That is, magnetic materials with a good degree of crystallinity will present a large saturation magnetization. However, the addition of chitosan polymer (poor crystallinity) leads to a decrease of magnetic response.

Surface modification processes, including immobilization of enzyme, were evaluated by Defaei et al. [33]. The authors synthesized magnetic nanoparticles coated with silica and functionalized with naringin (MNP@SiO2/NA). This material was employed as support to immobilize α-amylase (MNP@SiO2/NA/AA). After each modification process a decrease on saturation magnetization values was observed due to the increase of thickness of the shell layer on the magnetic nanoparticles. So, the saturation magnetization values were 38, 27, and 22 emu g<sup>−</sup><sup>1</sup> for MNP@SiO2, MNP@SiO2/NA, and MNP@SiO2/NA/AA, respectively.

### **3.2 X-ray diffraction (XRD)**

*Applied Surface Science*

ing (MRI) [30], and cellular therapy [31].

of the immobilized derivative.

**3. The chemistry of materials**

examples will be presented below.

**3.1 Magnetization measurements**

problems are some of the advantages of the use of nanoparticles as a matrix. Also better stability and performance of biocatalyst immobilized along with a low protein unfolding were also reported [25, 26]. Some disadvantages for the NPs (e.g., large-scale application and price of material preparation) have been presented [26]. On the other hand, magnetic nanoparticles (MNPs) have been used as a support of several biological molecules as well as present superparamagnetism (i.e., magnetic response is only observed after application of magnetic field) and can be recovered by the use of a magnet [27]. So, the magnetic property as a plus feature to the nanoparticles would make them more attractive not only for potential uses in biotechnology [14] but also for a lot of biomedical applications including magnetic hyperthermia [28], drug delivery [29], contrast agent in magnetic resonance imag-

As above mentioned (Section 2.2), some factors influence the size (micro or nano) of iron oxide particles. For instance, Maciel et al. [17] assessed the effect of the temperature and the nature of the precipitating agent (strong base) to produce iron oxide nanoparticles. The authors employed sodium hydroxide (NaOH) as the precipitating agent and carried out the synthesis at low temperature (50°C). Small magnetic nanoparticles with a diameter near to 15 nm were obtained and used as a matrix to immobilize trypsin. The magnetic bio-derivative displayed about 90% retention of specific activity after five reuses. Despite Cabrera et al. [2] have reported mDE@PANI nanoparticles (~12 nm) as a promising matrix to immobilize trypsin, the immobilized derivative (mDE@PANI-TRYP) retained 75 and 60% of its initial activity after five and nine cycles of reusability, respectively. The decrease of catalytic activity could be attributed to the loss of the magnetic bio-derivative (mDE@PANI-TRYP) during the washing process after each reusability cycle. After coating with polyaniline, the mDE@PANI showed better stability in suspension. Thus, working with very small particles and good colloidal stability can lead to loss

Therefore, the choice of micro or nanoparticles as support will depend on several factors including colloidal stability of the particles, operating conditions,

In this section, a brief presentation of differential physicochemical techniques used to characterize the support and/or to demonstrate the efficiency of the immobilization methodology will be described. The description of theoretical basis, equipment, and conditions of analysis of the techniques are not the purpose of this material. The main information obtained from each analysis method as well as some

In general, a material with magnetic property is analyzed by the magnetization measurement technique in order to quantify this property. The saturation magnetization (*M*s), remanent magnetization (*M*r), and coercivity (*H*c) are among the main data obtained by this technique. Additionally, the presence of a hysteresis loop, as well as the *M*r and Hc parameters, could help to assess the magnetic behavior of a material. For instance, a superparamagnetic material presents *M*r and *H*c values

The inclusion of materials into the magnetic particles decreases their magnetization power although they still can be attracted by an external magnet. The

and application of the immobilized derivative, among others.

**34**

near to zero.

X-ray diffraction (XRD) is an important method used for analyzing the intermolecular structure of ordered materials. However, this technique is not appropriate for quantifying the degree of order. Magnetic materials such as bare iron oxide particles as well as magnetic composites can be characterized by XRD analysis in order to evaluate the presence of different components in the sample. XRD can also be used to estimate the particle size by using the Scherrer equation, for example.

By using XRD technique, to differentiate between magnetite and maghemite is not possible since the iron oxides present similar standard XRD patterns. According to the International Center of Diffraction Data (reference code: ICDD 019-0629), the crystal planes at (111), (220), (311), (400), (422), (511), (440), (620), and (533) corresponding to the 2θ peaks at 18.44, 30.30, 35.67, 43.37, 53.80, 57.35, 62.97, 71.43, and 74.48° are attributed to both magnetite and maghemite [17]. For instance, Gregorio-Jauregui et al. [32] could not differentiate by XRD technique the iron oxides present in magnetic nanoparticles coated with chitosan. The authors attributed the presence of magnetite due to the black color of the magnetic composite. Furthermore, the coating with chitosan did not affect the crystalline structure of the magnetic nanoparticles.

Cabrera et al. [2] assessed by XRD the chemical composition (qualitative data) as well as the crystalline structure of magnetic diatomaceous earth coated with polyaniline (mDE@PANI) nanoparticles. The XRD pattern of the mDE@PANI sample displayed characteristic peaks for crystalline and amorphous silica along with albite, polyaniline, and magnetite. The iron oxide was the predominant crystalline phase. Additionally, the authors reported that the coating process with PANI did not affect the crystallinity degree of the magnetic sample since the narrow peaks were preserved.

Díaz-Hernández et al. [34] reported the use of magnetite nanoparticles coated with chitosan (Fe3O4@chitosan) as support for immobilization of enzymes. In spite of XRD pattern which displayed a low peak at 18° probably related to maghemite, the authors concluded that magnetite was present in the Fe3O4@chitosan nanoparticles. Therefore, the XRD technique revealed that the addition of chitosan polymer did not affect the crystal structure of the magnetic sample. Moreover, the authors carried out the XRD spectrum for enzyme immobilized by cross-linking. The XRD pattern for the immobilized derivative displayed broad peaks and with low intensity, but all peaks were in agreement with magnetite. This finding could be attributed to the amorphous nature of the biomolecule immobilized.
