**3. Results and discussion**

#### **3.1. Partial obtaining peroxidase enzyme from** *Euphorbia amygdaloides* **plant**

The data obtained in the purification process of peroxidase enzyme are given in **Table 1**.

There are many studies on the purification of peroxidase enzyme in the literature. Plants such as wheat seeds, barley and wheat, soybeans, fava beans, sorghum, watermelon seeds, red beets, cotton, pearl millet seedlings, Asian rice, lettuce, wild radish, and pearl barley hybrids were used for the purification of peroxidase enzyme [25–29].

Peroxidase enzyme was purified by ammonium sulfate precipitation from *Euphorbia amygdaloides* plant with CM-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration chromatography. According to the data obtained in 75% ammonium sulfate precipitation step, the enzyme was purified with purification coefficient of 6.4 and 51.6 for 10 mL volume [23]. In our study, the enzyme was purified with a purification coefficient of 149.5 and a yield of 29.5 for 20 mL volume, according to the order of 60–80% ammonium sulfate precipitation step.


SEM (scanning electron microscope) basically works on the basis of obtaining the images of the surface morphology which are scanned with the help of electrons. The electrons sourced from tungsten tip are sent to the surface to be scanned. After that, the emitted electrons are captured by the detector and the image is formed. SEM analysis images of ZnO and

may be a soft structure. It has been determined that these nanoparticle structures obtained by the surface characterization process have an average size of 30–80 nm. The SEM image for ZnO nanoparticle was taken at 5 μm. In this image, the nanoparticle structure exhibits a wavier surface. Generally, the liquid containing peroxidase enzyme prevents the formation of ZnO nanoparticle formations composed of dust and flake-like structures. However, it appears that the powder and flake structures combine in some regions and exhibit a wavy appearance after this enzyme liquid is evaporated. After the surface characterization analysis of ZnO nanoparticles, it was found that these structures vary between 60 and 80 nm on

The Fourier Transform Infrared Spectrophotometer (FTIR) is used to determine the bond formation between the elements present in the structure to be measured. In this direction, the structural formation that the sample possesses can be understood by measuring the vibrations of the bond occurrences in the structure at certain frequencies. This helps in the determination of the functional groups in the material being measured. The FT-IR spectrums of ZnO

When looking at **Figure 3A**, it is possible to observe the absorbance at 510–564 cm−1, which is indicative of Zn-O band formation in this analysis which was carried out to detect the biomolecule by revealing the stabilization ability and bandwidth of the metal nanoparticles synthesized by green synthesis [38]. Geetha et al. [30] synthesized ZnO nanoparticles by green synthesis using the *Euphorbia spindle* plant. They applied FT-IR analysis in their characterization studies

nanoparticles in this study taken in this way are shown in **Figure 2**. The SEM image

The Investigation of Removing Direct Blue 15 Dye from Wastewater Using Magnetic *Luffa sponge* NPs

nanoparticle structure resembled a dust particle, suggesting that it

http://dx.doi.org/10.5772/intechopen.73216

213

Fe<sup>3</sup> O4

average.

and Fe<sup>3</sup>

O4

**Figure 2.** SEM analysis images of ZnO and Fe<sup>3</sup>

O4

nanoparticles.

recorded for the Fe<sup>3</sup>

O4

nanoparticles are given in **Figure 3**.

**Table 1.** Purification process of peroxidase enzyme from *Euphorbia amygdaloides* plant.

#### **3.2. Characterization of Fe3 O4 and ZnO NPs**

The results of the optimization studies for the green synthesis of nanoparticles are given in **Table 2**.

No research has been found in the literature on the green synthesis of Fe<sup>3</sup> O4 and ZnO NPs from the *Euphorbia amygdaloides* plant. *Euphorbia milii* was used for the synthesis of ZnO nanoparticles [30]. A study on the green synthesis of the Fe<sup>3</sup> O4 nanoparticle using the *Euphorbia amygdaloides* plant is not available in the literature. However, studies on the green synthesis of Pd/Fe<sup>3</sup> O4 nanoparticles using *Euphorbia condylocarpa M. bieb* root extract and *Euphorbia stracheyi Boiss* root extract have been carried out [31]. Our study provides a contribution to the literature. The highest peak in the optical absorption spectrum of the ZnO nanoparticles synthesized by pulsed laser ablation was at 300 nm [32]. In our study, the highest peak value of ZnO nanoparticles was read at 304 nm (**Table 2**). The sharpness of ZnO absorption indicates the uniform nanoparticle distribution [33, 34]. Fe<sup>3</sup> O4 nanoparticles exhibit an absorption band in the range of 330–450 nm of the UV–Vis spectrum in the literature and Fe<sup>3</sup> O4 nanoparticles peak observed at 330 nm in a work [35]. The highest peak value of the Fe<sup>3</sup> O4 nanoparticles used in our study was read as 481 nm. This range is above the range available in the literature. Nagarajan and Kuppusamy [36] studied the optical properties of ZnO nanoparticles obtained from marine algae of Mannar Bay in India. They did not observe any peak between pH 5.0–7.0 and pH 9.0–10.0 in their pH optimization studies. The maximum yield was obtained at pH 8.0. In our study, pH 6.0 was determined as the optimum pH for ZnO nanoparticle synthesis. Optimum pH for Fe<sup>3</sup> O4 nanoparticles is found at 8.0. Manouchehr et al. [37] reported that they performed the synthesis of Fe<sup>3</sup> O4 nanoparticles in the pH 7.0–9.0 environment. These values are close to the values we have obtained in our study. While the highest absorbance values were obtained at a concentration of 5 mM ZnCl2 in the synthesis of ZnO nanoparticles, the highest absorbance values were obtained at a concentration of 1 mM FeCl2 -Fe2 Cl<sup>3</sup> in the synthesis of Fe<sup>3</sup> O4 nanoparticles.


**Table 2.** The results of the optimization studies for the green synthesis of nanoparticles.

SEM (scanning electron microscope) basically works on the basis of obtaining the images of the surface morphology which are scanned with the help of electrons. The electrons sourced from tungsten tip are sent to the surface to be scanned. After that, the emitted electrons are captured by the detector and the image is formed. SEM analysis images of ZnO and Fe<sup>3</sup> O4 nanoparticles in this study taken in this way are shown in **Figure 2**. The SEM image recorded for the Fe<sup>3</sup> O4 nanoparticle structure resembled a dust particle, suggesting that it may be a soft structure. It has been determined that these nanoparticle structures obtained by the surface characterization process have an average size of 30–80 nm. The SEM image for ZnO nanoparticle was taken at 5 μm. In this image, the nanoparticle structure exhibits a wavier surface. Generally, the liquid containing peroxidase enzyme prevents the formation of ZnO nanoparticle formations composed of dust and flake-like structures. However, it appears that the powder and flake structures combine in some regions and exhibit a wavy appearance after this enzyme liquid is evaporated. After the surface characterization analysis of ZnO nanoparticles, it was found that these structures vary between 60 and 80 nm on average.

The Fourier Transform Infrared Spectrophotometer (FTIR) is used to determine the bond formation between the elements present in the structure to be measured. In this direction, the structural formation that the sample possesses can be understood by measuring the vibrations of the bond occurrences in the structure at certain frequencies. This helps in the determination of the functional groups in the material being measured. The FT-IR spectrums of ZnO and Fe<sup>3</sup> O4 nanoparticles are given in **Figure 3**.

When looking at **Figure 3A**, it is possible to observe the absorbance at 510–564 cm−1, which is indicative of Zn-O band formation in this analysis which was carried out to detect the biomolecule by revealing the stabilization ability and bandwidth of the metal nanoparticles synthesized by green synthesis [38]. Geetha et al. [30] synthesized ZnO nanoparticles by green synthesis using the *Euphorbia spindle* plant. They applied FT-IR analysis in their characterization studies

**Figure 2.** SEM analysis images of ZnO and Fe<sup>3</sup> O4 nanoparticles.

**3.2. Characterization of Fe3**

**Volume (mL)**

212 Iron Ores and Iron Oxide Materials

distribution [33, 34]. Fe<sup>3</sup>

**Table 2**.

**Enzyme fraction**

Crude extract

(NH<sup>4</sup> )2 SO<sup>4</sup> (60–80%)

of Fe<sup>3</sup> O4

Fe<sup>3</sup>

tion of 5 mM ZnCl2

**O4**

[30]. A study on the green synthesis of the Fe<sup>3</sup>

O4

of the UV–Vis spectrum in the literature and Fe<sup>3</sup>

a work [35]. The highest peak value of the Fe<sup>3</sup>

obtained at a concentration of 1 mM FeCl2

 **and ZnO NPs**

**Activity (EU/mL) Total activity** 

50 236.4 ± 1.0 11.82/100 3.82×102

**Table 1.** Purification process of peroxidase enzyme from *Euphorbia amygdaloides* plant.

**(EU) 103 /%**

No research has been found in the literature on the green synthesis of Fe<sup>3</sup>

The results of the optimization studies for the green synthesis of nanoparticles are given in

20 174.3 ± 1.02 3.49/29.5 1.88 ± 0.16 92.71 149.54

**Protein (mg protein) (mL)**

the *Euphorbia amygdaloides* plant. *Euphorbia milii* was used for the synthesis of ZnO nanoparticles

O4

nanoparticles using *Euphorbia condylocarpa M. bieb* root extract and *Euphorbia stracheyi Boiss* root extract have been carried out [31]. Our study provides a contribution to the literature. The highest peak in the optical absorption spectrum of the ZnO nanoparticles synthesized by pulsed laser ablation was at 300 nm [32]. In our study, the highest peak value of ZnO nanoparticles was read at 304 nm (**Table 2**). The sharpness of ZnO absorption indicates the uniform nanoparticle

O4

O4

481 nm. This range is above the range available in the literature. Nagarajan and Kuppusamy [36] studied the optical properties of ZnO nanoparticles obtained from marine algae of Mannar Bay in India. They did not observe any peak between pH 5.0–7.0 and pH 9.0–10.0 in their pH optimization studies. The maximum yield was obtained at pH 8.0. In our study, pH 6.0 was determined as the optimum pH for ZnO nanoparticle synthesis. Optimum pH for Fe<sup>3</sup>

nanoparticles is found at 8.0. Manouchehr et al. [37] reported that they performed the synthesis

have obtained in our study. While the highest absorbance values were obtained at a concentra-


ZnO 304 4 6.0 60 5

O<sup>4</sup> 481 4 8.0 30 1

**Table 2.** The results of the optimization studies for the green synthesis of nanoparticles.

**Nanoparticle Wavelength (nm) Contact time (h) pH Temperature (°C) Metal ion concentration (mM)**

nanoparticles in the pH 7.0–9.0 environment. These values are close to the values we

in the synthesis of ZnO nanoparticles, the highest absorbance values were

in the synthesis of Fe<sup>3</sup>

plant is not available in the literature. However, studies on the green synthesis of Pd/Fe<sup>3</sup>

O4

nanoparticles peak observed at 330 nm in

nanoparticles used in our study was read as

O4

nanoparticles.

nanoparticle using the *Euphorbia amygdaloides*

**Specific activity (EU/mg)**

± 0.7 0.62 —

nanoparticles exhibit an absorption band in the range of 330–450 nm

and ZnO NPs from

**Purification coefficient (EU/**

**mg)**

O4

O4

structures are located at 2θ angles of 31.77, 34.40, 36.22, 47.61, 56.58, 62.85, 66.41, 67.93, 69.08, 72.54, and 76.85° corresponding to the plane distances of the atoms present in the structure of the nanoparticle. In this case, the XRD chart confirms that the nanoparticle we are analyzing

The Investigation of Removing Direct Blue 15 Dye from Wastewater Using Magnetic *Luffa sponge* NPs

Nearly all of the characterization studies for ZnO nanoparticles synthesized by different methods in the literature are discussed according to XRD analysis results. In the analyses, the planes are generally observed at the highest peak 101 with the planes of 100, 002, 101, 102, 110, 103, 112 [30, 39]. In parallel with these studies in the XRD spectrum we obtained in our study,

nanoparticle structure is obtained in the green synthesis process carried out. 2θ values specific to this nanoparticle were determined as 30, 33, 44, 53, 56, and 62°.The distances between the planes determined in this direction are 220, 311, 400, 422, 511, and 440 respectively. In the light

O4

The data obtained for the immobilization of nanoparticles on the LS are given in **Table 3**.

Analytical methods used for quantitative analysis require calibration. Calibration is a process for accurately determining the relationship between the signal measured at the output of any device and the concentration of the material causing the signal. The curve obtained is a

As seen in **Figure 5**, the DB15 azo dye had value of about qe = 80 mg/L with all membrane forms in the first 15 min. Measurements at 30, 45, and 60 min resulted in 80–100 mg/L qe. However, since the highest values were noted at 45 min, optimum contact time for this azo

of many azo dyes [40, 41]. The LS-ZnO membrane form provided slightly lower adsorption

O4

**Absorbance before immobilization**

ZnO 304 0.147 0.012 91.83

O<sup>4</sup> 481 0.188 0.015 92.02

O<sup>4</sup> 209 0.202 0.014 93.07

O4

nanoparticles reveal that the desired

http://dx.doi.org/10.5772/intechopen.73216

215

nanoparticle structure is in a spherical crystal

value is 0.9941, the slope is assumed to be 0.0094.

NPs have been used to study the remodeling

**% immobilization**

NPs membrane form. The highest adsorption was obtained with

**Absorbance after immobilization**

is a ZnO nanoparticle.

structure.

the 101 plane has the highest peak.

**3.3. Immobilization efficiency**

**3.4. Adsorption studies**

compared to the LS-Fe<sup>3</sup>

O4

LS-ZnO/Fe<sup>3</sup>

**Nanoparticle solution**

Fe<sup>3</sup>

ZnO/Fe<sup>3</sup>

The peaks obtained in the XRD spectrum of the Fe<sup>3</sup>

of this information, it is determined that the Fe<sup>3</sup>

straight line. Since the calibration curve R2

dye was accepted as 45 min. ZnO and Fe<sup>3</sup>

**Wavelength (nm)**

**Table 3.** Data of immobilization efficiency.

O4

NPs membrane form.

**Figure 3.** FTIR spectrum of (A) ZnO and (B) Fe<sup>3</sup> O4 nanoparticle.

in the range of 400–4500 cm−1. The researchers noted that the observed transmittance band at 435 cm−1 corresponds to ZnO bonding, confirming the formation of ZnO particles.

In the FT-IR spectrum of the Fe<sup>3</sup> O4 nanoparticle structure shown in **Figure 3B**, there are oscillations of bond structures that oxygen forms with iron. It is known that this nanoparticle structure oscillates between 200 and 650 cm−1. In this direction, as shown in the graph, the Fe<sup>3</sup> O4 nanoparticle structure obtained by green synthesis exhibited oscillations indicating specific bonds between iron and oxygen elements between 256 cm and 636 cm−1.

XRD (X-ray diffraction) method was used for the analysis of crystallized structures of nanoparticles used in the study. In this method, since the diffraction pattern to be produced by each structure will be different, the planar structure of the elements arranged symmetrically or periodically can be determined. The graphs obtained by XRD analysis of ZnO and Fe<sup>3</sup> O4 nanoparticles are given in **Figure 4**.

Plots of 100, 002, 101, 102, 110, 103, 200, 112, 201, 004, and 202 were determined in the XRD analysis graph to show the crystallized structure of the ZnO nanoparticle. ZnO nanoparticle

**Figure 4.** XRD spectrum for (A) ZnO and (B) Fe<sup>3</sup> O4 nanoparticle.

structures are located at 2θ angles of 31.77, 34.40, 36.22, 47.61, 56.58, 62.85, 66.41, 67.93, 69.08, 72.54, and 76.85° corresponding to the plane distances of the atoms present in the structure of the nanoparticle. In this case, the XRD chart confirms that the nanoparticle we are analyzing is a ZnO nanoparticle.

Nearly all of the characterization studies for ZnO nanoparticles synthesized by different methods in the literature are discussed according to XRD analysis results. In the analyses, the planes are generally observed at the highest peak 101 with the planes of 100, 002, 101, 102, 110, 103, 112 [30, 39]. In parallel with these studies in the XRD spectrum we obtained in our study, the 101 plane has the highest peak.

The peaks obtained in the XRD spectrum of the Fe<sup>3</sup> O4 nanoparticles reveal that the desired nanoparticle structure is obtained in the green synthesis process carried out. 2θ values specific to this nanoparticle were determined as 30, 33, 44, 53, 56, and 62°.The distances between the planes determined in this direction are 220, 311, 400, 422, 511, and 440 respectively. In the light of this information, it is determined that the Fe<sup>3</sup> O4 nanoparticle structure is in a spherical crystal structure.
