4.1 Structural analysis
The crystal structure, crystalline phases, and crystallite size of synthesized pure Fe3O4 and composite nanoparticles such as CS coated Fe3O4 and FA coated CS-Fe3O4 were identified by using an X-ray diffractometer. Fig.1. presents XRD pattern of prepared magnetic nanoparticles 1(a) Fe3O4 NPs, 1 (b) CS- coated Fe3O4 NPs, 1 (c) FA-CS-coated Fe3O4 NPs. The obtained characteristic peaks of NPs at 2 θ = 33.24o, 35.71o, 43.53o, 57.57o, and 62.49o correspond to Bragg reflection planes (220), (311), (400), (333), and (440). The obtained diffraction peaks are indexed as an inverse spinel structure of Fe3O4 NPs having lattice parameter a = 8.39 Å and results are in good agreement with the standard data (JCPDS: 19-0629) [21]. Manifestation of other peaks at different 2θ values is the evidence for the conjugation of chitosan and folic acid. The Williamson-Hall method was carried out to determine the crystallite size and the strain-induced in powders [22]. The instrument broadening (β) corrected for each diffraction peak was estimated by the relation
βD = [(β2) measured- (β2)instrumental ]1/2 (1)
On the other hand, the crystallite size of the nanoparticles was calculated by Debye- Scherrer equation as
D=Kλ/βD cosθ (2)
Where D is particle size (nm), K is the grain shape factor (0.9), λ is the wavelength (Å), θ is the Brag angle, and βD is the peak width at half-maximum intensity.
The strain induced in powder associated to the crystal imperfections was determined by,
ε = βs/4 tanθ (3)
βs = 4ε tanθ (4)
The above equation (4) indicates the strain-induced line broadening.
Βhkl = βs+ βD (5)
Βhkl = (4ε tanθ) + (Kλ/D cosθ) (6)
By rearranging Eq. (6)
Βhkl cosθ = (Kλ/D cosθ) + 4ε sinθ (7)
The above Eq.(7) is known as the Williamson-Hall equation. Therefore, this equation represents the UDM (Uniform Deformation Model), where the strain was assumed to be uniform in all crystallographic directions. The plot is drawn with 4 sinθ along the x-axis and Βhkl cosθ along the Y-axis. The crystalline size was calculated from the Y-intercept and the strain (ε) from the slope of the linear fit to the data. The UDM analysis results for the nanoparticles are shown in Figure 2 (a-c). The crystallite size of the nanoparticles was calculated as 10.35 nm (a. Pure Fe3O4), 14.87 nm (b. CS-coated Fe3O4), and 15.77 nm (c. FA-CS-coated Fe3O4) respectively. Table 1. represents the Crystallite size, microstrain, and intercept value of pure Fe3O4 NPs, CS-coated Fe3O4 NPs, and FA-CS-coated Fe3O4 NPs using the W-H method.
4. 2 FTIR analysis
Conjugation of Chitosan and Folic acid with Fe3O4 NPs is very important for biotechnological purposes. Functional groups of prepared MNPs are analyzed by FTIR spectrometer. Fig. 3 depicts the FTIR spectra which confirm the chemical composition of naked Fe3O4 NPs and the surface-modified Fe3O4 NPs. In Fig. 3(a) the obtained strong band at 558 cm-1 attributed to the Fe-O group [23]. Fig. 3(b) presents broadband at 3414 cm-1 assigned to the –OH group and the peaks manifested at 2922 cm-1 and 2868 cm-1 corresponds to the C-H group. The weak band around 1064 cm-1 is the stretching vibrations of the C-O bond in the spectrum of the composite NPs [24]. Fig. 3(c) confirms the conjugation of FA with CS-coated Fe3O4 NPs by the characteristic bands around 817cm-1, 1270 cm-1, and the spectrum at 1604 cm-1 assigned to the aromatic ring of FA [25]. The new strong band at 1626 cm-1 is the perfect indication for the unification of Chitosan and Folic acid with Fe3O4 MNPs [26] and a weak C=C stretching band was found at 1421 cm-1 [27].
4.3 Morphological analysis
Morphology and elements presented in the synthesis products of pure Fe3O4, CS-coated Fe3O4, and FA-CS-coated Fe3O4 nanoparticles were depicted in Fig. 4(a-c) & 5(a-c) respectively. According to SEM images, there is no agglomeration for pure Fe3O4 nanoparticles [ Fig. 4(a)] and it presents the spunch like structure. From Fig. 4(b) & 4(c) it was identified that CS-coated Fe3O4 NPs and FA-CS-coated Fe3O4 NPs possess agglomerated spherical structures.
Elements presented in the nanoparticles were evaluated by Energy dispersive X-ray analysis (EDAX). From the EDAX spectra of prepared NPs, it is crystal clear that the peaks are around 0.8. 4.5, 6.3, and 7 keV are assigned to the binding energy of Fe [28]. There is a peak approximately at 0.7 keV, corresponding to O. All the three EDAX spectrums of synthesized NPs not only exhibit Fe and O, however Mn, but Cl and Cs also manifested in less mass percentage.
4.4 Optical Study
The absorbance of chemical substances was determined by utilizing the quantitative technique UV/ Vis Spectroscopy. Fig. (6) represents the absorption spectra (fig. 6a-c) and Tauc’s plot ( fig. 6 e-f)of MNPs. This experiment was conducted between the wavelength 100 - 800 nm. The resultant cut of wavelength for all the three samples such as Pure Fe3O4 (6a), CS-coated Fe3O4 (6b), and FA-CS-coated Fe3O4 (6c) NPs were obtained around 200 nm. These samples reveal sharp absorption edges. Tauc’s plot is a significant tool to determine the optical band gap energy, which is drawn by using Tauc’s relation:
(αhν)n = A(hν-Eg)
where α is the absorption coefficient, A is a constant, ν is the photon frequency, h is Plank’s constant, and Eg is the bandgap energy. The absorption coefficient (α) was calculated for each value based on the absorption data [29]. The exponent n's value is determined by the type of transition like n = 1/2 for indirect bandgap and allowed transition; n=2 for direct transition; and n=3/2 for directly forbidden transition. A plot was drawn between (αhν)1/2 along Y-axis and hν along X-axis to obtain the Eg value of nanoparticles. The determined bandgap energy of the MNPs by using the Tauc’s plot is shown in Fig. 6 (e-f) and the calculated values are 4.7 eV, 4.3 eV, and 3.4 eV respectively. From these resultants, it is known that the Eg value of CS-Fe3O4 and FA-CS-Fe3O4 nanoparticles have been reduced than that of pure Fe3O4 nanoparticles. This is due to the change in the particle size concerning the surface modification with chitosan and folic acid [30].
4.5 Thermal study
Thermogravimetric analysis was performed to identify the change in mass of materials as a function of temperature. The prepared samples were subjected to up to 900 oC in an air atmosphere for estimating the chemical composition of prepared magnetic nanoparticles. TGA curve and 1st derivative TG (DTG) curve of pure Fe3O4 and CS-coated Fe3O4, FA-CS-coated Fe3O4 nanoparticles were shown in Fig. 7(A) & 7(B). Multistage decomposition was observed from the TGA analysis. From the TGA curve, it is found that the decomposition of the compound begins at 46o C approximately. The first derivative curve was plotted to detect the distinct degradation event where it begins and ends. From the observation, it is clear that there is no degradation at any point of temperature for pure Fe3O4 NPs [curve an in Fig. 7(A) & 7(B)] [31]. From the attained thermogram, it is obvious that there are several endothermic peaks are exhibited at different temperatures [Fig. 7(B)]. The endothermic peaks for the samples CS-coated Fe3O4, FA-CS-coated Fe3O4 nanoparticles around 100 oC are due to the removal of water absorbed by the nanoparticles with a minimum weight loss [curve b & c in Fig. 7(A) & 7(B)] [32]. On the other hand, the broad endothermic peaks between 200-500 oC and the strong & broad endothermic peaks at 690 oC and 750 oC are the indication of decomposition of the physisorption and the chemisorption of the surface modification agent such as Chitosan and Folic acid [curve b & c in Fig. 7(A) & 7(B)] [33]. Significantly, the inflection point was observed at 690 oC which describes the greatest rate of change on the weight loss. [curve b in Fig. 7(B)]. Above 780 oC rapid decomposition takes place. From these resultants of the TG curve, it was analyzed that Fe3O4 MNPs are coated with chitosan and folic acid at an appropriate level.