3.1 Yields of Locally Produced Chitosan
During the demineralization process excessive undesirable foams are produced due to the CO2 generation (CaCO3 + 2HCl → CaCl2 + CO2 + H2O) which was also reported by No and Hur [27].The demineralized and de-proteinized chitin has a light pink colour due to the presence of astaxanthin pigment; this pigment was eliminated during the decolourization step to yield cream white chitin powder which was also obtained by No and Meyers [11]. Yield was calculated as the dry weight of chitosan obtained from 400 g of crab shell. Chitosan yield was 22.75% which is comparatively higher than those reported in literature.Fernandez-Kim [23]reported 16.7–18.8% yield of chitosan from crawfish and No and Meyers [11] reported approximately 23% of chitin from crab shell.Brzeski [26]reported about 14% yield of chitosan from krill andAlimuniar and Zainuddin [3] was 18.6% from prawn waste.
3.2 Characterization of Chitosan
3.2.1 Solubility Test
It is commonly justified that main physical differences between chitin and chitosan is the ability of chitosan to be soluble in organic acid such as acetic acid or dilute hydrochloric acid. Chitosan with higher content of protonated amino group readily form well-ordered arrangement in Van der Waals force and hydrogen bond which exceed its tendency for intramolecular chemical bonds[21, 28]. The developed chitosan and the commercial chitosan both dissolved in 0.1 M hydrochloric acid within 30 minutes demonstrating excellent solubility.
3.2.2 Fourier Transform Infrared Spectrometer (FTIR)
Fourier Transform Infrared Spectrometer (FTIR) was used to probe the surface characteristics of the chitosan. the peak appearing was assigned to various functional group according to their respective wave number as reported in literatures hydroxyl group (OH) peaks appears at wavelength of 1350 cm− 1 the Amines group (R-NH2) peaks appears at 3400–3500 cm− 1. The FTIR spectrum of Figs. 3 and 2 for commercial and locally developed chitosan respectively wererelating with FTIR absorption bands. A wide absorption band at 3438 cm− 1 for Figs. 3 and 2 respectively indicates the presence of –OH stretching while peaks at absorption band featuring bending vibration of N-H from R-NH2 was observed at 3450 cm− 1 while C-H was displayed with stretching vibration of 2916.1 cm− 1, 2858.3 cm− 1and bending vibration of 1415.7 cm− 1, 1375.2 cm− 1. It was observed that the peaks were at the same frequency for both commercial and local developed chitosan but the locally developed chitosan has high absorbance as compared to commercial.
3.2.3 Raman Spectroscopy (RS)
Raman spectra were obtained for half of the films from deposition A before and after neutralization for determining chitosan’s functionality. Raman spectra were analyzed for fingerprint and group frequency peaks. Group frequency peaks tend to occur above 1500 cm− 1, while fingerprint modes are unique to the specific molecule and are usually found below 1500 cm− 1 [29]. Before starting the analysis, the chemical structures of chitosan were examined for the groups they contained in order to know what to expect. The functional groups identified are presented in Tables 1 and 2 for the commercial and locally developed chitosan respectively. Examining the molecules, chitosan contains 5 methine (C-H) groups per repeat unit and 1 methylene (CH2) group. According to Wojtkowiak and Chabanel [29] methyl CH3 bending can be found at 1460 ± 10 cm− 1 and CH3 deformation at 1375 ± 10 cm− 1. These peaks are presented in the Figs. 4 and 5 for both commercial and locally developed chitosan repectively.
Table 1
Wave numbers of the bands observed in the Raman spectra for commercial chitosan and their assignment to the respective normal vibrations.
RS | Assignments |
3362w | v(OH)HB |
3308w | |
2932vs | v(CH3) |
2885vs | v(CH2) |
2818shm | v(CH3) |
2743w | v(CH) |
1654w | v(CO) |
1591 m | |
1458 m | δ(CH) + ѡ(CH2) + δ(OH) |
1411 m | δ(CH3) + δ(CH) |
1325 | v(CN) + δ(CH) |
1263 | v(C-O) + δ(CH) + ρ(CH2) |
Φ,pyranoid ring; v, stretching; δ, in-plane bending vibrations; γ, ѡ, out-of plane bending; HB, hydrogen bond |
Table 2
Wave numbers of the bands observed in the Raman spectra for locally developed chitosan and their assignment to the respective normal vibrations.
RS | Assignments |
1146 | v(C-O-C) + v(ϕ) |
1114 | v(C-OH) + v(C-CH2) |
1093 | ρ(CH) + ρ(CH2) + ρ(CH3) |
1044shm | ρ(CH3) + δ(CH) + δ(OH) |
991shm | v(ϕ) + δ(CH) |
936 m | v(CN) |
896 m | v(ϕ) + ρ(CH2) |
703w | ѡ(NH2) + δ(ϕ) |
566shm | γ(NH) + γ(C = O) + ѡ(CH3) |
493 m | γ(CO-NH) + δ(C-CH3) |
479 m | γ(COC) |
444 m | γ(OH + γ(ϕ) |
424 m | γ(OH + γ(ϕ) |
285 m | δ(C-NH-C) + γ(OH) |
Φ,pyranoid ring; v, stretching; δ, in-plane bending vibrations; γ, ѡ, out-of plane bending; HB, hydrogen bond |
Raman spectroscopy is very helpful for distinguishing amines from alcohols because the N-H stretch is distinctly stronger than the O-H stretch [29]. Also, hydrogen bonding has less of an effect on amines than alcohols, which changes the spectra completely. There is one primary amine in the chitosan repeat unit the NH bend occurs at 1500 cm− 1.The bands from the range 500–1500 cm− 1 can be assigned tothe vibrations: δs(CH3,CH2) at 1429 cm− 1, δs(CH3,CH2) at 1360–1372 cm− 1, δ(CH) at 1319 and 1336 cm− 1v(C-C) and v(C-O) inthe range 1200–1300 cm− 1, δ(ϕ -OH) at 1163 cm− 1, vas(C-O-C)in the range 1000–1160 cm− 1, γ(CH) in the range 850–1000 cm− 1, and δ(ϕ) in the range 500–720 cm− 1(Socrates, 2001).
3.2.4 Scanning Electron Microscopy (SEM)
The morphology of commercial and locally developed chitosan from Figs. 7 and 6 was obtained using the scanning electronic microscope at four different magnifications. The locally developed chitosan has a rough surface characterized with holes, and has a porous spongy structure while commercial chitosan has similar characteristic as the locally developed chitosan but with less holes and pores.
3.2.5 Electron Dispersive Spectroscopy (EDS)
Electron Dispersive Spectroscopy (EDS) is a test to examine the presence of elements through amplitude of wavelength for the x-ray emitted after the electron was hit by the electron beam. For the emission of x-ray, the atoms must contain minimum of K-shell and L-shell where the electron is allowed to dislodge from shell to shell. Therefore, hydrogen being the only elements in the periodic table with only K shell is not detectable with EDS[30, 31]. Figure 10 is the EDS image of commercial chitosan, the spots represented with + in the Figureshows the points whose elemental composition are presented, Fig. 11a represent spot 2 and Table 3 presents the weight composition of the elements. Figures 11b and 11c depicts the spectrum of spot 3 and spot 6 respectively and Tables 4 and 5 presents the elemental composition of spot 3 and spot 6 for commercial chitosan. Figure 12 is the EDS image of locally developed chitosan and Figs. 13a and 13b represents spot 2 and spot 6 respectively and Tables 6 and 7 presents the elemental compositions for spot 2 and 6. Tables 3 and 4 contains Fluorine which is not part of elemental weight composition of chitosan and also Tables 6 and 7 contains Sodium which might be present as a result of inadequate washing during synthesis stage.
Table 3
Elemental weight composition of spot 2 for commercial chitosan
Atomic number | Element symbol | Element name | Confidence level | Concentration percentage | Certainty percentage | Error percentage |
7 | N | Nitrogen | 100 | 47.8 | 99.2 | 0.8 |
8 | O | Oxygen | 100 | 27.6 | 97.4 | 2.6 |
9 | F | Fluorine | 100 | 14.7 | 96.7 | 3.3 |
6 | C | Carbon | 100 | 9.9 | 99.2 | 0.8 |
Table 4
Elemental weight composition of spot 3 for commercial chitosan
Atomic number | Element symbol | Element name | Confidence level | Concentration percentage | Certainty percentage | Error percentage |
7 | N | Nitrogen | 100 | 49.6 | 99.1 | 0.1 |
8 | O | Oxygen | 100 | 26.6 | 97.3 | 2.7 |
9 | F | Fluorine | 100 | 12.8 | 96.4 | 3.6 |
6 | C | Carbon | 100 | 11 | 99.2 | 0.8 |
Table 5
Elemental weight composition of spot 6 of commercial chitosan
Atomic number | Element symbol | Element name | Confidence level | Concentration percentage | Certainty percentage | Error percentage |
7 | N | Nitrogen | 100 | 40.3 | 97.5 | 2.5 |
6 | C | Carbon | 100 | 31.6 | 93.4 | 0.7 |
8 | O | Oxygen | 100 | 28.1 | 95.3 | 4.6 |
Table 6
Elemental weight composition of spot 2 for locally developed chitosan
Atomic number | Element symbol | Element name | Confidence level | Concentration percentage | Certainty percentage | Error percentage |
8 | O | Oxygen | 100 | 39.7 | 98.3 | 1.5 |
11 | Na | Sodium | 100 | 38 | 99 | 1 |
6 | C | Carbon | 100 | 12 | 93.4 | 1.1 |
7 | N | Nitrogen | 100 | 10.3 | 97.5 | 2.4 |
Table 7
Elemental weight composition of spot 6 for locally developed chitosan
Atomic number | Element symbol | Element name | Confidence level | Concentration percentage | Certainty percentage | Error percentage |
8 | O | Oxygen | 100 | 39.6 | 98.3 | 1.6 |
11 | Na | Sodium | 100 | 38 | 99 | 1.2 |
6 | C | Carbon | 100 | 14.9 | 93.4 | 0.9 |
7 | N | Nitrogen | 100 | 13.5 | 97.5 | 2.2 |
3.2.6 X-Ray Diffraction (XRD)
The XRD spectrum of chitosan has low crystallinity. X-ray diffraction pattern for pure chitosan has peaks at 2θ = 10° and 20.09° for pure chitosan confirms the semi crystalline nature[32]. The XRD pattern of commercial and locally developed chitosan are shown in Figs. 14 and 15 respectively. Both showed broad diffraction at 2θ = 20° and that symbolizes semi crystalline chitosan. This was supported byYen, Yang and Mau [25] as the two characteristic crystalline peaks of chitosan at 9–10° and 19–20° with comparable crystallinity.