The Physicochemical Characterisation and Computational Studies of Tilapia Fish Scales as a Green Inhibitor for Steel Corrosion ^†

Abstract

The effect of increased corrosion in re-enforcement structures has led to the need to identify and develop more inexpensive, non-toxic, eco-friendly and readily available inhibitors from natural resources. Extensive research and development have led to the discovery of new classes of green corrosion inhibitors. In this work, Tilapia fish scales (FSs) were used as a green corrosion inhibitor as they are abundant in both organic components, such as collagen (C₁₂H₁₉N₃O₅), and inorganic components, such as hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). The FSs were subjected to a maceration process to extract all the inorganic and organic compounds. The FS extract was then characterised using an X-ray diffractometer (XRD), a scanning electron microscope (SEM) and Fourier transform infra-red (FTIR). Quantum computational studies were conducted in order to determine parameters such as the energy of the highest occupied molecular orbital (EHOMO) and the energy of the lowest occupied molecular orbital (ELUMO). The Gaussian 09 program density functional theory at the 6-311++(d,p) basis set was used to investigate the interaction between the organic and inorganic molecules, therefore examining both interaction energies. The XRD results confirmed that a large amount of hydroxyapatite was present in the extract, with a high diffractive peak at 32θ and small amounts of collagen picked up between 13θ and 25θ. SEM results showed the percentage weight of atoms, such as carbon (19.8%), calcium (27%), oxygen (41.3%) and phosphate (11.9%), which were found to be present in both the organic and inorganic part of the FS sample. FTIR results confirmed the presence of hydroxyl (3200–3500 cm⁻¹), carbonate (1620–1700 cm⁻¹) and phosphate groups (1200–800 cm⁻¹). The computation studies showed that hydroxyapatite was the most reactive molecule, as it had the highest EHOMO of −0.2076 eV compared with that of collagen at −0.2470 eV. The interaction energy of the FS molecule was −615 kJ/mol.

1. Introduction

Tilapia fish scales include between 40% and 55% organic material (collagen, lipids, proteins, and vitamins), as well as about 30% of inorganic substances like calcium and phosphates as biomaterial. Ten (10) grams of fish scales, which can be found on one or two fish, can yield about 200 milligrams (mg) of collagen. Since fish scales are frequently discarded in landfills, obtaining them is quite inexpensive. Without labour, it costs slightly over USD 4 to extract 100 mg of collagen from fish scales in a scientific setting [1]. The State of World Fisheries and Aquaculture 2016 report [2], released in 2016 by the Food and Agriculture Organisation of the United Nations, states that it contributes to global food security and dietary diversity [3,4]. Tilapia fish scales are safe to use because of the following factors: it is cheap and biocompatible, with no risk of diseases, such as foot and mouth disease, high absorbability, and no religious hindrances. Global food production and consumption have considerably expanded along with the rise in population. Since it is now recognised as one of the primary sources of animal protein in the majority of countries, fish farming has grown in popularity among other forms of food production [5]. The processing of aquatic products has recently been determined to be approximately 67 million tonnes per year on average. Around 5% of the spent (waste) material is made up of fish scales, which is more than 30% of the total amount of aquatic products [6].

Calcium hydroxyphosphate, is also referred to as hydroxyapatite, has a chemical formula of Ca₅(PO₂)₃(OH), but it is typically written as Ca₁₀(PO₄)₆(OH)₂. Fish scales, eggshells, and crab shells are examples of natural waste materials that can be used to make hydroxyapatite. Experts from around the world are interested in this inorganic material, since it is ideal for implant use and has a chemical structure similar to human bones and teeth [7]. The structure of the fish scale and bone is identical, and the fish collagen dermis has gradually changed into its derivatives. The three polypeptide chains that make up the collagen’s basic structure, which is coiled into a left-handed helix with hydrogen bonds acting as structure-directing agents, each contain the whole sequence of amino acids [8].The present paper demonstrates how the fish scale molecules are good corrosion inhibitors by studying the organic and inorganic compositions, thereafter comparing the experimental data with the quantum simulation studies.

2. Materials and Methods

2.1. Preparation of Tilapia Fish Scale

Tilapia fish scales were collected from a local fish market. The fish scales were washed with distilled water and dried with the use of a LASEC-EMF 260 oven from LASEC Group South Africa, at a temperature of 70–80 °C for 7–8 h. An electric grinder was used to crush the dried fish-scale sample to powder form and then it was sieved in order to separate the crushed fish-scale sample into uniform particle size by using a laboratory sieve of 300 µm in size. The grinded fish scales were then taken through the maceration process, which is carried out to extract the required molecules, whereby ethanol (98%) was measured in a conical flask to have a ratio of 1:5 sample to ethanol. The flask was closed with a cap, cello-tape was used to make it airtight, and it was thereafter left for 3–4 days to allow for good residence time. The mixture was then poured into a sieve of about 100 µm, allowing the thick, muddy material to remain behind. The filtrate was then poured into a rotary evaporator running at 80 °C in order to remove the ethanol. The sample remaining in the evaporator, which quickly turned into a powder-like form, was then taken out and stored in sample bottles. Figure 1 shows the preparation of the Tilapia fish-scale sample.

2.2. Physicochemical Characterisation of Tilapia Fish Scales

2.2.1. X-ray Diffraction (XRD)

The processed Tilapia fish-scale sample was analysed using the D8 Advance diffractometer equipped with a flip stick automated multi-position sample stage with a Cu-radiation (wavelength of 1.5406 Å). The scanning was carried out between 12° and 100° (2θ) with a 0.02° step size at 4 s. The sample was analysed using the standard open polymethyl methacrylate (PMMA) sample holders. The qualitative identification of the crystallographic phases was conducted using the search/match technique, where the measured diffraction peaks (intensities and peak positions) are matched against the ICDD’s PDF-4+ 2020 database entries using the Sieve+ software. Data treatment for the search/match procedure comprised subtracting the background contribution and stripping the kα2 X-ray beam contamination from the as-measured diffraction patterns. The quantification-proposed phase and crystallinity determination were carried out using the diffract-topas software v6 that employs the Rietveld refinement method [9].

2.2.2. Fourier Transform Infra-Red (FTIR) Spectroscopy

The FTIR sample holder was first cleansed with ethanol, and a small amount of the fish-scale sample was then placed inside. The FTIR Perkin Elmer Spectrum 100 spectrometer supplied by PerkinElmer SA, was employed to identify the functional groups within the fish scales at a range of 550 to 4000 cm⁻¹ with the spectra resolution maintained at 16 cm⁻¹. Thirty-two scans were co-added and averaged in order to achieve an acceptable signal-to-noise ratio. In all cases, spectra resolution was maintained at 16 cm⁻¹.

2.2.3. Scanning Electron Microscope (SEM)

Approximately 0.05 g of fish-scale extract was placed in the sample holder, and then carbon coated in the coating machine before it could be placed in the SEM instrument. The SEM instrument used is EDAX ELITE PLUS at a scale of 300 µm and sampling region of 155×, 20 kV. The system was operated in a low vacuum. The pressure was allowed to decrease in the equipment chamber, and the sample holder was moved to a good position where the FS sample was clearer. When the pressure was stable, the chemical analysis of the fish scale was analysed.

2.3. Quantum Chemical Studies

All calculations were performed using Gaussian16 revision C01 software together with the B3LYP level of theory and 6-311++G(d,p) basis set [10,11]. The simulation process involved conducting an optimisation of each of the molecules studied (collagen, hydroxyapatite and a combination thereof). The minimum energy conformer of each molecule was confirmed with the aid of normal mode analysis, which produced no negative/imaginary frequencies for the structures.

Various properties, such as the absolute electronegativity (ꭓ), the fraction of electrons transferred (ΔN), the energy of the highest occupied molecular orbital (E_HOMO), the energy of the lowest unoccupied molecular orbital (E_LUMO), the ionisation potential (I), the electron affinity (A), the hardness (η), and the softness (S) were all computed for the minima that where identified above. Ionisation potential (I), electron affinity (A), electronegativity (ꭓ), and global hardness (η) can all be expressed in terms of the E_HOMO and the E_LUMO, according to Koopman’s theorem [12,13,14]. All these chemical terms have been deduced by applying the equations below [15].

I = −E_HOMO

(1)

The amount of energy released when a proton is introduced to a system is known as electron affinity (A) and this is expressed as the negative form of the LUMO:

A = −E_LUMO

(2)

The ability of an atom or collection of atoms to draw electrons to itself is known as electronegativity (ꭓ). [15]; it can be estimated by using Equation (3):

$$\chi ={\displaystyle \frac{1+A}{2}}$$

(3)

A measure of an atom’s resistance to a charge transfer is called chemical hardness (η), which is estimated by using Equation (4):

$$\eta ={\displaystyle \frac{1-A}{2}}$$

(4)

According to the Pearson theory [16], the fraction of transferred electrons (ΔN) from the fish-scale molecule to the steel atom can be calculated with Equation (5):

$$∆N={\displaystyle \frac{\chi Cu-\mathsf{\chi }\mathrm{I}\mathrm{n}\mathrm{h}}{2(\eta \mathrm{C}\mathrm{u}+\eta \mathrm{I}\mathrm{n}\mathrm{h})}}$$

(5)

ꭓ is the electronegativity of copper and inhibitor, respectively, while η is chemical hardness of copper and inhibitor, respectively.

3. Results and Discussion

3.1. Results of the Physicochemical Characterisation of Tilapia Fish Scales

3.1.1. X-ray Diffraction (XRD)

The processed fish-scale sample’s treated diffraction patterns are overlaid in Figure 2. The strength of the peaks, to a first order, provides a clue as to the weight percentage (wt.%) of the corresponding phases in the sample. As a result, the principal phases are indicated qualitatively by the intense peaks at low diffraction angles, whilst the minor phases are indicated by the low intensity peaks in the sample. The diffractive peaks (2θ) in Figure 2 are an indication of the presence of hydroxyapatite (Ca₅PO₄)₃OH from 25.9θ to 75θ, as well as the collagen atoms (C, O, N, H molecules as they were picked up during the XRD scan from 13θ to 25θ [17,18]).

3.1.2. Fourier Transform Infra-Red (FTIR)

From Figure 3, the transition band in point 1, given as 3285.93 cm⁻¹, is believed to be a hydroxyl group (OH⁻) which is found in the collagen and hydroxyapatite molecules. Point 2 also indicates the OH band, but at this point it is for water, which may be as a result of small traces of ethanol left on the processed fish scale or even moisture from the machine itself; the carbonate CO₃⁻², phosphate PO₃⁻ groups are also present, which can also be attributed to the presence of amides between points 2 and 3.

Table 1. Functional groups from the FTIR results.

Frequency Range (cm−1)	Functional Group
3200–3500	N-H aliphatic primary amine/OH
1620–1700	CO2−/C=C alkene
1400–1300	C–H group
1200–800	PO43− and C–H
550–600	PO43− and C=C

summarises all functional groups present in the Tilapia fish scale which indicate the content of hydroxyapatite and type I collagen.

3.1.3. Scanning Electron Microscope (SEM)

The SEM analysis was carried out by running spectra with six different points of reference on the sample as shown in Figure 4, in order to obtain the element compositions.

Table 2. Element compositions of the processed Tilapia fish scales.

Average Amounts	Weight %	Atomic %	Error %
C K	19.8	30.995	11.5
O K	41.3	49.09	10.9
P K	11.9	7.107	3.072
Ca K	27	12.685	2.2

shows the averaged element compositions of the processed FS sample. It is evident that the results support those of the FTIR and the XRD, showing the presence of hydroxyapatite and collagen represented by atom Ca, P, O and C.

3.2. Quantum Chemical Analysis Results

The theoretical research and results from laboratory experiments may be highly compatible or irreconcilable. In order to validate the theoretical approach by comparing experimental data, quantum mechanical approaches were used to evaluate data in theoretical computations. The monoclinic Ca₅(PO₄)₃OH (Figure 5b) and hexagonal Ca₁₀(PO₄)₆(OH)₂ (Figure 5c) structures, with energies of 6400 hartee and 10,778 hartee, respectively, were studied closely, and the choice was made to use the monoclinic structure as it was more stable for this study—the structure is smaller and was easier to optimise. The monoclinic hydroxyapatite was then combined with the collagen after their structures were optimised to form a whole fish-scale molecule as shown in Figure 6.

It is easier for an active molecule to provide electrons to the unoccupied d-orbital of the surface, resulting in a E_HOMO value [10,19].

Table 3. Computational quantum parameters obtained from B3LYP/6-31G(d,p).

No.	Quantum Chemical Parameters	Collagen	Hydroxyapatite	Fish-Scale Molecule
1.	Molecule formula	C12H19N3O5	Ca5(PO4)3OH
2.	Energy (RB3LYP) (au)	−1009.1930	−5381.2380	−6400.0110
3.	EHOMO	−0.2470	−0.2076	−0.1804
4.	ELUMO	−0.0235	−0.1141	−0.0632
5.	∆E (eV)	0.2240	0.0900	0.1172
6.	Dipole moment (µ)	2.6695	20.5643	26.563
7.	Ionisation potential (I) (eV)	2.4700	0.2076	0.1804
9.	Electron affinity (A) (eV)	0.0235	0.1142	0.0631
10.	Electronegativity (x)	0.5110	0.5570	0.5320
11.	Chemical hardness (η)	0.4883	0.4430	0.4680
12.	Chemical softness (S)	2.0480	2.257	1.6030
13.	Fraction of electrons transferred (∆N)	4.4060	4.8115	4.5810
14.	Electronic energy (EE)	1009.1930	−1009.1930	6400.0110
15.	Polarisation (α)	175.9433	218.2313	383.2970

contains the calculated E_HOMO and E_LUMO values for collagen, hydroxyapatite and the fish-scale molecule. The monoclinic hydroxyapatite was used to construct the fish-scale molecule, as it was found to be more stable than the hexagonal one [20]. Data analysis reveals that collagen has the lowest energy E_HOMO compared to hydroxyapatite. The collagen, on the other hand, stabilised the HOMO level by 0.0394 eV. The energy gap (∆E), given by the below equation, is a crucial quantity as it is typically characterised by strong chemical activity and low kinetic stability [21,22].

E = (E_HOMO − E_LUMO)

(6)

According to Table 3, hydroxyapatite has the lowest energy gap (0.09 eV), while inhibitor collagen has the biggest energy gap (0.224 eV), which suggests that the hydroxyapatite molecule is the most reactive. The hydroxyapatite dipole moment in our study is 20.564 eV, while the collagen dipole moment is 2.669 eV. The hydroxyapatite’s high dipole moment value indicates greater reactivity/effectiveness [18]. The ionisation energy values of the collagen and hydroxyapatite are 0.247 eV and 0.208 eV, respectively. The low ionisation energy 0.208 eV of hydroxyapatite indicates the high reactivity and absorbability. In the current investigation, hydroxyapatite has a lower hardness value of 0.443 eV compared to collagen at 0.624 eV. A soft molecule has a modest energy gap compared to a hard molecule, as has been shown with the energy gaps discussed above. The percentage of electrons transferred (∆N) also reveals if a molecule can donate electrons. Hydroxyapatite has a high level of reactivity and adsorption effectiveness, with the largest percentage of electrons transferred at 4.812 eV, whilst the collagen is at 1.351 eV.

From Table 3 it is shown that the fish-scale molecule has a donor–acceptor capability which is fairly evenly distributed, as shown by the HOMO and LUMO energies. Figure 7 shows the HOMO and LUMO structures for the FS molecule, hydroxyapatite and collagen.

The interaction energy was determined by first taking the optimised structures of the hydroxyapatite and collagen and aligning them according to the HOMO and LUMO active sites. About six combinations were constructed, with each being optimised, and the resulting interaction energy was computed.

Table 4. The Gaussian calculated energy values.

Structure	Fish Scale	Collagen	Hydroxyapatite	Einteraction (Hartee)	Einteraction (kJ/mol)
1	−6400,5313	−1009,1757	−5391,2311	−0,1245	−326,8700
2	−6400,4592	−1009,1899	−5391,2133	−0,0560	−146,9729
3	−6400,4781	−1009,1919	−5391,1999	−0,0863	−226,5597
4	–6400,6190	–1009,1929	–5391,1917	–0,2343	–615,2623
5	−6400,4460	−1009,1931	−5391,2252	−0,0277	−72,7053
6	−6400,4644	−1009,1898	−5391,2057	−0,0990	−181,0518

outlines the results from the optimised structures in Figure 7.

The interaction energy was calculated by the following equation:

E_int = E_total − (E_col + E_HA)

(7)

where E_int is the interaction energy, E_col is the energy of collagen and E_HA is the energy of the hydroxyapatite.

                            E_int = − 6400.5831 − (1009.172 + 5391.191)
= −0.2 hartee
     = −615.23 kJ/mol

The binding energy is given by:

E_binding = −E_interaction

From the above calculation, we can therefore say that it takes 615.23 kJ/mol for the fish-scale molecules to interact with each other.

The DFT parameters were also used to create an IR spectrum which was obtained from the vibration of the optimised structures. This was used to compare the laboratory results from the FTIR.

Figure 8 exhibits similar peaks for the OH, Ca and P atoms with the peaks from 500 to 1500 cm⁻¹ representing strong characteristics of hydroxyapatite and picks from 1500 to 3500 cm⁻¹ representing those of collagen.

Table 5. Functional groups from the IR spectrum using DFT parameters.

Frequency Range (cm−1)	Functional Group
3200–3500	N-H amide/OH
1630–1700	CO2−/C=C alkane
1200–1450	C-H group
500–1200	PO33− and C=H

outlines the same functional groups as those reported from the FTIR results from the processed FS obtained from the laboratory experiments.

4. Conclusions

In the presented research work, Tilapia fish scales were processed and characterised. Fish scales were characterised using the XRD, FTIR, and SEM analyses. The results from all three techniques show that the presence of significant molecules like phosphate (PO₃⁻⁴), carbonate (CO₃⁻²), and hydroxyl groups (OH⁻) groups makes fish scales an essential waste material to use in many industries. The XRD showed a high presence of hydroxyapatite, while the FTIR showed the combination of hydroxyapatite and collagen, together with other components such as amino acids and amide. SEM confirmed the presence of both collagen and hydroxyapatite. These groups can be found in the molecules of collagen and hydroxyapatite, which are currently used mostly in the medical field to treat cancer and skin conditions. Quantum chemical studies show that hydroxyapatite is the most reactive compound through the studied parameters, and the IR spectrum confirmed the presence of both collagen and hydroxyapatite as illustrated by the FTIR results. The results from both the experimental and computational studies show that Tilapia fish scales can be used as a green corrosion inhibitor due to the presence of collagen and hydroxyapatite as well as some amino acids. Future work can involve the study of the inorganic and organic molecules of the fish scales separately, as corrosion inhibitors.