1. Introduction

The antimicrobial bio-polymers have generally considered as key to the enormous desires for the last years, in decent wound [1], engineer tissue [2], medical textile [3], packaging [4] and water treatment [5]. Mid all natural bio-polymers, chitosan is easily available, found in several insects and microorganisms and considered one of the most effective antimicrobial bio-polymers[6, 7 ]. By simple deacetylation process, chitosan can be easily extracted from chitin via deacetylated unit (\(\beta\)-(1-4)-linked d-glucosamine) copolymer as characterized by its basic character and simplified its solubility in the acitic acid, non-toxic, anti-bacterial activity, biodegradability and excellent biocompatibility [8, 9, 10]. So, it widely used in the bio-medical applications as a drug carrier [11], antimicrobial [12], antioxidant [13], antitumor [14], and a wound dressing agent [15, 16].

In addition, chitosan can be chemically modified for widen its applications via grafting with functionalized monomers [17, 18, 19, 20, 21, 22]. As it is well known that chitosan has a decent antimicrobial activity against some types of bacteria, and these activities are interconnected to the quantity of the adsorbed chitosan on the cells of bacteria due to the non-stop mutations of microorganisms coupled with new antimicrobial agents [23, 24]. Accordingly, scientists are struggling to synthesize novel antimicrobial chitosan derivatives to obstruct the wound infections. Several studies have reported the significant effect of antimicrobial bio-polymers and chitosan itself to enhance the wound healing process through preventing the wound infections that result in tissue maceration [25, 26, 27]. It has been reported that chitosan modified diisocyanate (DIMC) [28], O-quaternary ammonium N-acyl thiourea chitosan [29], chitosan-thioglycolic acid [29, 30], kanamycin-chitosan nanoparticles [31], crosslinked chitosan [32] and O-amine functionalized chitosan [33] exhibited better antibacterial activity degrees against Escherichia coli, Staphyloccocus aureus and other microorganisms. Likewise, chitosan Schiff base derivatives are considered one of the best choices for increasing antimicrobial activity of chitosan, since carbonyl groups of aldehyde or ketone can efficiently couple with \(NH_2\) groups of chitosan to form the corresponding chitosan Schiff base with imine characteristic group (-RC=N-) [34]. This leads directly to altering chitosan molecular structure, enhancement its hydrophilicity as well as increasing the positively charged ions, which results in better antibacterial activity compared to the unmodified chitosan.

The present work reports the development of antimicrobial chitosan derivatives that could accelerate the wound healing via hindering the microbial infections [35, 36, 37]. Herein, Spiroquinazolinone-chitosan (SQC) was synthesized, characterized and verified using different characterization tools. Furthermore, their antimicrobial activities were examined against various pathogenic microorganisms that frequently irritating infections.

2. Results and discussion

2.1. Chemistry

It has been synthesized by reaction of anthranilic acid with phthalic anhydride to afford N-benzoyl-anthranilic acid. Refluxing of N-benzoyl-anthranilic acid in acetic anhydride afforded 2-phenyl-4H-3,1-benzoxazin-4-one (1) as outlined in following Scheme [El-Hashash, \emph{et.al} 2018, 2017]. The reaction of the anthranilic causes the ring opening of the phthalic anhydride to afford the corresponding anthranil intermediate followed by ring closure to the afford the 2-benzoxazinyl-benzoic acid drug.

2.2. The DFT study of the benzoxazinone derivative

In the same way, DFT simulation of the benzoxazinone deivative outlines the electrophilic sites of the optimazid structure that has LUMO energy -3.73eV to interact with the nucleophilic amino group of bacteria and inhibit its enzyme. So, the benzoxazinone is considered good antibacterial reagent. In the same problem occur in which the benzoxazinone drug can scavenge all the active enzyme in the human body and so it must inhibit this drug immidiately after finishing from the harmful bacteria by the chitosn moieties.

2.3. Inhibition of the drug by chitosan

Reaction of the amino group of the chitosan with 2-benzoxazinyl benzoic acid afforded the spiro-quinazolinone derivative[ El-Koly A, Rizk S; 2019; El-Hashash M, Rizk S, 2017] as outlined in the following Scheme. Formation of spiro benzofuranon-quinazolinone product can confirmed the chitosan has basic character that outlined the following Scheme.

DFT simulation still confirmed that HOMO of chitosan is prefered from the HOMO of active enzyme. At this moment, the chitosan is suitable until now to inhibit the synthesized drug. The results of antibacterial and antifungal in the biological part are good agreement with the DFT study of the synthesized organic material.

Moreover, the chitosan itself has antimicrobial activity but, when it reacts with the drugs, loss the all activity of the chitosan and drug for microbe. The authors concluded that chitosan and drugs alone has become antimicrobial agents but when they mix chimically in one pot promoted ultrasound reaction, become no activity towards the bacteria and fungi.

2.4. Degree of deacetylation (DD)

Actually, there are several common methods have been adopted for determination the degree of deacetylation (DD) of chitosan via measuring its free amine groups. Since, it's considered one of the most essential parameters, which directly affect the properties and applications of chitosan. These methods have been conducted by using FT-IR [38], NMR [39], UV-spectrophotometric analysis [40], colloidal titration [40], and potentiometric titration [41, 42]. The degree of substitution could be estimated via determination the content of free amine groups in chitosan before and after reaction. Table 1 represents the calculated values of DD for chitosan and its two Schiff base derivatives.

Table 1. DD and DS values obtained from potentiometric titration method for chitosan and its Spiro derivative.

Sample	DD%	DS%
Chitosan	93.15	---
Spiro chist quin (SQC)	81.1	12.05

Moreover, the degree of substitution (DS) was \(12.05\%\) for Spiro quinazolinone-Chitosan.

2.5. FT-IR analysis.

Figure 2 demonstrates the FT-IR spectral analysis of neat chitosan and its two benzoxazinyl benzoic acid and Spiro chitosan quinazolinone derivative. The spectrum illustrates a typical band of polysaccharides such as common bands between 3200-3400 \(cm^{-1}\) corresponding to hydroxyl and amine groups. 2960 and 1933\(cm^{-1}\) refer to stretching vibration of \(CH\) and \(CH_2\). 1745 and 1565 \(cm^{-1}\) assigned to C=O spirofuranone with flattened with chitosan. The band at 1370\(cm^{-1}\) corresponding to NH2 bend vibration, where multipacks between 1200-1000 cm\(^1\) associated with glycoside C-O, C-O-C and C-C bond [21].

2.6. Electronic spectra.

Figure 6 investigates the UV-visible spectra for chitosan, BBA and its novel SQC. It was obvious in case of chitosan that the apparent absorbance band at maximum 230 nm could be attributed to \(n-\sigma^*\) transition of amine free electrons [43]. Conversely, in case of BBA(I) and SQC(II), the increase in the peak intensity as well as, the observed shift to the higher wavelengths 247 and 361 nm, respectively, could be explained by increasing in donor ability of the substituent to stabilize the excited state [44]. BBA and SQC bonds display a red-shift, which refers to the attachment benzoxazinone and conjugated Spiro precursor respectively to amine groups along chitosan backbone [45]. Furthermore, generation of new bands at higher wavelength from 259.6 to 300nm for BBA (I) and multibands from 361 to 421 nm for SQC (II) could be ascribed to the formation of spiroquinzolinone furanone, which creates a new transition \(n-\sigma^*\) at a higher wavelength.

3. Antimicrobial assay using Agar-well diffusion method

Over the recent years, Antimicrobial activities of chitosan and its derivatives have been drawing considerable attention. In addition, mutations of microorganisms to resist the action of antimicrobial materials have enhancing scientists to develop new antimicrobial materials have potential activity against the new version of pathogenic microorganisms. Antimicrobial activities of chitosan, BBA and the developed SQC were initially determined using agar-well diffusion technique against representative Gram-positive bacteria (S. aureus and B. cereus) and Gram-negative bacteria (E. coli, P. aeruginosa, and Salmonella sp.) in addition to yeast strain. For bacterial strains, the activity of chitosan and its derivatives were compared with erythromycin as reference antibiotics, while, nystatin was considered the positive control for yeast strain. The inhibition zones of antimicrobial activities were measured as given in Table \ref{t2}.

Although the results showed that P. aeruginosa, S. aureus and B. cereus were resistant to the erythromycin, chitosan and its derivatives presented remarkable activities against these tested bacteria. The results exhibited the significant efficacy of the two chitosan and BBA against the indicator microorganisms more than the SQC biopolymer. We could deduce from these findings that BBA (I) demonstrated the highest activity in the presence of all examined microorganisms regardless their structures. The antimicrobial action of chitosan might be differed according to several intrinsic factors including chitosan source, the molecular weight that influences the penetration inside microorganisms, and the synthesis of new chitosan derivatives with novel characteristics that usually diminish the antimicrobial action of chitosan. Three principal mechanisms have been posited to elucidate chitosan interaction with various kinds of microorganisms, which vary based on the cell wall structure and metabolic process [46]. First mechanism is chemical interaction via electrostatic manner between \(+ve\) charge of amine groups (NH3+) of chitosan with -ve charges on the cell wall of various microorganisms. 2nd mechanism shows the chitosan Mwt which controls its diffusion into the nuclei of microorganisms and binding with DNA to prevent the protein synthesis. 3rd mechanism depends on the bulk chelating of chitosan to metal ions which are vital constituents for microbial growth and metabolic pathways such as spore formation in Gram\(-+ve\) bacteria. Accordingly, we propose that the previous mechanisms could be donated together to implement the antimicrobial potency of the synthesized chitosan derivatives.

Earlier studies have reported the BBA compound income its antimicrobial activity, and the newly derivatives showed excellent antibacterial, antifungal, antiparasitic and anticancer activities [47, 48]. This illustrates the synergistic boost of chitosan activities against the examined microorganisms compared to the synergistic effect of Spiroquinazolinone furanone moiety to the prime chitosan. Significance surprising of these results, MIC, bactericidal and fungicidal activities of the novel chitosan SQC derivative were reduced. The authors can be explain combination of the BBA with chitosan amalgamation that antimicrobial activities in both as blocker for each other to decrease the active sites \((NH_2)\) lactonic group and COOH by forming spiroquinazolinone-furanone of chitosan product SQC.

Table 2.Inhibition indices of chitosan and chitosan Schiff bases (I \& II) against E. coli, P. aeruginosa, Salmonella sp., S. aureus, B. cereus, and C. albicans. Values are expressed as mean \(\pm\) SD (\(n = 3\)).

Bacteria	Erythromycin	Chitosan	BBA	SQC
E. coli	11 \(\pm\) 0.2	11.5 \(\pm\) 0.19	17.7 \(\pm\) 0.16	3.7 \(\pm\) 0.03
P. aeruginosa	-ve	12.6 \(\pm\) 0.13	17.2 \(\pm\) 0.24	-ve
Salmonella sp.	12.5 \(\pm\) 0.17	13 \(\pm\) 0.17	17.1 \(\pm\) 0.15	2.7 \(\pm\) 0.04
S. aureus	-ve	13.9 \(\pm\) 0.19	18.9 \(\pm\) 0.21	-ve
B. cereus	-ve	13.6 \(\pm\) 0.16	18.1 \(\pm\) 0.2	-ve
Fungi	Nystatin	---	---	---
C. albicans	15.2 \(\pm\) 0.22	11.4 \(\pm\) 0.14	15.8 \(\pm\) 0.25	3.5 \(\pm\)\ 0.18

4. Determination of MIC

In the current research, minimum inhibitory concentrations (MICs) could be defined as the lowest concentrations of chitosan, BBA and SQC that prevent the growth of the tested strains after overnight incubation as presented in Figures [\ref{f7}-\ref{f9}]. These completely hindrance of microbial growth have not attained using the unmodified chitosan and modified SQC (II). With regard to SQC, MIC value was observed at concentration of 50 µg/ml for all examined microorganisms excluding B. cereus and C. albicans that required 300\(\mu\)g/ml for inhibiting their growth. The overall results of Spiro chitosan quinazolinone SQC revealed lower activity than the original chitosan; therefore, amino group motivated the antimicrobial activities of chitosan. Moreover, we derived that BBA has the strongest activities against whole indicator microorganisms and this action could be elucidated via boosting the chitosan behavior that clearly showed a synergistic influence on chitosan. This renders SQC the most acceptable candidate for further investigations.

5. Material and Methods

5.1. Materials

Chitosan (MW. 100,000-300,000 Dalton) was obtained from Across Organics. (New Jersey, USA), 2-benzoxazinylbenzoic acid, (purity \(97\%\); M.W. 267.15) was acquired from Sigma Aldrich (Germany). Sodium hydroxide pellets (purity \(99-100\%\)), Sulfuric acid (\(98\%\)), Ethanol (\(99.9\%\)) were supplied from International co for Supp & Med. Industries, (Egypt).

5.2. Microorganisms

Representative Gram-negative bacteria [Escherichia coli (E. coli.), Pseudomonas aeruginosa (P. aeruginosa) and Salmonella sp.] and Gram-positive [Staphylococcus aureus (S. aureus), and Bacillus cereus (B. cereus)] bacteria in addition to Candida albicans (C. albicans) were utilized to study the antibacterial and antifungal performance of the development materials. All strains were revived from glycerol stocks via growing overnight at \(37^oC\) and 150rpm in LB broth medium containing (peptone 1%, yeast extract \(0.5\%\), \(NaCl 1\%\)).

5.3. Synthesis of 2-(4-oxo-4H-benzo[d][1,3] oxazin-2-yl)benzoic acid

A mixture of 2g anthranilic acid (0.002 mol) and 1.8g phthalic anhydride (0.002 mol) in 20ml n-butanol was refluxed for 3h. After cooling the obtained solid was collected, dried and recrystallized from n-butanol to give 1.61g 2-(3-carboxybezamido)-5-benzoic acid in yield \(76\%\) as deep brown crystals: m.p. \(180-182^oC\). A solution of 2g 2-(3-carboxy-bezamido)-5-benzoic acid (0.01 mol) in 15ml acetic anhydride was refluxed for 1h, after cooling the obtained solid was filtered off and crystalized from ethanol to give 1.72g of 2-benzoxazinyl-benzoic acid in \(75\%\) yield as white crystals: m.p. \(148-150^oC\); IR (KBr)v': 3443(broad), 1757, 1707, 1607 cm-1; 1H NMR (CDCl3, 300 MHz)\(\delta\):7.2-8.8 (ArH), 4.9 (s, 1H, OH exchangeable with D2O ); MS (70eV) m/z (100%): 392.95 (100.0%), 393.95 (16.2%), 394.96 (1.2%). Calculated Elemental Analysis for C15H9NO4 (MW 267): C, 45.83; H, 2.05; N, 3.56; found C, 45.79; H, 2.32; N, 3.46.

General Procedure for the preparation of the chitosan-spiroquinazolone

An equimolar mixture of 2-benzoxazinyl-benzoic acid (3.03 g 0.01 mol) and chitosan (0.01 mol) in 50 mL boiling ethanol. The reaction mixture was refluxed for 3h. The solid that separated after cool was filtered off, washed by petroleum ether (b.p \(40- 60^oC\)),dried and then, crystallized from DMF. Yield \(70\%\). m.p. 300-302. IR(KBr)v 3333, 3225(NH), 3056 (CHAr), 1804, 1670 (CO) of spirofuranone and quinazolinone. 1HNMR (DMSO):\(\delta\) multiplet at 7.23-7.72 assigned for 8ArH aromatic protons, singlet peaks at 5,54 and 6.23 a acidic NH and OH a protons which exchanged in D2O.

5.4. Physicochemical characterization

The changes in the chemical structures and surface morphologies of the prepared chitosan, BBA and SQC were investigated by Fourier Transform Infrared Spectrophotometer (FT-IR; Model 8400 S, Shimadzu, Japan) was examined. Moreover, the electronic spectra of the developed chitosan Schiff bases were identified via UV-Vis Spectrophotometer (Ultrospec 2000 Pharmacia BiotechCo., Cambridge, England) in scanning ranged using \(0.2\%\) chitosan or its new Spiroquinazolinone derivatives. The electronic absorbance was investigated in the scanning range 200-500nm [43].

5.5. Antimicrobial assay using Agar-well diffusion method

Agar-well diffusion approach was adopted to assess the antimicrobial activities of pure chitosan, BBA and chitosan SQC derivative against various pathogenic microorganisms (E. coli, P. aeruginosa, Salmonella sp., S. aureus, B. cereus and C. albicans) as previously described. Old overnight cultures of the tested microorganisms were diluted 10-fold in LB broth free medium and their turbidities were adjusted to be equal the McFarland 0.5 standard via measuring at 625nm, where bacterial strains were \((1-2 \times 108)\)CFU/ml, while C. albicans was \((1-5 \times 108)\) CFU/ml. Then, \(50 \mu l\) of the cell suspensions were spread over the surface of LB agar plates using glass spreader. The Agar plates were bored with a metal cork borer to produce wells of 6 mm in diameter, and \(50\mu l\) of the examined biopolymers were loaded. Afterwards, the Petri dishes were kept in the fridge for 2h to diffuse the materials into the agar.

The plates were aerobically maintained at \(37^oC\) for 18h, and the antimicrobial activities were then determined via estimating the inhibition zone of microbial growth. Our previous report proved the efficiency of chitosan to inhibit the growth of several microorganisms; therefore, it considers positive control. Moreover, reference antibiotics including erythromycin \(15\mu g/ml\) and nystatin (100 units) discs were applied for bacteria and Candida experiments, respectively. This assay was adopted as a screening approach to determine whether the prepared BBA and SQC products have siginficant inhibition against the indicator microorganisms to apply the further studies or the further should be neglected. Therefore, this antimicrobial evaluation for the investigated biopolymer was implemented in triplicate.

5.6. Determination of minimum inhibitory concentration (MIC).

Microtiter plate method is one of the most effective approaches for determining the MIC for various antimicrobial agents. Consequently, it was conducted to investigate the impact of different concentrations of chitosan and the new derivatives on the growth of the indicator microorganisms as previously demonstrated. The tested biopolymers were sterilized by 0.22\(\mu\) m syringe filter previous to use. The overnight bacterial cultures were diluted 100-folds in LB broth free medium to optical densities of 0.9 for all microorganisms via measuring the bacterial turbidity at 600nm. Afterward, 20\(\mu\) l of the bacterial culture suspensions were inoculated into a sterile 96-well microplate, and various levels of filtered native chitosan and modified chitosan (25, 50, 100, 150, 200 and 250\(\mu\)g/ml) were incorporated. The wells were then completed with LB broth free up to 200 \(\mu\) l followed by mixing well employing a bench shaker for 2 min at 100 rpm and incubated aerobically at 37\(^o\)C for 24h.

6. Conclusion

Chitosan with different function groups (i.e.; hydroxyl and amine groups) has a promising structure to prepare new derivatives. Here, Carbonyl groups of the compound BBA and was coupled with chitosan amine group to produce SQC respectively. The formation of compound SQC was confirmed by monitoring modifications in the chemical structure using spectroscopic analysis, FT-IR, abd elemental analysis. The findings of the antimicrobial activities revealed that antimicrobial activities of chitosan and BBA were significantly boosted by amino and lactonic groups respectively. Furthermore, the cellular toxicity assay pointed to the safety of SQC. The present findings lead us to further this study via preparing a membrane of SQC for investigating in vivo as a promising wound dressing candidate to promote the wounds and burns healing process.

Author Contributions

All authors contributed equally to the writing of this paper. All authors read and approved the final manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

1. Introduction

To reduce our dependence on fossil fuels and reduce the emission of carbon dioxide, a large scale transition toward sustainable energy sources is necessary [1]. In this regards Hydrogen could play an important role in our modern life. It is a promising energy carrier, which could have a low impact on the environment and its energy content is 10 times greater than fossil fuels. Hydrogen production through the water splitting is a cheapest and clean source of energy [2]. On large scale production of hydrogen from the water splitting, largely depend upon the catalysts that are require to overcome the challenging requirement thermodynamically and kinetics of this half reaction [3, 4]. From an electrochemical point of view, it can be split into the two half reactions:

The oxygen evolution reaction (OER) is a key reaction and often the rate-determining step in electrochemical and Photoelectrochemical water splitting, which is a promising route to carbon-free hydrogen production. For this process we require the OER catalysts which are close to the state- of art catalysts. Iron oxide (\(\alpha\)-hematite) because it has been identified as a suitable water oxidation catalyst [5, 6, 7, 8, 9, 10], conjugating an overall good performance in both electrolysis and solar water splitting schemes with the advantage of being earth-abundant, non-toxic and environmentally safe.

Pulsed-laser deposition (PLD) of thin films is a technique that employs high-energy-density laser pulses to generate, in the regime of phase explosion, ablated material from a solid target, consisting of a mixture of vapor/liquid nanodroplets. PLD could present some significant advantages over the methods listed above: precise control of the quantity of the deposited material, enhanced adhesion due to the energetic nature of the process, and, most importantly, the possibility of nanostructuring the surface by the deposition of nanoparticles (NPs) [11, 12]. Additionally, being essentially a physical deposition method, it is suitable to all kind of substrates. The main drawback of the PLD technique is the need of specialized equipment, although this is already employed in industrial applications.

2. Materials and Methods

The target for the Pulse laser deposition consisted of the cold pressed with a pressure of about \(450-500Kg/cm^2\) for two times consecutively powders of Fe and Boric acid. Two sets of electrodes, fabricated on the FTO substrates with different thickness by changing the number of pulses: 5000 pluses for the thinner samples and \(10000\) pulses for the thicker samples. The fluency of the laser was fixed at 3\(J/cm^2\) and the deposition was carried out in a reactive oxygen atmosphere, at a pressure of \(1.5\times10^{-2}\) mbar. The number of pulses set at \(5000\) and the repetition rate at \(20Hz\). The coatings were deposited on FTO substrates, with a target - substrate distance of \(4.5 cm\). All the sample were subjected to a post-deposition annealing treatment, carried out at different Temperature i.e. \(500^oC, 600^oC\) and \(700^oC\) for 4 hours with heating rate of \(5^oC/min\) and one sample subjected at \(600^oC\) for 4 hours and again at \(800^oC\) for 1hours with heating rate of \(5^oC/min\). We represent the differently annealed sample with the following symbols:

Sample	Temperature
Thin sample annealed at \(500^oC\) for 4 hours	ANN\(@500^oC\)
Thin sample annealed at \(600^oC\) for 4 hours	ANN\(@600^oC\)
Thin sample annealed at \(700^oC\) for 4 hours	ANN\(@700^oC\)
Thin sample annealed at \(600^oC\) and \(800^oC\) for 4 hours	ANN\(@600^oC+800^oC\)
Thin sample annealed at \(500^oC\) for 4 hours	ANN\(@500^oC\) thick
Thin sample annealed at \(600^oC\) for 4 hours	ANN\(@600^oC\) thick
Thin sample annealed at \(700^oC\) for 4 hours	ANN\(@700^oC\) thick
Thin sample annealed at \(600^oC\) and \(800^oC\) for 4 hours	ANN\(@600^oC+800^oC\) thick

The following SEM images show the morphology of the thin films. Samples ANN\(@500^oC\) Figure 1 and ANN\(@600^oC\) Figure 2 show the presence of urchin-like structures, increasing the surface of the catalyst and thus the amount of active sites for the water oxidation.

The lengths of needles are not homogeneous, dense and aggregated nanoparticles are found at \(600^oC\) annealing temperature. Furthermore, SEM images show the nano needle with a non-uniform morphology. Samples show spherical nanoparticles covered with sparse nano flowers. The flower consists of needles petals grown radially from the surface. Probably the roughness present on the core surface is the starting point for the formation of these features. Overall SEM images confirm the urchin like structure on samples ANN\(@500^oC\) and ANN\(@600^oC\).

Sample ANN\(@600^oC + 800^oC\) Figure 3, show that there is no developed urchin like structures. At \(700^oC\) Figure 4 samples show spherical nanoparticles covered with sparse nano flowers. The flower consists of needles petals grown radially from the surface. Probably the roughness present on the core surface is the starting point for the formation of these features. As will be shown in the following sections, the electrochemical performance of these samples is not good. Here I discuss the thin sample SEM images because these are similar to the thick sample.

Figure 4 shows the Raman spectra of the \(Fe_2O_3\) annealed at different temperature. From the Raman spectra it is confirmed that the hematite modes are present. The peak present at 1070 might be an artifact or an impurity present on the surface of the sample after annealing at 500C for 4h. With increasing temperature the peaks became more sharp and visible; clearly indicating the crystallization of the samples.

2.1. Result and Discussion

Electrochemical water oxidation experiments were performed in a three electrode configuration with a working electrode (Iron oxide thin film on FTO), a platinum mesh as counter electrode and saturated calomel electrode (SCE) as a reference electrode, with Gamry Interface 1000 potentiostate. All the electrodes are immersed in a KOH electrolyte solution with pH=13. At this pH, oxygen evolution is expected at potential EO2/H2O = 1.23 - (0.059pH) = 0.463 V (463mV) versus reversible hydrogen electrode (RHE). The Figure 5 compares the anodic response of various thin electrodes annealed at various temperatures.

The oxygen evolution catalysts are required to speed up the reaction rate and reduce the overpotential. Thin Samples ANN@500oC and ANN@600oC shows the response toward the water oxidation at about 0.80 V vs RHE, with overpotentials of 343.9 mV and 370 mV respectively (taken at 0.2 mA/cm2 current density). Moreover, the Tafel slope of the electrode ANN\(@500^oC\) (81.6+0.6 mV/dec-1) is better than that of the electrode ANN\(@600^oC\) (109.8+0.2 mV/dec-1), showing good kinetic towards water oxidation. The electrode ANN\(@700^oC\) shows the response toward water oxidation at 0.90 V vs RHE. Moreover, it's provides the 455.6 V overpotential at 0.2 mA current density, with Tafel slope 135.8+0.5. So the efficiency toward the water oxidation is low. Electrode ANN\(@600^{o}C\)+\(800^{o}C\) shows the bad catalyst behavior towards the water oxidation (Figure 6).

The samples ANN\(@500^{o}C\) and ANN\(@600^{o}C\) show good catalytic performance, in term of overpotential. Large overpotential is associated with slower kinetics for the OER on hematite and with the electronic and structural characteristic of the hematite/electrolyte and hematite/substrate interfaces. After increasing the thickness of the film, then again film ANN\(@500^oC\) thick and ANN\(@600^oC\) thick showed good catalytic performance, both in term of overpotential and tafel slope as compared to the thin films (Table 1). Other samples ANN\(@700^oC\) and ANN\(@600^oC\)+\(800^oC\) display the bad catalytic enactment. The detrimental in water oxidation activity for the samples annealed more than \(600^oC\) could be due to the losing of the conducting properties of FTO. Since, it is clear known that FTO losses its conductivity above \(550^oC\). Thereby, it is clear that the decreased activity is mainly due to the losing FTO conductivity at high temperature annealing.
The overpotential is calculated as $$Overpotential=V_{Applied}+0.241-E^oO_2/H_2O$$ Where \(E^oO_2/H_2O = 463\) mV at pH = 13 and the 240 mV term is required to convert the applied potential Vapplied from SCE to RHE (Reversible Hydrogen Electrode). Samples overpotential and Table slopes are shown in Table 1.

Table 1. Overpotentials at various current densities and Table slopes of the thin and thick films annealed at various temperatures.

Sample	Overpotenital	Overpotenital	Overpotenital	Tafel slope
	at 0.2(mA)	at 1(mA)	at 10(mA)	mV/dec\(^{-1}\)
Ann\(@500^o C\) thin	343.9	412.4	769.8	81.6+0.6
Ann\(@600^o C\) thin	370.0	457.0	631.1	109.8+0.2
Ann\(@700^o C\) thin	455.6	534.5	699.9	135.8+0.5
Ann\(@600^o C+800^o C\) thin	708.9	103.9	---	373.7+2.9
Ann\(@500^o C\) thin	329.1	364.5	416.5	50.2+0.09
Ann\(@600^o C\) thin	398.8	436.6	495.6	56.0+0.1
Ann\(@700^o C\) thin	463.5	526.4	414.4	83.2+0.7
Ann\(@600^o C+800^o C\) thin	615.2	865.0	---	258.8+2.8

The significant difference in table slopes between thin and thick samples ANN@5000C and ANN@600oC could either depend on a different composition of the material (e.g. quantity of magnetite present) or simply be an effect of the different morphology, more porous in the case of thick samples.

3. Tafel analysis

The Tafel slope is also an important measure of electrode performance, because it accounts for changes in mechanism at different overpotential.

We calculate Tafel slope according to the Tafel equation Figure 7, which applies in the voltage region where the forward reaction (OER) is predominant compared to the backward reaction (HER). Figure 7 show the Tafel slope of best performing ANN\(@500^oC\) thick electrode.

4. Chronoamperometries

A good OER catalyst that use in the water splitting device must satisfy the two basic requirement. First it must be highly active towards its respective reaction. Secondly it must be stable in the oxidation condition and maintain the efficiency over the time scale for commercial use.

The 12 hours chronoamperometries were performed for checking the long term stability of the best performing samples: ANN@500oC thick and ANN\(@600^oC\) thick (Figure 8). In the first 2.8 hours the current density of the ANN@600oC thick electrode is increased, then it remains stable at 6mA/cm2 for next 9.2 hours. In the case of the ANN\(@500^oC\) thick electrode, in the first 6 hours the current density fluctuates between7 mA/cm2 and 5 mA/cm2 and then gradually decreases from 5.5 mA/cm2 to 5mA/cm2. It could be due to the bubbles formation on the surface of the electrode.The same pattern is detected when repeating the experiment a second time on the same electrode.

I am raw html block.
Click edit bBefore and after water oxidation the miro-Raman spectra for all the samples are represented in the Figure 9. It is indicating that after water oxidation the peak intensities were reduced, the probable reason could be due to the surface coverge of the electrolyte. However, the phase and crystallinity of the all the samples were well preserved ever after water oxidation, showing that the prepared thin film samples are highly stable to the water oxidation experiments.

5. Conclusion

We investigated that the Iron oxide (\(\alpha\)-hematite) was showing promising catalytic activity towards the oxygen evolution reaction and can be employed as anode in electrocatalytic water splitting techniques for hydrogen production. The best performing electrodes showed an overpotential@0.2 mA/cm2 of about 330 mV and a Tafel slope of 50mV/dec. These electrode metrics are in line with those of current state-of-the-art materials like IrO\(_2\) or CoPi. We observe that the deposition of more material, e.g 10000 instead of 5000 pulses, leads to better overpotentials and Tafel slopes, indicating that the performance can be enhanced simply by increasing the catalyst loading. That is normally not possible for bulk-like thick films or even thin-films where the surface coverage is already complete and in the present case it is likely resulting from the hierarchical 3D structure.

Author Contributions

All authors contributed equally to the writing of this paper. All authors read and approved the final manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.utton to change this html

1. Introduction

Corrosion is a common term germane the industry of crude oil refining similarly generate a few of problems on such ferrous metals. Upon the hole the corrosion is the formation of metal oxides, sulfides, hydroxides or other certain compound on the surface of such a metal yet the rate of corrosion is depend on the conditions of the surrounding and chemical composition of such metal as well [1]. As an essential requirement of the corrosion the metal need to be exposed either some kind of stronger oxidizing agent than \(Fe_2+\) or any environment consisting water and oxygen also the process will be modified by the acids and salt present in the medium. Beside of that the mercaptans and elemental sulfur play a dominant role in the cause of metallic corrosion [2]. Those compounds are found in crude oil in various amounts. Therefore, the crude oil can be emphasized as a corrosive admixture especially in the industry of crude oil refining because of the vast applications of ferrous metals with regard to different units such as the distillation column, storage tanks and transportation tubes. In the current research there were expected to investigate the effect of different two types of crude oils on the rates of corrosion in seven different types of ferrous metals and the variations of the initial hardness of such metals due to the corrosion.

2. Materials and methodology

2.1. Materials

2.1.1. Ferrous metals

Seven different types of ferrous metals were selected as the samples which are having foremost applications in the industry of crude oil refining frequently. A batch of samples was consisted with three different types of carbon steels, three different types of stainless steel and Monel.

2.1.2.Crude oils

It was selected two different types of crude oils which were used in the Sri Lankan crude oil refining industry and also those are slightly different in their chemical composition including essential properties such as corrosive properties.

2.2. Methods

2.2.1.Experiments on crude oils

The sulfur content, Mercaptans content, acidity and the salt content of both crude oils were determined. Because those are the dominant corrosive properties found in crude oils. A descriptive summary of the relevant procedures of experiments for such properties has been given in the Table 1.

Table 1. Procedures of the experiments for the corrosive properties in crude oils.

{Property}	{Method}	{Readings}
Sulfur content	Directly used	Direct reading
Acidity	Each sample was dissolved in a mixture of toluene and isopropyl and titrated with potassium hydroxide.	End point
Mercaptans content	Each sample was dissolved in sodium acetate and titrated with silver nitrate.	End point
Salt Content	Each sample was dissolved in organic solvent   and exposed to the cell analyzer.	sample

There were determined the ferrous concentration of crude oil samples which were exposed to the carbon steels and stainless steels by the atomic absorption spectroscopy (AAS). It was also tested the copper concentrations in crude oil samples which were exposed to Monel by the same instrument because of the confirmation evidences for the corrosion eventually.

2.2.2.Experiments on metals

The elemental composition of each type of metal was determined by the XRF detector due to the importance of the chemical composition on the rate of corrosion. According to the working principles of the XRF detector it was able to detect the compositions of each metal and even though most of nonmetals without carbon. A batch of similar sized metal coupons was prepared from seven different types of ferrous metals. Those metal coupons were immersed homogeneously in separate crude oil samples as shown in the Figure 1.

The corrosion rates of metal a batch of metal coupons were determined by the weight loss method after 15 days from the immersion at once. A batch was consisted seven types of metal and the same procedure was repeated for another two similar batches of metal coupons in order to after 30 days and 45 days from the immersion while observing the corroded surfaces through the 400X lens of an optical microscope and the corroded metal surfaces were cleaned by sand papers and isooctane. Also the initial weight and weight after removing corroded particles were measured by analytical balance as a necessity of the calculations [3]. The mathematical expression of the weight loss method is given in the Equation 1. Where, \(W\) = weight loss in grams, \(k\) = constant (22,300),\(D\) = metal density in g/cm\(^{3}\), \(A\) = area of metal piece (inch\(^{2}\)), \(t\) = time (days), \(CR\)= Corrosion rate of metal piece. The initial hardness and the hardness after the corrosion were tested in each metal coupon by the Vicker's hardness tester eventually. According to the working principles of the Vicker's hardness tester there were tested at least three particular points on each metal coupon at once and the average value was interpreted as the hardness of the metal [1]3}.

3. Results and Discussion

3.1. Chemical Compositions of Metals

According to the readings of XRF detector the elemental compositions of ferrous metals are given in the Table \ref{t2}.

Table 2. Chemical compositions of metals.

Metal	Fe (\%)	Mn (\%)	Co (\%)	Ni (\%)	Cr (\%)	Cn (\%)	P (\%)	Mo (\%)	Si (\%)	S (\%)	Ti (\%)	V (\%)
Carbon Steel  (High)	98.60	0.43	-	0.17	0.14	0.7	0.12	0.086	0.09	-	-	-
Carbon Steel (Medium)	99.36	0.39	-	-	-	-	0.109	-	0.14	<0.002	<0.04	-
Carbon Steel  (Mild Steel)	99.46	0.54	<0.30	-	<0.07	-	-	-	-	-	<0.19	<0.07
410-MN: 1.8 420-MN: 2.8 (Stainless Steel)	88.25	0.28	-	0.18	10.92	0.10	0.16	-	0.11	-	-	-
410-MN: 1.7 420-MN: 1.7 (Stainless Steel)	87.44	0.30	-	-	11.99	-	0.18	-	0.09	-	-	-
321-MN: 1.4 304-MN: 1.9 (Stainless Steel)	72.47	1.44	-	8.65	17.14	-	0.18	-	0.12	-	-	-
Moneal 400	1.40	0.84	0.11	64.36	<0.04	33.29	-	-	-	-	-	-

The results showed relatively higher percentage of ferrous in carbon steels, moderate amount of ferrous in stainless steels and trace amount of ferrous in Monel metal whilst composed significant amounts of nickel and copper. Beside of that there were found some trace amounts of nickel and chromium in especially stainless steels based on the enhancements of their properties such as the hardness, stress and reduction of the corrosion rate as well [1, 2, 3].

3.2. Corrosive Properties of Crude Oils

According to the quantitative analysis of the corrosive properties the numerical values for those properties of both crude oils are given in the Table \ref{t3}.

Table 3. Corrosive properties of crude oils.

Property	Murban	Das Blend
Sulfur Content (Wt. W%)	0.758	1.135
Salt content (ptb)	4.4	6.3
Acidity  (mg KOH/g)	0.01	0.02
Mercaptans content (ppm)	25	56

It was shown higher amount of organic acids in Das Blend crude oil than the Murban crude oil. Acidity is the parameter which interprets the total amount of organic acids presents in a certain crude oil also called "naphthenic acids" which is having a general formula of RCOOH also play a major role in the metallic corrosion [2, 5, 6]. In some of recent previous researches there were interpreted some relationships between the acidity and the rate of corrosion of metals. The general mechanism of the formation of the metallic corrosion is given in the Equation (2), Equation (3) and Equation (4). According to the obtained results it was found some higher amount of salts from Murban crude oil than the Das Blend crude oil. The summation of the \(NaCl\), \(CaCl_2\) and \(MgCl_2\) in a particular crude oil is known as the total salt content of that particular crude oil. At some higher temperature conditions those salts tend to be broken into HCl even though does not behave as corrosive compounds in the gas state [7]. When reducing the temperature some of molecules tend to be reacted with the moisture and finally produce hydrochloric acids also formed H2S. That is an approach of the metallic corrosion as explained in the Equation (5), Equation (6) and Equation (7). According to the results there were found higher amounts of both elemental sulfur and active sulfur compounds in Das Blend crude oil than the Murban crude oil. Sulfur compounds consisted with some fraction or a functional group which can be reacted with metallic compounds and tend to be formed the corrosion usually called as the "sulfidation" Those compounds are known as active compounds including elemental sulfur, mercaptans and hydrogen sulfides although highly depended on the temperature. Mercaptans also identified as a corrosive compound which is having a formula of RSH. The typical temperature for a proper sulfidation process is nearly 2300C [2, 8, 9]. The general chemical reaction of sulfidation is given in the Equation (8). There found some higher corrosive strength from Das Blend crude oil than the Murban crude oil because it was composed higher amounts of sulfur, mercaptans and organic acids than Murban while higher amount of slats composed in Murban crude oil than Das Blend crude oil.

3.3. Corrosion rates of metals

According to the determinations of weight loss method the average corrosion rates of metal coupon are given in the Figure 2.

According to the distribution of the corrosion rates there were observed relatively higher rates of corrosion from different three types of carbon steels and relatively lower corrosion rates in stainless steels including the least corrosion rates in both crude oils from 321-MN:1.4 304-MN:1.9 (Stainless Steel). Also the Monel metal showed moderate corrosion rates in both crude oils. When referring the chemical compositions of stainless steels those metals were composed at least 12 chromium with sufficient amount of nickel and also the combination of nickel and chromium tend to be formed much useful corrosive protection film on the relevant metal itself [1, 4,5, 10]. Therefore, the stainless steels showed relatively lower corrosion rates in both crude oil than the corrosion rates of other metals.

Regarding the effect of crude oils on the rate of corrosion in seven different types of ferrous metals there were found higher corrosion rates from four types of metals in Murban crude oil than the Das Blend crude oil since Das Blend was ahead in corrosive properties because of the requirement of higher temperature for a proper "sulfidation" process [2, 8, 9].

The variations of the corrosion rate of each metal coupon with the exposure time accordance to both crude oils are given in the Figure 3.

According to the above distribution there were identified the gradual reduction of the rate of corrosion in each metal coupon with the exposure time in front of the crude oils. Also there cannot be emphasized a recent sequence or series regarding the variation although it can be proved the inversely proportional relationship between the corrosion rate and the exposure time period by referring the above distribution [3].

3.4. Microscopic Review

According to the microscopic analysis there were identify some of distinguish properties of corrosion compounds based on their visible features foremost the color [4]. The important observations are highlighted in the Figure 4.

The optical features interpret some important corrosive compounds and observations which are explained the behaviors of the corrosion.

• Ferrous Sulfides
• Corrosion Cracks
• Pitting Corrosion
• Trace Compounds

Some of special features of corrosion compounds and recent observation from the current research have been finalized in the Table 4.

Table 4. Properties of corrosion compounds.

Compound	Appearances	Observations
FeS	Black, brownish black, property of powder, pitting, cracks	Observed most of features in
Fe\_2O\_3	Rusty color	Observed rarely
Cus	Dark indigo/ dark blue	Unable to specify

According to the obtained results there were observed most of above features on most of metals and also there were observed corrosion cracks on most of stainless steels. By analyzing the specific properties of corrosive compounds there can be concluded the formation of FeS on the surfaces of stainless steels and carbon steel. Also there can be assumed that the formation of CuS on Monel metal surfaces although it's impossible to specify CuS along with the optical features.

3.5. Decay of Metals

During the corrosion rate analysis there were found some invisible weight loss from most of metal coupons apart from specific corrosion compounds. The major objective of the analysis of atomic absorption spectroscopy (AAS) was to investigate the decay of metals into crude oils. The obtained results for the AAS analysis are shown in the Figure 5.

The results showed some considerable amount of ferrous in both Murban and Das Blend crude oil samples which were exposed to high carbon steels and medium carbon steels also found highest corrosion rates from such metals and simultaneously found some higher copper concentration in both crude oil samples which were exposed to Monel metal also it showed moderate corrosion rates. The corrosion compounds are tended to remove from the metal surfaces once it has been formed on the metal surface because of the repulsive and attractive forces in between successive electrons and protons [4]. The invisible weight loss of metals can be explained under this concept can be emphasized the mechanism of the decay of metals.

3.6. Reduction of the Hardness in Metals

The distribution of the variations between initial hardness and hardness after corroded has been shown in the Figure 6.

There were identified slight reductions of the initial hardness of metal coupons due to the corrosion accordance to both crude oil even though unable to specify any relationship or any sequence regarding the variations of the hardness with respect to any crude oil or even any type of metal. The hardness of metals may be varied with the position where it will be tested and after the formation of corrosion compounds the stability of the metal surfaces will be reduced due to the tendency of removing those particular compounds from the metal surface [1]1,2}. According to the obtained results there can be concluded the formation of the corrosion compounds on each metal surface also proved the interaction between metals and crude oils.

4. Conclusion

According to the obtained results the least corrosion rates were found from 321-MN: 1.4 304-MN: 1.9 (Stainless Steel) in both crude oils while finding relatively corrosion rates from carbon steels and Monel as well. Because 321-MN: 1.4 304-MN: 1.9 (Stainless Steel) is composed 18 chromium and 8 nickel which is tend to create a stable corrosive protection layer on the metal surface itself. It was found higher corrosion rates of four types of metals in Murban crude oil while other three types were showing higher corrosion rates in Das Blend since Das Blend crude oil was composing higher amount of sulfur, mercaptans and organic acids although lower amount of salts than the Murban crude oil also it can be concluded the effect of salts is stronger than the overall effect of organic acids, sulfur and mercaptans at the room temperature. Apart from that there were identified black color corrosive compound, corrosion cracks and pitting corrosion accordance the microscopic analysis. Also found some significant ferrous concentrations in crude oil samples regarding carbon steels and significant amount of copper in crude oil samples regarding Monel since unable to find any amount of ferrous in crude oil samples regarding stainless steel. The slight reductions of the initial hardness of metal coupons were observed due to the corrosion eventually.

Author Contributions

All authors contributed equally to the writing of this paper. All authors read and approved the final manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.