Chitosan Molecular Weight Effects on The Synthesis of Gold Nanoparticles and Catalytic Degradation of Environmental Pollutants

Gold nanoparticles (Au NPs) was synthesized with Chitosan different molecular weight (MW) using a microwave as a heating source. Since, Chitosan acts as a reducing and stabilizing agent. The as-synthesized Au NPs were characterized by transmission electron microscopy (TEM) images and selected area electron diffraction patterns (SAED). Furthermore, the Au NPs fabrication was ascertained by UV–Visible spectroscopy (UV–Vis) through the detection of the localized surface plasmon resonance (LSPR) characteristic peak, X-ray powder diffraction (XRD), and energy dispersive X-Ray Spectroscopy (EDS). The formation of the Au NPs was confirmed by the detection of a LSPR peak at 518–527 nm in the UV–Vis spectrum. In addition, the XRD studies depicted that the obtained Au NPs were highly crystalline with ‘face-centered’ cubic geometry. Moreover, TEM micrographs showed that the most Monodispersed AuNPs was synthesized with low molecular weight (LMW) Chitosan with particle size 4.48±0.09 nm. The synthesized Chitosan-Au nanocomposite exhibited an efficient catalytic property in the reduction of two organic environmental pollutants which are, 4nitrophenol (4-NP) and methyl orange (MO) dye in the presence of sodium borohydride (NaBH4).


INTRODUCTION
Many aromatic compounds, such as dyes and phenols, are released from industrial processes. Since, they are among the main causes that responsible for the severe environment contamination for either soil and surface water. Dyes pollutants are produced from printing, dying, dyestuff manufacturing, plastics, food, drugs, cosmetics, and textile industries. The effuents products released from these industries are accompanied by

Synthesis of AuNPs
In a typical experiment, 0.2 g of Chitosan were dissolved in 20 mL 1% acetic acid under vigorous stirring on a magnetic stirrer for about 3 hrs at 40˚C. Since High, medium, and low Chitosan MW were abbreviated as HMW, MMW, and LMW Chitosan, respectively. The effect of different experimental conditions of the synthesis of Au NPs, such as various molar concentrations of HAuCl 4 , microwave irradiation time, and microwave power was demonstrated. Therefore, in order to investigate the effect of the precursor HAuCl 4 salt different molar concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 mM), about 5.0 mL of HAuCl 4 solution, was added drop by drop using a pipette to 20 mL HMW Chitosan to ensure complete dissolving of the reactants. Then, the colloidal mixture was transferred to a boiling vessel and exposed for 3 min irradiation time and 1000 W microwave irradiation power as proposed in the schematic drawing in Fig. 1. The effect of microwave irradiation time and power on the synthesis of Au NPs, the colloidal solution was irradiated for different intervals (60, 90, 120, 150, and 180 s) and various microwave power (450, 600, 800, 900, and 1000 W) was tested for the appropriate HMW Chitosan solution. Therefore, the optimum experimental reaction conditions were defined. Later, the same procedure will be applied for both MMW, and LMW Chitosan-HAuCl 4 colloidal solution, respectively.

Catalytic activity of AuNPs for 4-NP reduction in the presence of NaBH 4
The catalytic activity test of Au NPs for 4-NP reduction was performed as follows: 2.0 mL of 0.08 mM stock solution of 4-NP, 0.5 mL of 0.08 M freshly prepared NaBH 4 solution in ice-cold water under continuous stirring and 0.3 mL of Au-Chitosan nanocomposite were mixed in a 3 mL standard quartz cuvette. Since, the catalytic activity of different gold Chitosan MW t was studied, where the colloidal solution concentration of HMW, MMW, LMW Chitosan capped Au NPs was 1.5 mM, 2.0 mM, and 1.5 mM (1%, w/v), respectively. To monitor the reduction of 4-NP, UV-Vis spectra of the samples at room temperature (25˚C±2˚C) were recorded in the range of 200-700 nm.The reaction rate constant (k) of catalytic reactions was estimated through measuring the absorbance decrease at 400 nm.

Catalytic activities of AuNPs for methyl orange reduction in the presence of NaBH 4
The catalytic activity of the synthesized Au NPs was investigated by degrading a hazardous dye, Methyl orange with the help of NaBH4. In general, 20 mg of the dye was added to 1.0 L of distilled water and used as a stock solution of 0.06 mM. After that, 2.0 mL of the prepared Methyl orange dye was added 0.5 (0.06 M) of ice-cold fresh prepared NaBH4. Thereafter, about 0.03 mL of the synthesized Au NPs stabilized with Chitosan was added to the previous mixture solution and mixed in a quartz cuvette of 1 cm path length ultrasonically for 15 min. Thereby, the rates of degradation of MO dye in the presence NaBH4 using Au NPs have been analyzed spectrophototometrically at wavelength 464 nm with constant time intervals (i.e., every 60 s). A control set was maintained without Au NPs for the dye and estimated for the absorbance. This was carried out for different, gold Chitosan MW as mentioned in the 4-NP catalytic activity procedure.

Characterization Techniques
UV-Visible (UV/Vis) Spectrophotometer (Thermo Scientific Evolution 201) with matching quartz cells of 1 cm was used during all measurements and scanning a wavelength range from 300 to 900 nm. The surface morphology of the nanocomposite was analyzed using a high-resolution transmission electron microscope (HRTEM) and selected area electron diffraction angle (SAED) JEOL JEM-2100 microscope, operating at 200 kV. The synthesized Au nanostructure were characterized using XRD (PANalytical X'Pert PRO powder diffractometer with CuKα (λ=1.5418 Ǻ). The chemical composition of the products was determined by energy dispersive x-ray spectroscopy (EDS) using a JEOL scanning electron microscope (SEM) model JSM 6510 LV instrument that supplied with Oxford X-Max 20. A household microwave (Samsung model MW1030WA, Korea) with a fixed frequency of 2.5 GHz and 1000 W power was used as an irradiation source.

UV-visible (UV-Vis) spectroscopy analysis
UV-Vis spectroscopy is a valuable tool used for studying, identifying, and characterizing of metallic NPs. Since, NPs made from certain metals, such as gold and silver, strongly interact with specific wavelengths of light and the proper unique optical characteristics of these materials are the basis for the field of plasmonics. In which, these NPs have vast optical properties which are so sensitive to shape, size, concentration, agglomeration state, and refractive index near the NPs surface according to the most popular form of Mie's theory for spherical NPs within quasi-static limit is given as (1) where C ext is the extinction cross section of the spheres, R is the radius of a homogeneous sphere ε m is the dielectric constant of the surrounding medium, λ is the wavelength of the radiation, and ε 1 and ε 2 denote the real and imaginary part of the complex dielectric function of the particle material, respectively. A resonance occurs whenever the condition of ε 1 = 2ε m is satisfied, which explains the dependence of the localized surface plasmon resonance (LSPR) extinction peak on the surrounding dielectric environment. For a small Au nanosphere within the quasi-static limit, its LSPR has an almost fixed resonance frequency and shows limited tenability [30]. In this study, the attempt to synthesize the nanometer gold using microwave irradiation method was carried out. Since, the effect of various variables such as Au + precursor concentration, microwave irradiation time, and power on the Au NPs was investigated. Figure. 2 shows the growth of the LSPR of different concentration of HAuCl 4 .3H 2 O (0.5, 1.0, 1.5, 2.0, and 2.5 mM). This could be ascertained by the enhancement of the LSPR peak at certain wavelength (λ max =523 nm) as shown in Fig. 2. Also, the increase of absorption peak intensity is related to the increase of Au 3+ concentration in the Chitosan/[AuCl 4 ]¯ colloidal solution [31]. Moreover, the change of mixture color which was pale yellow into a red color ( Fig. 2 inset) , ensures the formation of Au NPs. This could be attributed to the reduction of the metal salt mechanism which was suggested by Huang and Yang (2004) [32]. Since, they proposed that the Chitosan, a derivative of glucose, which acts as a reducing agent. This could be carried out by the hydroxyl (-OH), and amine (-NH 2 ) groups simultaneously [33]. Furthermore, it is suggested that the number of the reducing sugars could be enhanced by the elevated temperature during the microwave irradiation. Moreover, after the reduction of gold ions (Au 3+ ) to gold nanoparticles (Au 0 ), they were capped by -NH 2 and/or -OH functional groups as proposed in Fig.1. To investigate the effect of the microwave irradiation time on the fabrication of Au NPs, a solution of 20 mL HMW Chitosan (1%, w/v) mixed 5 mL (1.5 mM) gold chloride solution was chosen. This is due to, the UV-Vis spectrum of the aforementioned reaction mixture characterized with a narrow full width at half maximum (FWHM) which was 112 nm (λ max =523 nm) which is attributed to the particle size [27,34]. Since, the narrow FWHM, the smaller the particle size diameter. Figure. 3 represents the UV-Vis absorption spectra of the Chitosan/HAuCl 4 .3H 2 O (1.5 mM) colloidal solution at different irradiation time (60, 90, 120, 150, and 180 s). It can be observed the gradual increase of the absorption intensity of LSPR (λ max ≃526 nm) for the sample irradiation time (i.e., from 60 to 90 s). Whereas, a detected blue shift of the LSPR peak was estimated for 120 and 180 s irradiation to a shorter wavelength (λ max =523 nm). This could be correlated with the smallest particle size fabrication [35]. Finally, to assess the effect of microwave irradiation power on the Au NPs synthesis, the Chitosan-HAuCl 4 colloidal solution was exposed to microwave with power 450, 600, 800, 900, and 1000 W, respectively. Therefore, 5 mL of HAuCl 4 .3H 2 O (1.5 mM) was mixed with 20 mL Chitosan (1%, w/v) and irradiated for 180 s for different microwave irradiation power. Fig. 4 depicts the UV-Vis absorption spectra and reveals that the gradual increase of absorption intensity at LSPR about 523 nm. Since, it seems that the higher reaction radiation power resulted in an increase in the absorption peak, with increasing the microwave power. This results proposed that the growth of the Au NPs was enriched with the increase of microwave radiation power [18,29]. Hence, the obtained data confirm that the optimum reaction conditions for generating the Au NPs was 180 s microwave irradiation time, and 1000 W irradiation power. This is in contrast to the findings obtained by Ngo et al, (2016) [30], where they found that the Au NPs synthesized with short irradiation time and low irradiation power of the microwave. Since, it was investigated that both the higher microwave power during the ramping regions of the applied microwave power and the longer irradiation time (i.e., retention time of microwave ≃ 5 min) were the optimum synthesis conditions for Au NPs.
This could be owing to the faster formation of glucose units residues from Chitosan that accelerates the reduction of [Au Cl]¯ ions to form Au 0 nanoparticles. Furthermore, the gradual increase in the absorption peak with with a blue shift for 1.5 mM HAuCl 4 concentration (λ max =523 nm, 180 s irradiation time and 1000 W irradiation power) and more narrower FWHM ( Fig. 3 and 4) correlated to the fabrication of small size nanoparticles [30]. This will be confirmed later by transmission electron microscopy micrographs.
The UV-Vis absorption spectra of the gold chloride-MMW Chitosan colloidal solution are depicted in Fig. 5.
The solution of different gold chloride colloidal concentration (5 mL) mixed with 20 mL MMW Chitosan (1%, w/v) exhibited the absorption maxima around 527 nm. With the gold chloride concentration increases (0.5 to 2.5 mM), a plasmon absorption peak increased with a remarkable blue shift (from 527 to 520 nm) for 2.0 mM HAuCl 4 concentration. Moreover, a detected gradual increase in the absorption peak intensity (Fig. 5) confirmed that the fabricated Au NPs with small nanoparticles.
The UV-Vis spectra of LMW Chitosan-HAuCl 4 colloidal solutions have been shown in Fig. 6. Since, the Au NPs spectral peak, of 1.5 mM HAuCl 4 exhibited a gradual increase in the absorption peak with a blue shift of LSPR (λ max =518 nm) and narrow FWHM (110 nm) compared to other HAuCl 4 concentrations of 2.0 and 2.5 mM which were 113 and 115 nm, respectively. Therefore, the LMW Chitosan is more proper efficient rather than HMW and MMW Chitosan as reported before [36][37][38]. Furthermore, this result can be supported by the highest deacetylation degree (DD) value which is approximately more than 75-85% for LMW Chitosan. Where, the more free amino groups available for gold ion coordination and reduction, the higher yield of reduction [39]. In addition, in comparison with long-chain HMW Chitosan, the short-chain LMW is better in terms of water solubility and biocompatibility [36].  [40]. These major characteristic peaks of Au nanocrystals were attributed to the face centered cubic (FCC) crystalline structure [41]. In addition, according to the low content of Au NPs in the Au-Chitosan bionanocomposite, the diffraction intensity of Au nanocrystal was weak in the pattern. The average particle size of Au NPs can be estimated using Debye-Scherrer equation:

X-ray diffraction (XRD) analysis
where D hkl is the particle size perpendicular to the normal line of (hkl) plane, K is the shape factor constant (K≃0.9), β hkl is the width at half height of (hkl), θ hkl is the Bragg angle of (hkl) peak and λ is the wavelength of X-ray with a value 1.5418 Ǻ. From the Scherrer equation, the average crystalline size of Au NPs for Au-HMW, MMW, LMW Chitosan are found 17.25±3.9, 8.9±2.39, and 4.71±1.21 nm, respectively, which are consistent with the findings with the transmission electron microscopy results which will be discussed later.

Transmission electron microscopy (TEM) studies
The size and morphology of the fabricated Au NPs were investigated using transmission electron microscopy (TEM) analysis. Since, the photographs of Au NPs capped by various Chitosan MW (i.e., HMW, MMW, and LMW ) was revealed that the synthesized NPs were spherical in shape, but with different detected particle size diameter. It can be observed from Fig. 8 of Au NPs capped by a HMW Chitosan TEM image that a polydispersed NPs synthesized with size range 4-18 nm, where nearly more than 70% of the particles have size between 9-15 nm. Whereas, the size of MMW Chitosan-Au nanocomposite was 5-34 nm. For LMW Chitosan-Au nanocomposite micrograph, it can be noticed that the fabricated NPs of small and narrow particle size 4.48±0.09 nm. The high resolution TEM (HRTEM) and the nanodiffraction patterns were used to study the morphology and the internal crystalline structure of the synthesized Au NPs stabilized by various types of Chitosan MW. This was carried out using selected-area electron diffraction (SAED) patterns. The typical SAED patterns recorded in the area represented in Fig. 8d, 9d, and 10d are shown in Fig. 8e, 9e, and 10e. Since, they clearly exhibited a bright circular diffraction spots/rings that could indexed to (111), (200), (220), and (311) planes. Since, the spots are predominantly oriented along the (111) plane with a space lattice fringe in the HRTEM image of 2.35 Ǻ and 2.34 Ǻ corresponding to the Au NPs synthesized and stabilized by HMW and MMW Chitosan nanocomposite, as shown in Fig. 8e and 9e, respectively. Whereas, Fig. 10d represents the HRTEM images with clear lattice fringes having a d-spacing of 0.23, 0.20, and 0.14 nm revealed that the FCC crystal lattice with (111), (200), and (220) planes [42]. Moreover, these findings confirm the results obtained by XRD patterns. In addition, the fast Fourier transformation (FFT) process was used to obtain the Fourier spectra that equivalent to optical diffraction patterns. FFTs were typically employed to recognize the orientation of NPs with respect to the electron beam. The obtained FFT patterns of different Au single crystal synthesized by various Chitosan MW were represented in Figs. 8f, 9f, and 10f. Such FFTs patterns confirmed that the synthesized Au nanocrystal corresponds to FFC crystals [43].

Energy Dispersive X-Ray Spectroscopy (EDS) analysis
EDS is a Microanalysis technique equipped in addition to scanning electron microscopy (SEM) to determine the elemental structure of certain examined samples. The EDS analysis of Au-Chitosan different MW samples nanocrystallites showed an optical absorption band peak at approximately 2.2 keV [44] in the three studied samples as shown in Fig 11a-c. Since, a clear peak for Au atoms was seen in the spot directed EDS spectrum of all the Au NPs samples. In addition, By comparing relative peak sizes it can be concluded that the presence of Au is really detected. Also, The presence of carbon, and oxygen atoms signals were observed, where this is likely to be due to X-ray emission from the Chitosan structure. Furthermore, the elemental mapping analysis of the Au NPs showed a tremendous distribution of metallic Au of the Au NPs pellet solution.

Catalytic reduction of 4-nitrophenol in the presence of NaBH 4
The efficacy of the synthesized Au NPs stabilized by different Chitosan MW was evaluated by the catalytic reduction of 4-nitrophenol (NP) into 4-aminophenol (AP) in the presence of sodium borohydride (NaBH 4 ). The catalytic degradation of 4-NP to 4-AP was monitored by UV-Vis spectroscopy. Since, the color of 4-NP is light yellow, and exhibits a maximum absorption peak at λ max = 317 nm as shown in Fig. 12a After then, NaBH 4 was added and the color was converted to yellow green with a red-shift of the absorption peak from 317 to 400 nm (Fig. 12a inset). This is correlated to the formation of 4-nitrophenolate ion. To the reaction mixture, 0.3 mL of Au NPs stabilized by various Chitosan MW was added. The reaction course was monitored every 60 seconds by tracking the gradual decrease of the absorption spectra at λ max = 400 nm that attributed to 4nitrophenolate ion, with a noticeable growing a new peak 293 nm which is ascertained to 4-AP formation as shown in Fig. 12b. The catalytic reduction of the reaction follows the Langmuir-Hinshewood (LH) model [45]. This reaction correlates to the pseudo-first-order kinetics, and the rate constant (k) of the catalytic reaction can be determined using the following relation: Where are the concentration of 4-NP at the beginning and the time t of the reaction, while are their corresponding absorption, respectively. Figure. 12c and 12d represent the catalytic reduction of 4-Np via MMW and LMW Chitosan-Au nanocomposite with a reduction time 9 and 7 min, respectively. Moreover, the rate constant values obtained using various Au NPs catalysts were represented in table.1. Figure. 12e [47].

Catalytic reduction of methyl orange in the presence of NaBH 4
The MO dye degradation with the aid of NaBH 4 in the absence and presence of Au nanocatalyt was recorded using UV-Vis absorption spectra as shown in Fig. 13a. This catalytic reduction is achieved via Au NPs, which acts as an electron relay and helps in the transfer of electrons from the BH4¯ ions to the dyes. This is followed by a successive degradation of the dye and an observed gradual decrease in the absorption intensity of the UV-Vis spectrum as shown in Fig. 13b. The chemical reduction of MO by NaBH 4 in the presence of Au NPs as catalyst is shown in Fig. 13b-d for different Chitosan MW-Au nanocatalyst. It is known that NaBH 4 individually is unable to reduce methyl orange in the absence of a catalyst, thus showing the catalytic efficacy of Au NPs [48]. From Fig. 13b-d, it is apparent that there is a rapid change in absorbance of MO in the occurrence of Chitosan-Au nanocomposite. The degradation is indicated by the progressive reduction absorption intensity and absorbance at k of of the UV-Vis spectra. , respectively as represented in the table. 2. This result consistent with the catalytic activity efficiency for 4-NP using LMW Chitosan-Au nanocatalyst in the presence of NaBH 4 . Therefore, one can conclude the Au NPs capped by Chitosan, which acts as both a reducing and a capping agent for gold salt, can be efficiently used as an effective catalytic removal agent for different environmental aromatic pollutants.

Conclusions
In this study, Au NPs were prepared using a green, cost effective, and rapid reduction method, where the microwave was used as a simple heating source. Since, for the synthesis Au NPs, the optimum reduction conditions such as concentrations of HAuCl 4 , irradiation, and microwave radiation power were investigated. This was carried out using a polymeric matrix of different Chitosan MW (i.e., HMW, MMW, LMW) that used as a reducing and capping agent. TEM micrographs showed that a nearly monodispersed Au NPs with a small particle size diameter 4.48±0.09 nm synthesized with LMW Chitosan-Au nanocomposite. Whereas, it was 13.45±5.58 and 7.63±0.79 nm for HMW and MMW Chitosan -Au nanocomposite, respectively. The rapid degradation of 4-NP and MO aromatic pollutants was carried out with a high reaction rate constant LMW rather than HMW, and MMW Chitosan -Au nanocatalyst. This is due to that the small NPs size enables a high surface to volume ratio for the rapid catalytic degradation. Therefore, Chitosan-Au nanocomposite is strongly suggested as an adequate nanocatalyst for the removal of the environmental aromatic pollutants.