CHAPTER 3

3. Materials and methods

This chapter details the materials and methods employed in the synthesis,
characterization, and application of various zinc oxide (ZnO)-based nanocomposites for
photocatalytic dye degradation. The synthesized materials include pure ZnO, copper-doped
ZnO (Cu-ZnO), copper-reduced graphene oxide supported ZnO (Cu-rGO/ZnO), and
palladium-reduced graphene oxide supported ZnO (Pd-rGO/ZnO). The preparation of these
nanocomposites involved meticulous synthesis processes, followed by a series of analytical
techniques to characterize their structural, morphological, and optical properties. The
photocatalytic activities of these materials were evaluated through dye degradation studies,
exhibiting their potential applications in environmental remediation.

3.1 Materials
KMnO4 (AR 99.5%), NaNO3 (ACS 99%), H2SO4(98%), HCl (98%), graphite powder
(<20 μm,(98%)), Palladium (II) Chloride pure 98% were received from SRL chemicals. Zinc
acetate di hydrate (AR 99.5 %). 98% H2SO4, graphite oxide powder 98% assay, sodium
nitrate extrapure AR ACS 99%, Cupric Acetate Monohydrate pure 98% and Potassium
Permanganate extrapure AR 99.5% were procured from SRL chemicals. All the chemicals
were bought from Sigma Aldrich and used without further purification. Analytical grade
solvents and reagents were used throughout the experiment. Millipore water (18.2 MΩ·cm at
25ºC) was used for all the solution preparation.

3.2 Preparation of ZnO nanoparticles

The undoped ZnO was prepared by facile hydrothermal method. For ZnO preparation, Zinc
acetate dihydrate (0.1 M), and Cetyltrimethylammonium bromide (0.01M) were taken in a
250ml glass beaker and stirred for 10 min to obtain homogenous solution. The pH of the
obtained reaction mixture was increased to 9 by adding 1M NaOH. The whole reaction
mixture was carefully transferred to Teflon lined stainless steel autoclave and kept at 130°C
for 3 h. The obtained precipitate was thoroughly washed with DI water, followed by ethanol
to eliminate unwanted impurities. The obtained ZnO precipitate was then centrifuged, dried at
70°C, sintered at 350 °C for 2 h and stored in a clean glass vial for further studies.

3.3 Preparation of graphene oxide

 Graphene oxide (GO) was prepared using the Modified Hummer’s method
(Hummers Jr & Offeman, 1958)
. Briefly, 27ml of sulphuric acid, and 3ml of phosphoric acid were gently
mixed and stirred for 10 minutes. Then, 0.250 g of graphite powder was added into above
mixture with constant stirring. 1.5g of KMnO 4 was added to the above reaction mixture while
maintaining the temperature less than 20°C to prevent explosion. The whole reaction mixture
was continuously stirred at 32 °C for 24 h. Then, the reaction mixture was diluted to 500 ml
deionized water under vigorous stirring. To remove excess KMnO 4, 30% H2O2 was added
slowly and stirred for 15 minutes. The obtained precipitate was washed with 5% HCl acid
and deionized water and centrifuged at 6000 rpm for 10 minutes. The resultant precipitate
was dried in oven at 90 °C for 24 h to obtain graphene oxide.

3.4 Preparation of the photocatalyst (Cu-rGO/ZnO)

The GO suspension was prepared by adding GO (1.5g) into distilled water and a few
drops of N2H4 (6 drops) were added under constant stirring. Zinc acetate dihydrate (0.1 M),
Cetyltrimethylammonium bromide (0.01M) and 0.01M solution of CuNO3 were taken in a
separate beaker and stirred for 15 minutes. To this solution, GO suspension was added
gradually and stirred for 20 minutes. The pH of the reaction mixture was adjusted to 9 by
adding 1M NaOH solution under constant stirring. Finally, the reaction mixture was
transferred to a Teflon-lined stainless steel autoclave and kept at 130°C for 3 h. The obtained
precipitate was centrifuged and washed with DI water followed by ethanol to get an
impurities-free catalyst. After that, the obtained material was dried at 70°C, sintered at 350 °C
for 2 h in a muffle furnace with the air contact and denoted as Cu-rGO/ZnO nanocomposite.
The percentage ratio of each component in the catalyst Cu-rGO/ZnO nanocomposite is
0.1:10:1 respectively.

3.5 Preparation of Pd-doped rGO/ZnO Nanocomposite

The preparation of the Pd-rGO/ZnO nanocomposite involved a hydrothermal process.

Initially, 90 ml of 0.1 M zinc acetate dihydrate, 10 ml of 0.01 M palladium chloride, and 2 ml
of 0.01 M CTAB were combined in a beaker and stirred for 30 minutes to ensure thorough
mixing. Subsequently, 10 ml of the aqueous GO suspension (1.5 mg/ml) was added to the
reaction mixture. The pH of the mixture was adjusted to 9 by carefully adding 1 M NaOH
solution while stirring continuously for an additional 10 minutes.
 The reaction mixture was then transferred to an autoclave, which was placed in a
furnace and maintained at 130°C for 3 hours to facilitate the formation of the nanocomposite.
After the hydrothermal reaction, the mixture was centrifuged at 12,000 rpm to separate the
composite precipitates from the liquid phase. The obtained precipitate was washed several
times with water and isopropyl alcohol to remove any impurities. The washed precipitate was
then dried in an oven at 60°C. Finally, the dried material was sintered at 350°C for 2 hours to
enhance its structural stability and properties. The resulting greyish powder was stored in a
glass vial for further analysis and experimentation.

3. 6 Preparation of rGO/ZnO:
A similar protocol to the above procedure was followed for preparing rGO/ZnO
without adding PdCl2. This process will result in the synthesis of rGO/ZnO composite. This
step is essential to compare the efficiency of the Pd-rGO/ZnO nanocomposite against the
rGO/ZnO composite. The obtained nanocomposites can be further characterized and used for
various applications, as described in the experimental section.

3.7 Characterization of prepared nanoparticles (ZnO, Cu-ZnO, rGO/ZnO,

Pd-rGO/ZnO and Cu-rGO/ZnO)

Structural properties of prepared samples were determined by X-ray diffraction

(XRD) analysis (Model: Bruker, D-8 Advance, K = 1.5406 Aº, Cu Ka radiation). The
functional group of the prepared samples were analyzed by Fourier transform infrared
spectroscopy (FTIR) (Model: Perkin Elmer spectrum II). A photoluminescence study for the
samples was performed on a spectrophotometer (Agilent-Cary eclipse; Model:
FL1201M002). Morphological features of synthesized materials were done using a scanning
electron microscope (SEM) (Model: Quanta FEG-250). X-ray photoelectron spectroscopy
(XPS) analysis was done for the samples to determine the surface chemical profile (XPS, Phi
– 5300 esca system). The surface properties of the samples were analyzed using the BET
analytical technique (Quantachrome® ASiQwin™ Instrument version 5.0). A thermal
analysis method called thermogravimetric analysis (TGA) is used to measure a sample's total
mass as a temperature-dependent attribute. Thermogravimetry (TGA) analysis was performed
on an instrument NETZSCH STA 2500 STA2500A-0061-N in the temperature range of
20°C/20.0 (K/min)/1000°C

XRD
 Figure: X-ray diffraction working principle

X-ray Diffraction (XRD) was employed to investigate the crystalline structure and phase
purity of the synthesized materials, including ZnO, Cu-ZnO, Cu-rGO/ZnO, and Pd-rGO/ZnO
nanocomposites. The preparation of samples for XRD analysis involved several meticulous
steps to ensure accurate and reliable results. First, the dried nanocomposite powders were
finely ground using an agate mortar and pestle to achieve a uniform particle size, ensuring the
sample was homogeneous and free of agglomerates that could affect the diffraction patterns.
A small amount of the finely ground powder was then placed on a clean, flat sample holder.
The powder was evenly spread and lightly pressed to create a smooth, flat surface,
minimizing any preferred orientation effects during the measurement. The prepared sample
holder was then carefully placed in the XRD instrument, and precise alignment was
performed to ensure optimal data acquisition. The XRD analysis provided crucial information
about the crystal lattice, phase composition, and crystallite size of the materials, contributing
to the overall understanding of their structural properties.

FTIR

Fourier Transform Infrared Spectroscopy (FTIR) was utilized to identify the functional
groups present in the synthesized materials and to confirm the presence of reduced graphene
oxide (rGO) in the composites.
 Figure: FTIR working principle (Source:
https://specac.com/theory-articles/introduction-to-atr-ftir-spectroscopy-part-1-the-
basics/)

The samples were prepared by mixing a small amount of the nanocomposite powder with
potassium bromide (KBr) to form a pellet. The FTIR spectra were recorded over a range of
wavelengths to detect characteristic absorption peaks corresponding to various functional
groups, thus providing insights into the chemical composition and bonding within the
materials.

SEM and EDAX

Scanning Electron Microscopy (SEM) was employed to obtain detailed images of the surface
morphology and particle size of the synthesized nanocomposites. The samples were prepared
by placing a thin layer of the nanocomposite powder onto a conductive adhesive tape, which
was then mounted on an aluminum stub. The samples were coated with a thin layer of gold to
enhance conductivity and prevent charging under the electron beam. SEM analysis provided
high-resolution images that revealed the surface texture, morphology, and particle size
distribution of the nanocomposites.
Figure: Scanning electron microscope (Source: https://www.tribonet.org/wiki/scanning-
electron-microscope/)

Energy Dispersive X-ray Spectroscopy (EDS) analysis was conducted to determine the
elemental composition of the synthesized nanocomposites. The samples were prepared
similarly to those for SEM analysis. EDS was performed in conjunction with SEM, allowing
for the identification and quantification of the elements present in the nanocomposites. This
analysis confirmed the successful incorporation of dopants and the presence of key elements
in the composites.

Figure: EDAX analysis – working principle (Source: (Long et al., 2021))

UV-Visible Spectroscopy

Figure: Working principle of UV-Visible spectroscopy (Source:

https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/uv-vis/uvspec.htm)

UV-Visible (UV-Vis) Spectroscopy was used to study the optical properties and band
gap energies of the synthesized materials. The samples were prepared by dispersing a small
amount of the nanocomposite powder in ethanol to form a homogeneous suspension. The
UV-Vis spectra were recorded over a range of wavelengths, and the absorption edges were
analyzed to determine the band gap energies. This analysis provided insights into the optical
behavior of the materials and their potential applications in photocatalytic processes.

Brunauer-Emmett-Teller (BET) Analysis

Brunauer-Emmett-Teller (BET) analysis was conducted to determine the specific surface area
and porosity of the synthesized nanocomposites. The samples were degassed under vacuum at
an elevated temperature to remove any adsorbed gases and moisture. Nitrogen adsorption-
desorption isotherms were measured at liquid nitrogen temperature. The BET surface area
was calculated from the adsorption data, providing valuable information about the surface
characteristics and porosity of the nanocomposites, which are crucial for their catalytic
performance.

X-ray Photoelectron Spectroscopy (XPS)

Figure: working principle of XPS analysis (Source: (Greczynski et al., 2023))

X-ray Photoelectron Spectroscopy (XPS) was employed to analyze the surface chemistry and
elemental composition of the synthesized nanocomposites. The samples were mounted on a
sample holder and introduced into the XPS chamber under high vacuum conditions. XPS
spectra were recorded by irradiating the samples with X-rays and measuring the kinetic
energy of the emitted photoelectrons. The obtained spectra provided information about the
oxidation states of the elements and the chemical environment on the surface of the
nanocomposites, confirming the successful doping and presence of functional groups.

Thermogravimetric Analysis (TGA)

Thermogravimetric Analysis (TGA) was used to evaluate the thermal stability and
composition of the synthesized nanocomposites.
 Figure: Image of TGA instrument

A small amount of the sample was placed in a TGA pan and heated at a controlled rate under
an inert atmosphere. The weight loss was recorded as a function of temperature, providing
insights into the decomposition temperatures, thermal stability, and the amount of organic and
inorganic components in the nanocomposites. This analysis helped to understand the thermal
behavior and composition of the materials, which is essential for their practical applications.

3.8 Photocatalytic measurement

CBR dye degradation study

The CBR dye degradation by the prepared samples was carried out in a photocatalytic
chamber. Known concentrations of prepared photocatalysts were mixed with aqueous CBR
dye solution (10 ppm) to evaluate the photocatalytic efficiency. The Xe arc lamp is used as a
source with a UV cut-off filter in the photocatalytic chamber. The source lamps 16W and
20W were used to evaluate the photocatalytic efficiency. The mixture containing CBR dye
and the photocatalyst in two different concentrations (0.1 g/L and 0.01 g/L) was placed in the
dark and ultrasonicated for 10 minutes to obtain adsorption-desorption equilibrium. The pH
of the solution was at 7.5. The experiment with two different concentrations of photocatalyst
and dye was carried out separately. The photocatalytic dye degradation experiment was
conducted for 150 minutes and centrifuged to remove the adsorbed photocatalyst. UV-Visible
double beam spectrophotometer (HITACHI, UH 5300) was used to obtain absorption spectra.

Bromophenol dye degradation study

 The photocatalytic performance of the prepared nanocomposite was evaluated by
photocatalytic degradation of bromophenol blue under UV light in a photocatalytic chamber.
The sample Cu-rGO/ZnO was added to the aqueous solution of bromophenol to determine
the photocatalyst effect. The Xe arc lamps, 20W (with ultraviolet cut-off filter), were utilized
as a light source. The bromophenol blue (10ppm) aqueous solution was prepared for each
experiment, and a known amount of photocatalyst was dispersed. The suspension
(bromophenol blue with photocatalyst) was ultrasonicated for 20 min in a dark room at
ambient temperature. The pH = 7.4 is observed during the degradation test. All experiments
were conducted at ambient conditions for 150 min, and all samples were filtered and
centrifuged to eliminate the photocatalyst substance. A UV-Vis double beam
spectrophotometer (HITACHI, Model: UH5300) was used to measure the UV-Vis absorbance
of samples.

3.9 Antibacterial activity

Antibacterial activity of Pd-rGO/ZnO nanocomposites

The antibacterial activity of rGO/ZnO and Pd-rGO/ZnO nanocomposites were tested by using
the agar well diffusion method. Bacterial strains such as Escherichia coli, Staphylococcus
aureus, Enterococcus faecium, and Pseudomonas aeruginosa are cultured for 24 hours to
attain a density of 106 colony-forming units (CFU) per millilitre. Ampicillin is a well-known
antibiotic and it was used as a control. Agar plates are prepared and spread with the 24-hour-
old bacterial cultures. Wells are created in the agar plates using a sterile borer. These wells
will hold different concentrations of the nanocomposites. Different concentrations of
rGO/ZnO and Pd-rGO/ZnO nanocomposites (50 µg/mL, 100 µg/mL, 150 µg/mL, and 200
µg/mL) are added to the wells in the agar plates. The inoculated agar plates are then
incubated at 35°C for 24 hours. This allows time for the antibacterial activity of the
nanocomposites to take effect. After incubation, the zone of inhibition around each well is
measured using a ruler. The zone of inhibition indicates the area where bacterial growth has
been inhibited by the nanocomposites. Measurements are typically taken in millimetres
(mm).

Antibacterial activity of Cu-rGO/ZnO nanocomposite

The antibacterial activity of Cu-rGO/ZnO nanocomposite was tested by using the agar well
diffusion method. For the study of antibacterial activity, the following gram-positive and
gram-negative bacterial strains have been used. Gram-positive bacteria: Enterococcus
faecium and Staphylococcus aureus. Gram-negative bacteria: Escherichia coli and
Pseudomonas aeruginosa. Bacterial strains (24 hours old) were spread on agar plate, where
the bacterial cell density was 106 CFU/ mL. The agar well was made using a sterile borer.
Different concentrations of nanocomposites (50 μg/mL, 100 μg/mL, 150 μg/mL, and 200
μg/mL) were added to the wells and inoculated plates were incubated at 35 °C for 24 hours.
Then the zone of inhibition was measured in mm scale.

2.10 The colony count analysis

The colony count analysis revealed that both rGO/ZnO and Pd-rGO/ZnO nanocomposite
exhibited a reduction in colony formation compared to the bacteria sample (without any
nanomaterial). This reduction indicates antibacterial activity against the tested bacterial
strains such as Escherichia coli, Staphylococcus aureus, Enterococcus faecium, and
Pseudomonas aeruginosa. Specifically, the Pd-rGO/ZnO nanocomposite demonstrated
superior antibacterial activity compared to rGO/ZnO. Pd-rGO/ZnO nanocomposite physically
interacts with bacteria, disrupting membrane integrity, which likely impedes bacterial growth.
Pd-rGO/ZnO nanocomposite can generate reactive oxygen species (ROS), which are known
to have antimicrobial properties. These ROS may facilitate the penetration of Pd +2 and Zn+2
into the bacterial cell wall, damaging the cell membrane and leading to bacterial cell death.