CHAPTER 2

EXPERIMENTAL METHODS

2.1 Introduction

The structural, morphological and optical properties of investigated Ag, TiO2, AC,

AC/Ag, Ag/TiO2, and AC/Ag/TiO2 nanocomposites were characterized using analytical

instruments. Scanning Electron Microscopy (SEM) with EDX was used to detect the surface

morphology, elemental composition, and topography information. High-Resolution

Transmission Electron Microscopy (HRTEM) was used to identify the morphology, particle

size, and crystallographic by Selected Area Electron Diffraction (SAED). X-ray Diffraction

(XRD) was used to identify the polymorphism, component composition, crystal size, and

crystal lattice structure. Spectroscopic characterization is utilized to determine the

intermolecular interaction and chemical reactions through intramolecular interactions. Fourier

transform infrared (FT-IR) spectroscopy is extensively applied to analyze inter-and intra-

molecular interactions. This chapter provides an overview of all characterization techniques

used in this study, as well as the required theory and instrumentation details.

2.2 UV-Visible spectrophotometer

Optical absorption is a more reliable way than photoluminescence to study the bandgap.

Absorption is a direct measurement of photoexcitation across the bandgap. Therefore, it does

not have the problem of exciton recombination. A beam of monochromatic light with a uniform

wavelength is shone on a sample in the absorption measurement. In contrast, the intensity of

the transmitted light is detected on the backside of the sample. When the photon energy is

greater than the bandgap of the material, the absorption of the photons can take place, and

electrons can be elevated in energy from the valence band to the conduction band, creating

27
electron-hole pairs. If one plots the square of the absorption coefficient as a function of photon

energy, the interband excitation appears as a linear absorption edge. The bandgap energy can

be determined from the intercept of the absorption edge and the baseline. Since the material

absorbs less and can transmit the photons having energy less than the bandgap of the

semiconductors, the energy is insufficient to create an electron-hole pair. The ability of a

material to absorb photons of a given wavelength is measured quantitatively by the optical

absorption coefficient (α), which is measured in units of reciprocal distance. There are two

major intrinsic absorption processes involved in determining whether they are direct and

indirect absorption. As a general rule, the larger the bandgap, the smaller the value of a given

wavelength, but the absorption coefficient depends on the density of the states in the conduction

and valence band (Perkampus et al., 2013). The optical absorption coefficient is related to the

energy bandgap, and the following equation gives it

(αhν)2 =A (hν-Eg)

Where α is absorption coefficient, A is proportionality constant; hυ is photon energy, and Eg is

optical bandgap. The optical band gap can be evaluated by plotting α2 versus hυ from the

absorption spectra using the relation (αhν)2 α hν-Eg and extrapolating the straight-line portion

of this plot to the energy axis. The variation of (αhν)2 versus hυ is the fundamental absorption

region is plotted on the right and bottom (Chander et al., 2018).

The direct optical absorption is the first-order process involving only the absorbed

photon and is represented approximately as the vertical line on the energy versus wave vector

plot. The absorption transition conserves energy so that ΔE = hѵ (where hѵ is the photon

energy), there is no change in wave vector (k) between the initial and final states except for the

small photon momentum. An indirect optical transition is a second-order process involving

both the absorbed photon and the simultaneously absorbed or emitted phonon, which occurs at

28
different values of the wave vector k at the minimum of the conduction band and the maximum

of the valence band. The absorption transition conserves energy by requiring ΔE = hѵ + Ep,

Ep is the photon energy, and the change in k between the initial and final states k is

equal to the value of k of the phonon involved. The spectrophotometer consists of continuous

change in wavelength and automatic comparison of light intensities of the sample and reference

material. The automatic operation eliminates many time consuming changes and provides a

rapid spectrogram. In the double beam-in-time arrangement, energy from a dispersed source

passes through the exit slit of the monochromator. It is alternated between reference and sample

compartments at 60 Hz (or other fixed frequency). These two beams alternately strike a single

detector where their optical energy is converted into an electrical signal. The sensors output is

an alternating signal whose amplitude is proportional to the difference in intensities in the two

channels. An automatic slit server system constantly maintains the reference signal that widens

or closes the slits to achieve a 100 % transmittance baseline. The switching between reference

and sample paths is done by rotating half sector mirror system, or by a vibrating mirror

assembly, or by a stack of thin horizontal glass plates that are silvered along their edges and

alternately oriented, or by a prismatic beam splitter (Clark et al., 1993).

29
 Figure 2.1 Schematic diagram of UV-visible spectroscopy

2.3 X-ray Diffractometer (XRD)

X-ray diffractometer is one of the most comprehensive analytical instruments to detect

polymorphism and crystal structure. Crystals are symmetrical arrays of atoms containing rows

and planes of high packing density so that they can be three dimensional diffraction gratings

(R.E. Reed-hill et al., 1994). When X-rays of a given frequency strike an atom, they interact

with its electrons, causing them to vibrate with the frequency of the X-ray beam. When a beam

of X-rays irradiates the regular interval spaced atoms, the scattered radiation undergoes

interference. In a specific direction, constructive interference occurs, and destructive

interference appears in other directions. When an electron strikes a metal target at high speed,

the electrons of the target are excited to raise a shell of higher energy level, and the outer

30
electrons fall to fill in the vacancy X-rays are emitted then. An electron emits k X-rays from

the L to the K shell, and an electron produces k X-rays from M to K shell. The k X-ray is used

for the characterization analysis. Each crystalline plane reflects a part of the X-ray beam and

permits it to pass through. When X-rays strike a crystal, the beam is from the surface layer of

atoms and from atoms underneath the surface to a considerable depth (Skoog et al., 2001,

Bunaciu et al., 2015).

The beam would be reflected not from just two lattice planes but many parallel planes.

The symbol d represents the spacing (or distance) between planes. The crystallite size D can

be evaluated using the Scherer equation, using the full-width at half-maximum (FWHM) value

of the film reflections

D = kλ/βcosθ,

D is the average crystallite dimension perpendicular to the reflecting phases, X is the

X-ray wavelength, k is the Scherer constant, which equals 0.9 for spherical particles but varies

depending on particle form (crystallite, domain) and diffraction reflection indices (h k l). It is

the full width at the half limit of the peaks (Rao et al., 2017). When the relationship is satisfied,

the reflected rays will phase, and constructive interference will result. X-ray diffractometer is

a powerful instrument to determine crystal structure, lattice parameters, crystal orientation

(degree of texture), crystal size, crystallinity, phase composition, residual stress, and

orientation relationships. The PXRD system used in this study was an XPert PANalytical

analytical Diffractometer. The source of XRD was Cu Kα1 radiation (λ=1.5460 Å) operated at

40 keV and 30 mA. The X-ray was passed through a 1mm slit, and the signal was passed

through a 1mm slit, a nickel filter, and another 0.1mm slit. The detector type was Xcelerator

type. The scanning rate is 0.05° step size and a collection interval of 2 seconds per step in the

range from 10° to 80°/140°. The quantity of the sample is around 25-50 mg. The analysis time

31
depends on the scanning rate and the total two theta ranges, and the time for a sample is

approximately 5 minutes to 2 hours.

Figure 2.2 Schematic diagram of X-ray diffraction technique

2.4 Fourier Transform Infrared (FT-IR) Spectroscopy

Fourier transform infrared (FT-IR) is a vibrational spectroscopic technique based on

the excitation of stretching and bending modes of chemical bonds collecting in infrared spectra.

Instead of recording the amount of energy absorbed, when the frequency of the infrared light

is varied (monochromator), the IR light is guided through an interferometer. After passing the

sample, the measured signal is the interferogram. A mathematical Fourier Transform is

performed on this signal results in a spectrum more identical to conventional infrared

32
spectroscopy. A beam of infrared light is passed through the sample to analyze a sample, and

the amount of energy absorbed at each wavelength is recorded. This may be finished by

scanning through the spectrum with a monochromatic beam, which changes the wavelength

over time, or by using a Fourier transform instrument to measure all wavelengths at once.

Nevertheless, the observed absorbance or transmittance spectrum shows the sample's

wavelength absorbs the IR region. The working principle of FT-IR is almost dependent on the

detection of the covalent bonds, molecular vibration, and bending of organic functional groups.

In addition, including hydrogen bonding, the inter- or intramolecular interactions are also

determined (Pavia et al., 2001, Skoog et al., 2001). FT-IR spectroscopy is sensitive to the short-

range order structure of molecular solids and is used to study fundamental molecular vibrations,

offering a fingerprint of a molecular solid. Nevertheless, the samples have a limit of high

sensitivity for infrared light. FT-IR is an analytical instrument to identify specific molecular

vibrational frequencies according to the changes in dipole moment. In our study, FT-IR can be

performed across an amorphous crystalline spectrum of materials. Therefore, the

conformational change on crystallization, the components of the compound, and

characterization of polymorphism are identified (Li et al., 2003). FT-IR spectroscopies are

more economical than conventional dispersive spectroscopies because building the

interferometers is easier than fabricating a monochromator. In addition, the measurement of a

single spectrum is faster for the FT-IR technique because the information at all frequencies is

collected simultaneously. This allows multiple samples to be collected and averaged together,

improving sensitivity (Mucciniet al., 2004). FT-IR spectrum was obtained by Perkin Elmer

(Spectrum RXI) and used to measure absorbance, reflection, and transmission of samples in

the wavenumber regions between 4000-400 cm-1. In each case, the chamber was allowed to

equilibrate with a flowing nitrogen purge after admitting the sample into the holder. Purge

times were typically at least one minute but two minutes before the background scan and the

33
sample measurement. About 500 mg of KBr was mixed with 1.5-2.0 mg of the sample. Then

the samples were measured in the regions 400-4000 cm-1 with a resolution of 2 cm-1.

Figure 2.3 Schematic diagram of FT-IR spectroscopy

2.5 Scanning Electron Microscope (SEM)

The scanning electron microscope (SEM) is an electron microscope capable of

providing high resolution, magnification, and depth of field images than optically attained.

SEM images have a characteristic three-dimensional appearance and help judge the surface

structure of the sample. The main applications of SEM are on topography, morphology, and

composition of samples (Skoog et al., 2001). In a typical SEM, electrons are thermionically

emitted from a tungsten or lanthanum hexaboride (LaB6) cathode and are accelerated towards

an anode; alternatively, electrons can be emitted via Field Emission (FE). Tungsten is selected

because of all metals' highest melting point and lowest vapor pressure, thereby allowing it to

be heated for electron emission. The electron beam, which typically has an energy ranging from

100 eV to 50 keV, is focused by one or two condenser lenses into a beam with an excellent

focal spot sized 1 nm to 5 nm. The incident beam passes through pairs of scanning coils in the

objective lens, which deflect the beam in a raster fashion over a rectangular area of the sample

34
surface. The primary electron beam effectively spreads and fills a teardrop-shaped volume

through these scattering events, known as the interaction volume, extending from less than 100

nm to around 5 m into the surface. Interactions in this region lead to the subsequent emission

of electrons which are then detected to produce an image and provide elemental information

(Reed-hill et al., 1994).

The spatial resolution of the SEM depends on the size of the electron spot, which

depends on the magnetic electro-optical system which produces the scanning beam. The size

of the interaction volume also limits the resolution. The spot size and the interaction volume

are both immense compared to the distances between atoms, so the resolution of the SEM is

not high enough to image down to the atomic scale, as is possible in the transmission electron

microscope (TEM).

Figure 2.4 Schematic diagram of Scanning Electron Microscope

35
2.6 High Resolution Transmission Electron Microcopy (HR-TEM)

High-Resolution Transmission Electron Microscopy (HR-TEM) is an imaging

technique whereby a beam of electrons is transmitted through a specimen. An image is formed,

magnified, and directed to appear either on a fluorescent screen or a layer of photographic film

or detected by a sensor such as a CCD camera. The primary function of TEM is involved in

studying the internal- and microstructure of materials by the diffraction pattern and chemical

composition analysis by Energy Dispersive X-ray Spectroscopy (EDS) and Electron Energy

Loss Spectroscopy (EELS).

2.6.1 The basic TEM operation

In TEM, there are two critical systems: (i) the illumination system that controls the

electron source (intensity) and (ii) the imaging system that collects the electrons scattered and

transmitted from the sample. Depending on the mode of operation, the electrons collected by

the imaging system can either form a diffraction pattern or an image. In diffraction mode, the

electrons that form the SAED pattern are elastically scattered by the sample. Thus, if the atomic

arrangement in the sample is periodic, the diffraction pattern consists of well-defined spots. In

contrast, a polycrystalline sample with less atomic order results in diffused spots, arcs, or even

rings. This particular TEM feature is essential and reveals information about the microstructure

of the sample.

Moreover, if the grain size is really fine, the resulting diffraction pattern is in the form

of continuous rings. So, it can be seen that the diffraction technique can also give an idea about

the relative grain sizes of the different samples. The SAED pattern taken from the interface

area provides the diffraction information from the sample, enabling the determination of the

exact relationship. TEM also allows a certain degree of freedom in the rotation of specimens.

If the sample is prepared thoughtfully, the entire crystal structure and the atomic arrangement

36
in more than one direction can be probed using the diffraction technique. A typical diffraction

pattern contains a bright central spot and slightly weaker diffracted spots. The TEM is used

heavily in material science/metallurgy and the biological sciences. The specimens must be

fragile for hundreds of nm and stand in the high vacuum environment and applied voltage

inside the instrument. Because the image formation in the transmission electron microscopy

depending on the diffraction of electrons, it is necessary to consider some elementary facts

about the diffraction. The wavelength of an electron is related to its velocity by the following

equation.

ℎ
λ = 𝑚𝑐

Where m is its mass, and h is the Planck constant. In the image mode, the image can be

formed using either the central bright spot (transmitted beam with a small amount of elastically

scattered electrons) or entirely from the scattered electrons. When the image is formed using

the central beam, it is known as a Bright Field (BF), whereas it is known as the Dark Field (DF)

if the diffracted beam or scattered electrons are used to form the image. Both types of images

are based on the amplitude contrast technique. However, DF images usually have better

contrast. Thus, the DF technique is generally used to resolve certain features in the

microstructure (defects, clusters, precipitates, grains, and grain boundaries). The grain size of

the samples has also been calculated using this technique. The imaging techniques are essential

in material structures. The lenses are magnetic, being composed generally of a current-carrying

coil surrounded by a soft iron case. The lenses are energized by direct current. The detail in the

image is formed by the diffraction of electrons from the crystallographic planes of the object

being investigated. Defects in materials influence the electronic and mechanical properties of

materials, so understanding how they behave gives a powerful insight. By carefully selecting

the orientation of the sample, it is possible not just to determine the position of defects and

37
crystal graphics but also to determine the type of defect and crystal graphic present. If the

sample is orientated so that one particular plane is only slightly tilted away from the most vital

diffracting angle (known as the Bragg Angle), any distortion of the crystal plane that locally

tilts the plane to the Bragg angle will produce extreme contrast variations. However, defects

that only displace atoms that do not tilt the crystal to the Bragg angle will not strongly contrast.

Bragg’s law is described as the following equation.

𝑛𝜆
θ = 2𝑑

Where θ is the incident angle of the electron beam and is very small, we should expect

the diffracted spots to be deviated through distances that are inversely proportional to the

interplanner spacing d.

Selected area electron diffraction (SAED) is a crystallographic experimental technique

that can be performed using TEM. In TEM, an ultra-thin specimen is subjected to a parallel

beam of high energy electrons. As TEM specimens are typically about 100 nm thick and the

electrons usually have high energy of 100-400 keV, the electrons pass through the sample

quickly. In this case, electrons are treated as wave like rather than particle like. Because the

wavelength of high-energy electrons is a fraction of a nanometer and the spacing between the

faces in a solid is only slightly larger, the faces serve as a diffraction grating to the electrons,

which are diffracted. If the sample is moved under the beam, bringing different sections of it

under illumination, the arrangement of the spots in the diffraction pattern will change. If the

sample is tilted, the same crystal will stay under illumination, but different diffraction

conditions will be activated, and other diffraction spots will appear or disappear. SAED is

referred to as "selected" because the user can easily choose the part of the specimen to be

obtained the diffraction pattern. SAED can be used to identify crystal structures and examine

crystal defects. SAED is mainly used in material science and solid-state physics to determine

38
crystallography and polymorphism. The size, composition and atomic structure of the NPs

were analyzed by High-Resolution Transmission Electron Microscopy (HR-TEM) and Energy

Dispersive Spectroscopy (EDS) using a 200 keV JEOL JEM 2100 microscope with a lattice

resolution of 0.14 nm and point to point resolution of 0.19 nm. For observation, only a tiny

amount of the sample was necessary. The selected area electron diffraction pattern evaluated

the crystallinity and crystal lattice parameters. The quality and half quantify the elemental

composition analysis was determined by energy dispersion spectroscopy (Mukasyan et al.,

2007).

Figure 2.5 Schematic diagram of Transmission Electron Microscope

39
2.7 Energy Dispersive Spectroscopy (EDS)

Energy-dispersive X-ray spectroscopy (EDS or EDX) is a compositional microanalysis

technique used with transmission electron microscopy (TEM) and SEM. Features as small as

1 m can be analyzed. EDS detects the characteristic X-rays emitted from the sample during

bombardment by an electron beam to characterize the elemental composition of the researched

volume. When the electron beam bombards the sample, X-ray photons produced by transitions

between deep atomic levels are ejected from the atoms comprising the sample surface. The X-

ray energy is the characteristic of the element from which it was emitted. X-ray strikes the EDS

detector creates a charge pulse proportional to the X-rays energy. The charge pulse is converted

to a voltage pulse (which remains proportional to the X-ray energy) by a charge sensitive pre

amplifier. The signal is then sent to a multichannel analyzer, where the pulses are sorted by

voltage. The X-ray energy spectrum versus counts is evaluated to determine the relative

abundance of emitted X-rays versus their energy. Hence, the elemental composition of the

sample is considered. Qualitative analysis: X-ray transition energy derived from the EDS

spectrum of the sample can be compared with known characteristic X-ray energy values for

determining the presence of an element in the sample. Elements with atomic numbers ranging

from beryllium to uranium can be detected. The minimum detection limits vary from

approximately 0.1 to a few atomic percent, depending on the element and the sample matrix.

Quantitative analysis: Quantitative results can be obtained from the relative X-ray counts at the

characteristic energy levels for the sample constituents. Semi-quantitative results are readily

available without standards using mathematical corrections based on the analysis parameters

and the sample composition. The accuracy of the non-standard analysis depends on the sample

composition. Greater accuracy is obtained using known standards with structure and

composition similar to that of the unknown sample.

40
Elemental mapping: Characteristic X-ray intensity can be measured relative to the lateral

position on the sample. Variations in X-ray intensity at any characteristic energy value indicate

the relative concentration of the applicable element across the surface. One or more maps are

recorded simultaneously using image brightness intensity to function the local relative

concentration of the components present. About 1 m lateral resolution is possible.

Line profile analysis: The TEM electron beam can be scanned along a preselected line across

the sample while X-rays are detected for discrete positions along the line. Analysis of the X-

ray energy spectrum of each position provides plots of the relative elemental concentration for

each element versus position along the line (Zhang et al., 2008).

Figure 2.6 Schematic diagram of Energy Dispersive Spectrum

2.8 Photocatalytic performance of nanoparticles

The photocatalytic studies of the prepared nanoparticles were performed in an open-air

atmosphere. In the photodegradation study, the as prepared photocatalyst was dispersed in

41
100 mL aqueous solution of MB dye. Prior to irradiation, the solution was ultra-sonicated and

stirred for 30 min in a darkroom to achieve adsorption/desorption conditions. Then, the solution

was allowed to expose in the direct sunlight under magnetic stirring. At every 10 min time

interval, 5 ml of suspension was sampled and centrifuged at 3000 rpm for 10 min. where the

reaction solution was maintained at 37° C. Then the photodegradation behavior was analyzed

using a UV–Vis spectrophotometer. The effects of photocatalyst dosage and dye concentration

were studied by adopting the same procedure. The degradation efficiency (DE) of MB dye is

estimated by the following equation:

DE (%) = (C0 − C) ∕C0 × 100%

Where C0 is the initial concentration of MB dye solution and

C is the final concentration of MB dye solution.

2.9 Antibacterial activity

Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Escherichia coli

(ATCC 8739), and Klebsiella Pneumonia (ATCC 10031) are obtained from the American Type

Culture Collection (ATCC) USA. Using the good diffusion method, the antibacterial activity

of synthesized NPs was tested against gram-positive (S. Aureus and Bacillus) and gram

negative bacteria (E. coli, and Klebsiella). The Muller–Hinton agar was poured into the

sterilized Petri plates and allowed to solidify. After solidification, fresh bacterial culture was

spread over the plates by the spread plate method. Six wells were made in the agar medium

using sterile cork borer and the diameter of each well is 5 mm. About 50 µg/ml of the

nanoparticle solution was loaded in the well and the plates were incubated for 24 h at 36 °C.

After the incubation, the formation of the zone of incubation around the well was observed and

42
scaled in mm. The formation of an inhibition zone indicates the antibacterial activity of

nanoparticles.

2.10 Conclusion

XRD, FT-IR, UV- Vis Spectroscopy, SEM, HR-TEM, and EDS were used to

characterize the physio-chemical properties of materials. The selection of the analytical

instruments was fast, economical, and sensible without complex procedures. Besides,

comprehensive technologies are also used for research and measurement of various domains.

The improvement of analytical approaches enhances the progress of science and engineering,

including quality and quantity.

43