GREEN ROUTE APPROACH STRUCTURAL

CHARACTERIZATION OF TITANIUM
DIOXIDE NANOPARTICLES USING
SYZYGIUM SAMARANGENSE LEAF EXTRACT

A PROJECT REPORT

Submitted by

PRIYADHARSHINI P S

RegNo:24083106112712107

In partial fulfillment for the award of degree of

MASTER OF SCIENCE
IN
PHYSICS

NANJIL CATHOLIC COLLEGE OFARTS AND SCIENCE

KALIYAKKAVILAI - 629153

MANONMANIUM SUNDARANAR UNIVERSITY,

TIRUNELVELI - 627012

APRIL-2026
 CERTIFICATE
This is to certify that the project report entitled “GREEN ROUTE
APPROACH STRUCTURAL CHARACTERIZATION OF TITANIUM
DIOXIDE NANOPARTICLES USINGSYZYGIUM SAMARANGENSE
LEAF EXTRACT” is a Bonafide and independent work done by the candidate
PRIYADHARSHINI P S [24083106112712107] in partial fulfillment for the
degree of Master of Science in Physics of Nanjil Catholic College of Arts and
Science under my supervision and guidance.

Supervisor
Dr.M. Amalanathan [Link].,[Link].,Ph.D

Head of the Department,

[Link] Dominic Christopher,
[Link].,[Link].,[Link](Phy).,[Link](Edn).,PGDCA.,Ph.D

External Examiners

Place: Kaliyakkavilai 1.

Date: 2.

ii
 DECLARATION

I hereby declare that the project entitled “GREEN ROUTE

APPROACH STRUCTURAL CHARACTERIZATION OF TITANIUM
DIOXIDE NANOPARTICLES USING SYZYGIUM SAMARANGENSE
LEAF EXTRACT” is the record of my project work as students of [Link]
Physics, Nanjil Catholic College of Arts and Science, done in the Department
of Physics Laboratory under the supervision and guidance of
[Link] [Link]., [Link]., Ph.D, Principal, Nanjil Catholic College
of Arts and Science.

Place: Kaliyakkavilai

Date: PRIYADHARSHINIP S

iii
 ABSTRACT

The green synthesis of titanium dioxide (TiO₂) nanoparticles using Syzygium

Samarangense, is an environmentally friendly and sustainable approach. The
bioactive compounds in the leaf extract act as natural reducing and stabilizing
agents, facilitating the formation of TiO₂ nanoparticles without the use of
hazardous chemicals. Structural characterization using X-ray diffraction (XRD)
confirmed the formation of a highly crystalline anatase phase, indicating
excellent lattice order and purity. UV–Vis spectroscopy revealed strong
absorptionin the ultraviolet region, suggesting notable optical properties suitable
for photocatalytic activity. The synthesized TiO ₂ nanoparticles exhibit potential
applications in diverse fields, including environmental remediation for the
degradation of pollutants, solar energy harvesting, antibacterial coatings, sensors,
and cosmetic formulations. This work emphasizes that Syzygium Samarangense-
mediated green synthesis offers a cost-effective, non-toxic, and efficient route
for producing functional TiO₂ nanoparticles with well-defined structural and
optical properties, making it a promising candidate for industrial and
environmental nanotechnology applications.

Keywords: Titanium Dioxide Nanoparticles, UV-Vis, XRD, Green Synthesis,

Syzygium Samarangense

iv
 ACKNOWLEDGEMENT

First of all, I express my deep sense of gratitude and thanks to the GOD
ALMIGHTY for the successful completion of this project work.

It is my duty to thank Rev. Fr. Dr.C. Steephen, [Link]., Ph.D., Secretary,

Nanjil Catholic College of Arts and Science, Kaliyakkavilai and Dr. M.
Amalanathan, [Link]., [Link]., Ph.D., Principal, Nanjil Catholic College of Arts
and Science, Kaliyakkavilai, for his kind support during my course of study.

I sincerely thanks to Dr. S. Antony Dominic Christopher, [Link]., [Link].,

[Link] (Phy)., [Link] (Edn)., PGDCA., Ph.D., Head of the department ofphysics
in Nanjil Catholic College of Arts and Science, Kaliyakkavilai for his valuable
guidance and facilities provided in the department to complete the project
successfully.

I wish to offer my immense gratitude and thanks to my project supervisor Dr.

M. Amalanathan, [Link]., [Link]., Ph.D., Principal, Nanjil Catholic College of
Arts and Science, Kaliyakkavilai, for his valuable guidance and comments and
timely advice in bringing out this project.

I express my thanks to all staff members and lab assistants of the Department of
Physics, Nanjil Catholic College of Arts and Science, for their assistance in
completing this project. Finally, I thank my parents and friends for their
continuous encouragement and useful suggestions for the completion of my
project work.

Priyadharshini P S

v
 LIST OF FIGURES

FIGURE TITLE PAGE

NO NO
1 Sol-Gel Method of Nanoparticle Synthesis 6
2 Evaporation Method Nanoparticles Synthesis 7
3 Laser Pyrolysis Method of Nanoparticle Synthesis 8
4 Schematic Representation of Bottom up and Top down 9

Approaches
5 Cancer Detection Using Nanoparticles 10
6 Nanotechnology in Agriculture 12
7 Photocatalytic and Self-cleaning Mechanisms of 13
TiO2 Nanocomposites
8 Schematic diagram representing various applications 19
of TiO2 Nanoparticles
9 Syzygium Samarangense Plant 23
10 Light Spectrum 27
11 Ultraviolet Visible Spectroscopy 29
12 Infrared Spectroscopy 35
13 X-Ray Diffraction Instrument 36
14 XRD Analysis 39
15 Scanning Electron Microscopy 41
16 Components of SEM 42
17 Absorption Spectrum of UV Visible Spectroscopy 65
18 Tauc Plot 67
19 XRD Analysis of TiO2 Nanoparticles 68
 TABLE OF CONTENT

CHAPTER TITLE PAGE

NO
I INTRODUCTION
1.1 Nanotechnology 1

1.2 Nanoparticles 2

1.2.1 Bottom-up Approach 4

1.2.2 Top-down Approach 7

1.3 Applications of Nanomaterials 10

1.3.1 Medicine & Healthcare 10

1.3.2 Electronics & Computing 10

1.3.3 Energy Storage & Production 11

1.3.4 Cosmetic & Personal Care 11

1.3.5 Food & Agriculture 11

1.3.6 Construction & Infrasturcture 12

1.4 Titanium Dioxide Nanoparticles 12

1.5 Importance of TiO2 Nanoparticles 13

1.5.1 Applications 14

1.5.2 Properties 15

1.6 Fundamental Properties and Biological Applications of TiO 2 17

Nanoparticles
1.6.1 Fundamental Properties 17

1.6.2 Biological Applications 18

1.7 Reducing Agents 19

1.8 Green Synthesis of TiO2 Nanoparticles 20

1.9 Key Merits of Green Synthesis Method 21

1.10 Key Herbal Component of the Study 22

1.10.1 Syzygium Samarangense 22

 II CHARACTERIZATION TECHNIQUES
2.1 Introduction 26

2.2 UV-Visible Spectroscopy 26

2.2.1 Principle 28

2.2.2 Instrumentaion of UV-Visible Spectroscopy 29

2.2.3 Applications 32

2.2.3 Advantages of UV-Vis Spectroscopy 33

2.2.5 Disadvantages of UV-Vis Spectroscopy 33

2.3 Infrared Spectral Analysis 34

2.3.1 Applications of Infrared Spectroscopy 35

2.4 X-Ray Diffraction Analysis 36

2.4.1 Working of XRD 38

2.4.2 Applications of XRD Analysis 39

2.5 SEM (Scanning Electron Microscopy) 40

2.5.1 Components and Working System of SEM 41

2.5.2 Application 43

2.3.3 Advantages 44

2.5.4 Limitations 44

2.6 Energy Dispersive X-Ray Spectroscopy (EDS) Analysis 44

2.6.1 Working Principle of EDS 45

2.6.2 Components of an EDS System 45

2.6.3 Data Interpretation and Elemental Analysis 45

III REVIEW OF LITERATURE

3.1 Introduction 48

IV RESULT AND DISCUSSION

4.1 Introduction 61

4.2 Materials and Methods 63

4.3 UV-Visible Spectroscopy 65

4.4 Tauc Plot 66

4.5 X-Ray Diffraction 67

V CONCLUSION AND FUTURE SCOPE

5.1 Conclusion 72

5.2 Future Scope 73

VI REFERENCES 75
 CHAPTER I

INTRODUCTION
 CHAPTER I

INTRODUCTION

1.1NANOTECHNOLOGY

Nanotechnology is the study of the control of matter on an atomic and

molecular scale. Nanotechnology deals with structures of size 100 nm or
atleast one dimension and involves developing materials within that size.
Nanotechnology is very diverse, encompassing numerous fields in the
natural sciences.

Nanotechnology has the potential to create many new materials and

devices with a vast range of applications in medicine and energy areas.
On the other hand, nanotech raises many of the same issues as with any
introduction of new technology, including concerns about the toxicity and
environmental impact of Nanomaterials and their potential effects on
global economics.

Nanotechnology is investigation of metals in size range of 1 to 100nm.

The world of nano science deviates from the larger world we are
accustomed to in our daily lives, centered on numerous aspects of
nanotech. When the length scale of materials decreases surface area
effects become more important and quantum effects emerge, leading to
modifications in the properties of materials, The quantity of atoms
appropriate for an object’s surface increases due to the compact nature of
metals.

1
 Nanotechnology is one of the major applications of nano science. Nano scale
fabrication provides the basis of integration and control on molecules and atoms.
DNA silicon chips are wonderful application of nanotechnology. It can be applied
in almost every field like in military defense for missile system, medicine,
astronomy and computing.

There are number of latest devices and materials such as eyeliner, suntan lotion,
sterile socks and self-cleaning glass have been synthesized and fabricated with
the help of nanotechnology. According to scientists, nanofood will be effective
for our stomach and prevents us from over eating.

Nowadays, nanotechnology is a novel and appealing scientific and research

field. Nanotechnology has emerged as one of the large and attractive areas of
research, offering unique features and extensive applications in various sectors
such as agriculture, food and biomedicine.

Modern nanotechnology research is gaining prominence due to its

groundbreaking and encouraging outcomes in numerous sectors. It is pioneering
the development of multifunctional Nanomaterials for various applications such
as energy storage, optical devices, medical devices, solar cells, biomedical and
drug delivery, foods, cosmetics and paints, textile industry etc.

1.2NANOPARTICLES

Nanoparticles are distinguished from microparticles(1–1000μm),"fine

particles" (sized between 100 and 2500 nm), and "coarse particles" (ranging
from 2500 to 10,000 nm),because their smaller size drives very different

2
physical or chemical properties, like colloidal properties and ultrafast optical
effects or electric properties.

Being more subject to the Brownian motion, they usually do not

sediment, like colloidal particles that conversely are usually understood to range
from 1 to 1000 nm.

Being much smaller than the wavelengths of visible light (400–700 nm),
nanoparticles cannot be seen with ordinary optical microscopes, requiring the
use of electron microscopes or microscopes with laser. For the same reason,
dispersions of nanoparticles in transparent media can be transparent, whereas
suspensions of larger particles usually scatter some or all visible light incident
on them. Nanoparticles also easily pass through common filters, such as
common ceramic candles, so that separation from liquids requires special
nanofiltration techniques.

The properties of nanoparticles often differ markedly from those of larger

particles of the same substance. Since the typical diameter of an atom is
between 0.15 and 0.6 nm, a large fraction of the nano particle's material lies
within a few atomic diameters of its surface. Therefore, the properties of that
surface layer may dominate over those of the bulk material. This effect is
particularly strong for nanoparticles dispersed in a medium of different
composition since the interactions between the two materials at their interface
also becomes significant.

Nanoparticles occur widely in nature and are objects of study in many

sciences such as chemistry, physics, geology, and biology. Being at the
transition between bulk materials and atomic or molecular structures, they often
exhibit phenomena that are not observed at either scale. They are an important
component of atmospheric pollution, and key ingredients in many industrialized
products such as paints, plastics, metals, ceramics, and magnetic products. The

3
production of nanoparticles with specific properties is a branch of
nanotechnology.

In general, the small size of nanoparticles leads to a lower concentration

of point defects compared to their bulk counterparts, but they do support a
variety of dislocations that can be visualized using high-resolution electron
microscopes. However, nanoparticles exhibit different dislocation mechanics,
which, together with their unique surface structures, results in mechanical
properties that are different from the bulk material. Non-spherical nanoparticles
(e.g., prisms, cubes, rods etc.) exhibit shape-dependent and size-dependent (both
chemical and physical) properties (anisotropy).

Non-spherical nanoparticles of gold (Au), silver (Ag), and platinum (Pt)

due to their fascinating optical properties are finding diverse applications. Non-
spherical geometries of nanoprisms give rise to high effective cross-sections and
deeper colors of the colloidal solutions. Anisotropic nanoparticles display a
specific absorption behavior and stochastic particle orientation under polarized
light, showing a distinct resonance mode for each excitable axis.

1.2.1 BOTTOM-UP APPROACH

The bottom-up approach utilizes physical and chemical processes that

function at the nanoscale to integrate main components into bigger
structures. A reduction in unit size, the approach yields a relevant
consummation to the top-down approach. Biological systems provide a
motivation for this approach in which natural forces of life harness their
chemical counterparts to produce the structures.

4
 Gold-palladium alloy nanoparticles which were based on carbon treated
with acid and breaking down hydrogen peroxide are formed from the
combination of white hydrogen and red oxygen when disallowing unwanted
decay.

This approach has been used in the production of nanoparticles from

condensation to coalescence of atomic vapors and liquids, respectively.

1. Chemical Techniques

a. Sol-gel Techniques

This allows the production of nanomaterials from alkoxides or colloidal

based solution. They take the form of monoliths, crystallized nano pigments,
and thin layers. They are centered on reactions in polymerization. The three
types of this are sol-gel, metal alkoxide, and pechini.

The sol-gel method focuses on the growth of chemical compounds in the

solution. Following movements between the species and liquid, the sol is
changed into some sort of three-sided channel increased in the solvent. The
process is then frozen and the sol-gels are transformed to deformed solid
material by the removal of liquid from the air-gel or by evaporation.

5
 Fig 1: Sol-Gel Method of Nanoparticle Synthesis

One benefit of the process is the ability to manage the size and oneness
of the material spread-out. They make the production of large parts of fibers
possible. But, one main disadvantage of the technique is that the precursors are
too pricy with very low gain; low weigh products and very dangerous to human
health.

b. Supercritical Fluid with a Chemical Reaction

The main goal of having a chemical conversion in a supercritical solvent is

to consistently improve the physical and chemical processes of the precursors
by improving the temperature on small mediums as well as the pressure. This
control allows easy control of the chemical reaction. The method permits the
production of divided materials of inorganic nature, such as oxide metal and
nitrate.

6
1.2.2 TOP-DOWN APPROACH

The top-down approach usually involves the following processes:

1. Evaporation/Condensation

This has to do with metal evaporation through heating and then condensing
of the obtained vapor for the purpose of obtaining nanopowders. This method is
based on the pressure caused by vaporizing the metal, which is its capacity to
vaporize depends on its chemical strength. The surface oxidation Fe, Ni, Co,
Cu, Pd, Pt equally create sufficient steam through radiation and inductive
heating (1200 and 2000 degrees), respectively. A certain amount of the material
is made in the laboratory.

Fig 2: Evaporation Method Nanoparticle Synthesis

Oxygen (A1, C8, T1, Zr) and refractory metals (with low pressure in
vapor, Mo, Hf, Ta, w) demand greater modes of heating. An example is electron
bombardment heating of 3000 degrees and combined with electric arc (3000 and
14000 degrees). If the atmosphere in which the metallic elements are placed

7
reactive (oxygen), the effect of this would be oxidation of the nanoparticles.
The challenge with this process is the loss of control of the nanometric size.

The powders are derived by swift cooling of the escaped vapor, making
sure the formation of a huge number of nanoparticles, then restricting their
growth and other processes. The technique is particularly used in factory
production of ceramic and metal nanopowders and production is made in very
large tons a year. The nanopowders are prone to fire and explosion in the air.

2. Laser Pyrolysis

The method is easy and effective in the synthesis of nanopowders. It is

produced by a form of exchange between the release of carbon dioxide laser and
the movement of reagents. The energy current between the two leads to a quick
increase in temperature in the reactive zone by arousing the vibration degrees of
molecules. There are disconnection of reactors and a flame that manifests where
the nanoparticles are produced and then go through quenching once the flames
disappear. The powders are deposited where they will be obtained.

Fig 3: Laser Pyrolysis Method Nanoparticle Synthesis

8
 The above technique is an easy way for particle synthesis of 15 – 20
nanometers at 100g/h. One of the benefits of this technique is the flow of
reaction. Other benefits are high level of purity, excellent chemical and physical
properties. Many nanopowders have been formed through this process.

3. Ionic/Electronic Irradiation

Through the use of irradiations, nanoparticles can be formed in an original

manner. The technique is hinged on the following:

The features of energy production by heavy ions facilitators, which

introduces local changes in the material in a circumference of 10nm around the
ion). The method is suitable in the production of polymer membranes
nanopowder. To achieve a ‘matrix’ production, the material can be developed in
the membrane pores of the polymer; irradiation makes the attainment of mass
possible.

Fig 4: Schematic Representation of Bottom-up and Top-down Approaches

9
1.3APPLICATION OF NANOMATERIALS

1.3.1 MEDICINE & HEALTHCARE

In the medical field, nanomaterials act as precision-guided vehicles

for therapy. By engineering nanoparticles—such as liposomes or gold
nanoshells—to recognize specific biomarkers on the surface of diseased
cells, doctors can deliver toxic drugs directly to a tumor while sparing
healthy tissue. Furthermore, "nano biosensors" allow for the detection of
diseases like cancer or Alzheimer’s at the molecular level, long before
traditional imaging could spot them.

Fig 5: Cancer Detection Using Nanoparticles

1.3.2 ELECTRONICS & COMPUTING

Electronics are being redefined by the transition from silicon to

nanomaterials like carbon nanotubes and graphene. These materials possess
extraordinary electron mobility, allowing for transistors that are not only
smaller but significantly faster and cooler than current technology.
Additionally, "Quantum Dots" are now used in high-end displays to produce

10
 more precise, vibrant colors by emitting specific wavelengths of light based
on their size.

1.3.3 ENERGY STORAGE & PRODUCTION

Nanotechnology addresses the efficiency gaps in renewable energy

by increasing the active surface area of materials. In solar cells,
nanostructured layers can capture a wider spectrum of sunlight and convert
it into electricity with less heat loss. For batteries, using silicon nanowires or
carbon-based nanomaterials for electrodes allows for more lithium-ion
storage and faster movement, resulting in batteries that charge in minutes
rather than hours.

1.3.4 COSMETICS & PERSONAL CARE

The most common application in cosmetics is found in sunscreens.

Traditional sunscreens used large particles of zinc oxide that left a thick, white
residue; however, by using "nanosized" versions of these minerals, the
sunscreen becomes transparent on the skin while providing even better UV
protection. Additionally, lipid-based nanoparticles are used in anti-aging creams
to help active ingredients penetrate deeper layers of the skin rather than just
sitting on the surface.

1.3.5 FOOD & AGRICULTURE

Nanotechnology in agriculture leads to "precision farming" by using

nano-encapsulated fertilizers and pesticides. These "smart" capsules are
designed to release their contents only in response to specific triggers, such as
preventing chemical run off into local water supplies.

11
 Fig 6: Nanotechnology in Agriculture

1.3.6 CONSTRUCTION & INFRASTRUCTURE

Nanomaterials are turning "dumb" materials like concrete into "smart"

materials. Adding nano-silica or carbon nanotubes to cement mixtures creates a
denser molecular structure, making the resulting concrete much more resistant
to water penetration and cracking. Some modern buildings even use titanium
dioxide (TiO2) coatings on their exteriors; when sunlight hits these
nanoparticles, they trigger a chemical reaction that breaks down smog and
pollutants in the surrounding air.

1.4TITANIUM DIOXIDE NANOPARTICLES

Titanium dioxide nanoparticles in the form of anatase have
received considerable attention in the construction industry in recent
years due to their potential to add new functionalities to infrastructures,
i.e., self-cleaning properties and the ability to remove air pollutants.

12
 When mixed with cement, it can photocatalytically degrade organic
pollutants that are, after neutralization, washed away through the
hydrophilic nature of the surface, maintaining at the same time the
aesthetic characteristics of concrete structures, particularly those
constructed with white cement (Cassar, 2004; Ruotet al., 2009).TiO2has
proven very efficient in the removal of pollutants such as NOx, aromatics,
aldehydes, and ammonia, and currently finds applications in various
infrastructure projects like pavements, tunnels, buildings, etc. (Guerrini,
2012).

Fig 7: Photocatalytic and Self-cleaning Mechanisms of TiO2

Nanocomposites

1.5IMPORTANCE OF TiO2 NANOPARTICLES

Titanium dioxide (TiO2) nanoparticles are indispensable in modern industry
due to their exceptional ability to manipulate light and catalyze chemical
reactions. By leveraging a high surface area and a specific band gap of
approximately (3.2 eV), these particles act as highly efficient UV filters in
sunscreens and high-performance pigments in coatings. Beyond surface
protection, their role as a potent photocatalyst allows them to break down
organic pollutants and bacteria when exposed to light, driving innovations in
self-cleaning building materials, air purification systems, and low-cost solar
energy.

13
1.5.1 APPLICATIONS

1. Transparent Sunscreens

In its "bulk" form, titanium dioxide is a thick white pigment. At the

nano-scale, however, it becomes transparent to visible light while still
maintaining its ability to block and scatter harmful UV rays. This allows for
high-SPF sunscreens that don't leave a chalky white residue on the skin.

2. Self-Cleaning Glass and Tiles:

Because TiO2 is a photocatalyst, it reacts with UV light (from the sun)

to break down organic dirt and grime. When applied as a coating to windows or
building exteriors, it decomposes pollutants, which are then easily washed away
by rainwater.

3. Air and Water Purification:

These nanoparticles are used to "scrub" environments.

 Air: They can neutralize Volatile Organic Compounds (VOCs) and

odours in indoor air filters.
 Water: They are used to break down complex organic pollutants and kill
bacteria in wastewater treatment systems without the need for harsh
chemicals.

14
4. Dye-Sensitized Solar Cells (DSSCs):

In the world of renewable energy, TiO2 nanoparticles act as a thin-film

semiconductor. They provide a massive surface area for dye molecules to sit on;
when light hits the dye, the TiO 2 captures the released electrons and funnels
them into an electrical circuit to generate power.

5. Antimicrobial Surface Coatings:

In high-traffic areas like hospitals or public transit, TiO 2 coatings are

used to create "active" sanitizing surfaces. When exposed to light, the particles
generate reactive oxygen species that can destroy the cell membranes of bacteria
and viruses, providing a layer of protection against germs.

1.5.2 PROPERTIES

1. Optical Properties

UV Absorption and Transparency

TiO2 is a wide-bandgap semiconductor (3.0 to 3.3 eV). At the

nanoscale, these particles are smaller than the wavelength of visible light
(400–700 nm), which allows visible light to pass through them, making the
material appear transparent. However, they remain highly efficient at
absorbing and scattering Ultraviolet (UV) radiation. This makes them ideal
for "clear" sunscreens and UV-protective coatings for wood or plastics.

High Refractive Index

Even as nanoparticles, TiO2 retains one of the highest refractive

indices of any transparent material (approx.2.5 for Anatase and 2.9 for

15
 Rutile). This property governs how effectively a material bends and scatters
light. In coatings and paints, this high index is what provides "hiding
power"—the ability to cover a surface with a completely opaque, bright
white finish using very thin layers.

Photoluminescence

Under specific conditions, TiO2 nanoparticles can exhibit

photoluminescence, where they absorb high-energy light and re-emit it at a
different wavelength. This property is frequently used in sensor technology
and bio-imaging to track the movement or concentration of particles within a
system.

2. Chemical Properties

Photocatalytic Activity

The most significant chemical property of TiO 2 is its ability to act as a

photocatalyst. When UV light hits the particle, it "excites" an electron from the
valence band to the conduction band, creating a hole and an electron.

 Oxidation: The holes react with surface water to create hydroxyl

radicals (OH).
 Reduction: The electrons react with oxygen to create superoxide
anions
2 (O -).

These reactive oxygen species are highly aggressive and can chemically
decompose organic pollutants, smoke, and even the cell walls of bacteria
into harmless CO2 and H2O.

16
Superhydrophilicity

Chemically, the surface of TiO2 is naturally populated with hydroxyl (-OH)

groups. When exposed to light, the surface undergoes a structural change that
increases the density of these groups, making the surface superhydrophilic
(water-loving). Instead of forming beads, water spreads into a uniform film.
This chemical shift is why TiO 2 coatings are used for anti-fogging glass and
self-cleaning building exteriors, as the water film carries away dirt.

Chemical and Corrosion Resistance

TiO2 is chemically inert and highly stable. It does not react with most acids or
alkalis at room temperature and is insoluble in water. This stability makes it an
excellent protective agent; when added to metals or polymers, it acts as a barrier
against environmental corrosion and thermal degradation.

1.6FUNDAMENTAL PROPERTIES AND BIOLOGICAL

APPLICATIONS OF TiO2 NANOPARICLES

1.6.1 FUNDAMENTAL PROPERTIES

Titanium dioxide (TiO2) nanoparticles are characterized by their
structural polymorphism, primarily existing as anatase, rutile, or brookite
phases, where the arrangement of their crystal lattice dictates their
chemical stability and energy levels. At the nanoscale, these particles
exhibit a high surface-to-volume ratio that enhances their reactivity and a
wide band gap (roughly 3.0 to 3.2 eV), which allows them to remain
transparent to visible light while effectively absorbing harmful ultraviolet
(UV) radiation.
UV light excites electrons to a higher energy state, creating "electron-
17
hole pairs" that react with surface moisture to generate hydroxyl radicals.
These radicals are highly oxidative, capable of breaking down organic
pollutants and pathogens into harmless substances. Furthermore, TiO 2
nanoparticles exhibit photo-induced superhydrophilicity, causing water to
spread into a thin, uniform film rather than beading, a property that, when
combined with their oxidative power, enables the creation of "self-
cleaning" and anti-fogging surfaces.

1.6.2 BIOLOGICAL APPLICATIONS

In the biological and medical fields, TiO 2 nanoparticles are
utilized for their exceptional biocompatibility and light-triggered
reactivity, serving as versatile agents in targeted drug delivery, cancer
therapeutics, and antimicrobial coatings. As drug carriers, their high
surface area allows for the attachment of ligands that direct
pharmaceuticals to specific diseased cells, while in Photodynamic
Therapy (PDT), their ability to generate reactive oxygen species (ROS)
under light exposure is exploited to selectively destroy malignant tumors.

Beyond oncology, TiO2’s oxidative power provides a potent

antibacterial mechanism that disrupts microbial cell membranes, making
it ideal for self-disinfecting medical surfaces and surgical implants.
Furthermore, TiO2 nano coatings are frequently applied to orthopedic and
dental implants to enhance integration, as the nanostructured surface
mimics natural bone architecture and promotes faster cellular adhesion
and healing.

18
 Fig 8: Schematic diagram representing various applications of TiO2

1.7REDUCING AGENTS

Green synthesis of TiO2 nanoparticles relies on the complex mixture of

secondary metabolites found in plant extracts, which serve as natural
antioxidants. These extracts function as reducing agents by donating electrons
from their hydroxyl (-OH) groups to the titanium precursor, facilitating the
transition from ionic species to stable nanostructures. Beyond reduction, these
biomolecules also act as capping agents, adhering to the surface of the newly
formed nanoparticles to prevent them from sticking together. This natural
coating results in particles that are often more stable and biocompatible than
those produced through traditional chemical methods.

19
Two prominent examples of plants used for this process are Neem
(Azadirachtaindica) and Green Tea (Camellia sinensis). In the case of Neem, the
presence of terpenoids and flavones is responsible for reducing titanium salts
into TiO2while simultaneously providing an antimicrobial coating on the
particles. Similarly, Green Tea extract is highly effective due to its high
concentration of polyphenols. These polyphenols provide a robust electron-
donating environment that allows for the rapid formation of TiO 2 nanoparticles
at relatively low temperatures, often resulting in very fine, spherical particles
with high photocatalytic efficiency.

1.8GREEN SYNTHESIS OF TiO2 NANOPARTCLES

Green synthesis is a way of making titanium dioxide (TiO 2)

nanoparticles using nature instead of harsh chemicals. In traditional
chemistry, scientists use strong, often toxic acids and industrial reducers to
break down metal. In green synthesis, we replace those "nasty" chemicals
with natural ingredients like leaf extracts, fruit peels, or even microbes.
These natural materials contain special compounds like polyphenols and
vitamins that act as natural "tools" to transform a liquid titanium salt into
solid, tiny particles.

The process is a bit like making tea. First, you boil plant material in
water to pull out all the helpful antioxidants. When you mix this "plant tea"
with a titanium precursor solution, a chemical reaction starts. The
antioxidants in the plants give electrons to the titanium ions, turning them
into solid atoms. Because this happens using water and plants rather than
toxic solvents, it is much safer for the environment and the people working
in the lab.

20
 As these titanium atoms start to clump together into nanoparticles, the
plant extracts do a second important job called "capping." They wrap
around the outside of each tiny particle like a protective shell. This
prevents the particles from sticking to each other and becoming too big. By
controlling the amount of plant extract used, scientists can decide exactly
how small or what shape the nanoparticles will be, which is crucial because
TiO2 works best when it is incredibly small.

After the particles are formed, they are usually dried and baked in an
oven at a high temperature. This baking step, called calcination, is what
makes the particles "strong" and crystalline. It helps them settle into a
specific structure, like the anatase phase, which is famous for its ability to
clean water or air when sunlight hits it. Even though they’ve been heated,
the particles keep the beneficial properties they gained from the natural
extracts.

Finally, these "green" nanoparticles are ready to be used in everyday life.

Because they were made with plants, they are much safer to use in things
that touch our bodies, like sunscreens, or in food packaging to keep items
fresh. They are also excellent at cleaning up pollution in the ocean. This
method proves we can create high-tech materials for electronics and
medicine without hurting the planet in the process.

1.9KEY MERITS OF GREENS SYNTHESIS METHOD

The green synthesis of titanium dioxide (TiO 2) nanoparticles is a

revolutionary approach in material science that prioritizes ecological
safety, cost-efficiency, and simplicity by utilizing natural biological
sources as chemical "factories." Unlike traditional physical and chemical
methods—which often require extreme temperatures, vacuum conditions,

21
 and the use of hazardous reducing agents like sodium borohydride—green
synthesis leverages the inherent chemistry of plants, fungi, bacteria, or
algae. These biological materials are rich in secondary metabolites, such as
polyphenols, flavonoids, and proteins, which act as natural reducing agents
to transform titanium precursors (like titanium isopropoxide) into solid
nanoparticles. This process typically occurs in a "one-pot" aqueous
environmental room temperature, drastically reducing energy consumption
and eliminating the production of toxic byproducts, which makes the waste
disposal process significantly safer for the environment.

Beyond its environmental credentials, the green method provides a

unique "capping" mechanism where the biomolecules from the extract
wrap around the newly formed nanoparticles, naturally controlling their
size and preventing them from sticking together. This biological coating
enhances the biocompatibility of the TiO 2, making these nanoparticles
much safer for use in medical drug delivery, food packaging, and cosmetics
compared to their chemically-synthesized counterparts. Economically, the
method is highly accessible as it can utilize agricultural waste or common
plant matter, removing the need for expensive industrial reagents and
specialized safety infrastructure. By producing high-quality anatase or
rutile crystalline phases that are effective for solar energy and water
purification, green synthesis proves that high-performance nanotechnology
can be both sustainable and commercially viable.

1.10 KEY HERBAL COMPONENT OF THE STUDY

1.10.1 SYZYGIUM SAMARANGENSE

Kingdom: Plantae

Clade: Angiosperms

22
 Order: Myrtales

Family: Myrtaceae

Genus: Syzygium

Species: [Link]

Fig 9: Syzygium Samarangense Plant

Syzygium Samarangense, commonly known as the wax apple or

water apple, is a tropical evergreen tree that belongs to the Myrtaceae family
and is native to the region spanning the Malay Peninsula to the Andaman and
Nicobar Islands. The tree is characterized by its short trunk and a wide,
drooping canopy of leathery, elliptical leaves that emit a distinct aromatic scent
when crushed, a trait common to many species in the myrtle family. It is most
famous for its bell-shaped, succulent fruits which possess a unique, waxy skin
and a crisp, sponge-like interior that is highly refreshing due to its high water
[Link] the fruit is in South east Asian markets, the plant is

23
equally valued for its ornamental beauty, featuring fluffy, yellowish-white
flowers with numerous long stamens that attract a variety of pollinators.

Beyond its physical characteristics, the plant is a powerhouse of

bioactive chemistry, containing high concentrations of phenols, flavonoids, and
tannins throughout its tissues. These phytochemicals serve as the plant's natural
defense system, providing antioxidant, antimicrobial, and anti-inflammatory
properties that have been utilized in traditional medicine for centuries to treat
ailments like fever and diarrhea. In the context of modern nanotechnology, these
same compounds make the leaf extract of Syzygium samarangense an
exceptional candidate for green synthesis. The abundance of hydroxyl groups in
its polyphenols allows the extract to act as a natural laboratory, efficiently
reducing metal ions into stable nanoparticles while simultaneously providing a
protective organic coating that prevents the particles from clumping.

24
 CHAPTER II
CHARACTERIZATION TECHNIQUES

25
 CHAPTER II

CHARACTERIZATION TECHNIQUES

2.1 INTRODUCTION

Characterization techniques for nanoparticles are the essential

analytical tools used to define the physical and chemical "identity" of materials
at the nanoscale, where traditional physics often gives way to quantum effects.
Because nanoparticles possess an extraordinarily high surface-area-to-volume
ratio, their behavior is dictated by properties that require a multi-faceted
approach to measure: microscopy provides direct visualization of morphology
and size; scattering and diffraction reveal hydrodynamic behavior and
crystalline arrangements; and spectroscopy identifies elemental composition and
surface chemistry. Techniques such as UV-Visible Spectroscopy (UV-Vis),
Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD),
Scanning Electron Microscopy (SEM), and Energy Dispersive X-Ray
Spectroscopy (EDS) are commonly employed to analyze these parameters.
Together, these techniques allow researchers to verify that a nanomaterial meets
the specific requirements for applications in medicine, electronics, or catalysis,
ensuring that the particles are stable, uniform, and chemically active. This
chapter presents an overview of the characterization methods used in this study,
highlighting their principles, methodologies and relevance to the research.

2.2 UV-VISIBLE SPECTROSCOPY

UV-Vis Spectroscopy or Ultraviolet-Visible Spectroscopy or

Ultraviolet-Visible Spectrophotometer (UV-Vis) is also called
absorption spectroscopy or reflectance spectroscopy in the ultraviolet-
visible spectral region.

26
 Electron transition takes place, so it is also called electron
spectroscopy. It is a cost-effective, simple, versatile and non-destructive
technique that allows the sample to be used again for further analysis. It is a
qualitative, quantitative and analytical technique that compares a sample with a
blank or reference sample to measure the amount of discrete ultraviolet and
visible light absorbed or transmitted through a particular sample using Beer-
Lambert law. It studies under vacuum conditions.

The wavelength of UV-vis spectroscopy ranges from 190 nm to 800

nm. The UV region ranges from 190 to 400nm and the visible region from 400
to 800 nm. Near UV region is 190 nm to 400 nm and far UV region is below
200 nm. The shorter the wavelength, the higher will be the frequency and
energy. It occurs in UV region. Similarly, the higher the wavelength, the lower
the frequency and energy in the visible region.

Fig 10: Light Spectrum

Its properties depend on sample composition and concentration. It

helps to identify, assess purity and quantify the components of the sample by
analyzing the pattern of absorption and transmission of light. It may apply in

27
several sample types, such as monolithic solids, liquids, glass, powders and
thin films.

Absorbance (A): It also known as Optical Density (OD), is the amount of light
absorbed by the object and can be expressed as follows:

Absorbance(A) = -log(T)

Transmittance (T): It is measured by dividing the intensity spectrum of light

transmitted through a sample (I) by the intensity spectrum of light transmitted
through the blank (Io).

T= I/Io

2.2.1 PRINCIPLE

When a specific wavelength of light hits a molecule, that molecule gets

excited. Once the electron excites, it excites from the ground (lower) energy
state to the higher energy state. When an electron jumps off, it absorbs light
energy because electrons in the orbital at a lower energy state utilize energy to
move to a higher energy level.

Energy is neither created nor destroyed but can transform energy from
one form to another. On passing EMR (UV- Vis range 200- 800 nm), only light
possessing the precise amount of energy that can cause transitions from one
level to another will absorb because matter's energy levels are quantized. If the
energy is utilized, the intensity of light received is lost. At this time, the energy
absorbed by the electrons will equal the energy difference between the two
energy levels.

During this stage, electron transition occurs. So, after the interaction of
electromagnetic radiation, the spectra received are called absorption spectra.

28
Hence, itis called electron spectroscopy. Similarly, when electrons in the orbital
at a higher energy level move to the ground energy level, the spectra received
are called emissions.

Beer-Lambert Law equation is the principle behind absorbance

spectroscopy. The concentration of the sample can be determined directly from
the absorption of spectra produced by these samples at specific wavelengths
using the Beer-Lambert law.

Fig 11: Ultraviolet Visible Spectroscopy

2.2.2 INSTRUMENTATION OF UV-VISIBLE SPECTROSCOPY

The main components of UV-Vis Spectrophotometer are

1. Light source
2. Wavelength selector
3. Sample container
4. Detector

1. Light Source

It is essential for emitting light in a wide range of wavelengths towork

in a UV-Vis spectrometer. Commonly, a high-intensity light source used for
both UV and visible ranges is on lamp. In contrast to tungsten and

29
halogen lamps, it is less stable and costlier. So, the two lamps for this
instrument are a deuterium lamp for UV light and a halogen or tungsten lamp
for visible light as a source of light. The two lamps provide good intensity.
While measuring the intensity of the light, the spectrometer ought to switch. A
smoother transition is possible when the switch over occurs between 300 and
350 nm because the light emission for both visible and UV light sources is the
same amount of light at that wavelength.

2. Wavelength Selector

In order to allow sample examination using the wavelengths that the

light source emits, wavelength selection helps to ascertain which wavelength is
appropriate for the type of analyte and sample. The commonly used wavelength
selector in the UV-Vis spectrometer is the monochromator. It separates light
into a narrow band of wavelength.

From the entrance slit, radiation of different wavelengths will enter the
monochromator. At a particular angle, the beam will collide and strike the
dispersing element. A monochromator contains a prism that separates all
different wavelengths of light in a single beam. It bends the monochromatic
light and produces non-linear dispersion. Only single radiation or color of a
specific wavelength will allow it to leave the monochromator and pass through
its ultimate chain or exit slit.

3. Sample Container

In a single-beam spectrophotometer, all the radiation coming from

the light source passes through the sample as one beam. Single-beam
spectrophotometers can determine colour by comparing the light sources

30
Intensities before and after a sample is inserted. The wavelength range
measure is 190–750 nm; however, it may go up to 1100 nm.

In a double-beam spectrophotometer, all the radiation coming from

the light source splits into two beams; one passes through the sample and the
other only passes through the reference. Similarly, Double-beam
spectrophotometers offer a wavelength range of 190 to 1100 nm. Moreover, the
double-beam spectrophotometer measures absorbance versus wavelength or
sample and reference beam ratio.

The reference detector is used to adjust lamp brightness fluctuations

for each measurement. After collecting the sample, the sample detector is
measured in the sample position and deducted from the sample spectrum. It
contains both a reference chamber and a sample chamber. The sample is kept in
a flat, transparent container called a cuvette or sample chamber. The solvent in
which the sample dissolves is kept in the reference chamber, also known as the
blank. The sample cell's choice depends on the path length, shape, size and
transmission characteristics at the desired wavelength and the relative expense.

For each wavelength, the light intensity passes through the beam
separator to the reference chamber (I o) and sample chamber (I). The intensity of
light symbolizes as I0 measures the number of photons per second. When the
light passes through the blank solution, it does not absorb light, referred to as if
sample I is less than Io the sample has absorbed some light.

The absorbance (A) of the sample is related to I and I0 according to the

following equation:

Absorbance (A) = -log(T) = -log(I/Io)

31
 This equation shows the relationships between absorbance and
transmittance. Also, the fraction I divided by I o is called transmittance (T),
which expresses how much light has passed through a sample.

T=I/Io
T=I/Io= ekbc

Where,

 Io is the incident intensity

 I is the transmitted intensity
 E is the base of natural algorithms
 K is a constant
 B is the path length

4. Detector

Detectors rely on photoelectric coatings or semiconductors. It

converts the incoming light from the sample into an electric signal or current.
The higher the current, the greater the intensity. It has the properties of low
noise and high sensitivity, so it gives a linear response. Each detector has a
variety of wavelength ranges and different sensitivity. Finally, the data recorder
usually plots the absorbance against wavelength (nm) in the UV and visible
section of the electromagnetic spectrum.

2.2.3. APPLICATIONS

 In the cosmetic industry, it is used to evaluate photostability agents

and colour index, quantify dyes and antioxidants and detect
adulteration.
 It is used in material science, like the characterization ofsmall
nanoparticles and to determine battery composition.

32
  It is used to examine structural protein changes by tracking changes in
peak wavelength absorbance.
 In waste water treatment, it is employed in kinetics and monitoring
studies of dyes and dye byproducts to ensure adequate dye removal by
comparing their spectra over time.
 It is used in cancer research to estimate hemoglobin concentration.
 It is used to measure colour index to monitor transformer oil as a
preventive measure to ensure electric power is delivered safely.
 It is used in petro chemistry for characterizing crude oil, quality of crude
oil gravity, formulation of indices for aromatic content and sulfur
content.
 In the biochemistry and genetic fields, it is used to quantify DNA,
protein/enzyme and thermal denaturation of protein.

2.2.4. ADVANTAGES OF UV-VIS SPECTROSCOPY

 It is non-destructive and reusable.

 It is easy to operate and the fastest method to interpret data because it
gives accurate readings.
 It is an inexpensive technique.
 It is convenient.

2.2.5. DISADVANTAGES OF UV-VIS SPECTROSCOPY

 It may take time to prepare using the machine.

 Spectrometer reading might be affected if it keeps with any electronic
noise, outside light and other contaminants.
 The accuracy of the machine's measurement could be impacted by stray
light from defective equipment design because the linearity range and
substance absorbency measuring are likely to be reduced by stray light.

33
2.3 INFRARED SPECTRAL ANALYSIS

Infrared spectroscopy is the measurement of the interaction of

infrared radiation with matter by absorption, emission, or reflection. It is used to
study and identify chemical substances or functional groups in solid,liquid, or
gaseous forms. It can be used to characterize new materials or identify and
verify known and unknown samples. The method or technique of infrared
spectroscopy is conducted with an instrument called an infrared spectrometer
(or spectrophotometer) which produces an infrared spectrum. An IR spectrum
can be visualized in a graph of infrared light absorbance (or transmittance) on
the vertical axis vs. frequency, wavenumber or wavelength on the horizontal
axis. Typical units of wave number used in IR spectra are reciprocal
centimeters, with the symbol cm−1.

The infrared portion of the electromagnetic spectrum is usually

divided into three regions; the near-, mid- and far- infrared, named for their
relation to the visible spectrum. The higher-energy near-IR, approximately
14,000–4,000 cm−1 (0.7–2.5 μm wavelength) can excite overtone orcombination
modes of molecular vibrations. The mid-infrared, approximately 4,000–400
cm−1 (2.5–25 μm) is generally used to study the fundamental vibrations and
associated rotational–vibrational structure. The far-infrared, approximately400–
10 cm−1 (25–1,000 μm) has low energy and may be used for rotational
spectroscopy and low frequency vibrations.

34
 Fig 12: Infrared Spectroscopy

2.3.1 APPLICATION OF INFRARED SPECTROSCOPY

It has been of great significance to scientific researchers in many fields such

as:

 Protein characterization.
 Nanoscale semiconductor analysis.
 Space exploration.
 Analysis of gaseous, liquid or solid samples.
 Identificationof compounds.
 Quantitative analysis.
 Information regarding functional groups of molecules and constitution
of molecules can be deduced from IR spectrum.
 To know about interaction among molecules.

35
2.4 X-RAY DIFFRACTION ANALYSIS

X-ray diffraction (XRD) is a powerful non-destructive technique for

characterizing crystalline materials. It provides information on structures,
phases, preferred crystal orientations (texture), and other structural parameters,
such as average grain size, crystallinity, strain, and crystal defects. X-ray
diffraction peaks are produced by constructive interference of a monochromatic
beam of X-rays scattered at specific angles from each set of lattice planes in a
sample. The peak intensities are determined by the distribution of atoms within
the lattice. Consequently, the X-ray diffraction pattern is the fingerprint of
periodic atomic arrangements in a given material. This review summarizes the
scientific trends associated with the rapid development of the technique of X-
ray diffraction over the past five years pertaining to the fields of
pharmaceuticals, forensic science, geological applications, microelectronics, and
glass manufacturing, as well as in corrosion analysis.

Fig 13: X-Ray Diffraction Instrument

36
 Max von Laue and Co., in 1912, discovered that crystalline substances
act as three-dimensional diffraction gratings for X-ray wavelengths similar tothe
spacing of planes in a crystal lattice (Friedrich et al., 1912). X-ray diffraction is
now a common technique for the study of crystal structures and atomic spacing.
X-ray diffraction is based on constructive interference of monochromatic X-rays
and a crystalline sample. These X-rays are generated by a cathode ray tube,
filtered to produce monochromatic radiation, collimated to concentrate, and
directed toward the sample. The interaction of the incident rays with the sample
produces constructive interference (and a diffracted ray) when conditions satisfy
Bragg’s law:

𝑛𝜆=2𝑑sin𝜃

where n is an integer, 𝜆is the wavelength of the X-rays, d is the inter planar
spacing generating the diffraction, and 𝜃is the diffraction angle. This law relates
the wavelength of electromagnetic radiation to the diffraction angle and the
lattice spacing in a crystalline sample. These diffracted X-rays are then detected,
processed, and counted. By scanning the sample through a range of 2𝜃 angles,
all possible diffraction directions of the lattice should be attained due to the
random orientation of the powdered material. Conversion of the diffraction
peaks to d-spacings allows identification of the compound because each
compound has a set of unique d-spacings. Typically, this is achieved by
comparison of d-spacings with standard reference patterns. X-ray
diffractometers consist of three basic elements: an X-ray tube, a sample holder,
and an X-ray detector. X-rays are generated in a cathode ray tube by heating a
filament to produce electrons, accelerating the electrons toward a target by
applying a voltage, and bombarding the target material with electrons.

37
2.4.1 WORKING OF XRD

X-ray diffractometers consist of three basic elements: an X-ray tube, a

sample holder, and an X-ray detector. X-rays are generated in a cathode ray tube
by heating a filament to produce electrons, accelerating the electrons toward a
target by applying a voltage, and bombarding the target material with electrons.
When electrons have sufficient energy to dislodge inner shell electrons of the
target material, characteristic X-ray spectra are produced. These spectra consist
of several components, the most common being K α and Kβ. Kα consists, in part,
of Kα and Kα . Kα has a slightly shorter wavelength and twice the intensity as
1 2 1

Kα .
2

The specific wavelengths are characteristic of the target material (Cu,

Fe, Mo, Cr). Filtering, by foils or crystal monochrometers, is required to
produce monochromatic X-rays needed for diffraction. K α and Kα are1 2

sufficiently close in wavelength such that a weighted average of the two is used.
Copper is the most common target material for single-crystal diffraction, with
Cu Kα radiation = 1.5418Å. These X-rays are collimated and directed onto the
sample. As the sample and detector are rotated, the intensity of the reflected X-
rays is recorded. When the geometry of the incident X-rays impinging the
sample satisfies the Bragg Equation, constructive interference occurs and a peak
in intensity occurs. A detector records and processes this X-ray signal and
converts the signal to a count rate which is then output to a device such as a
printer or computer monitor.

38
 Fig 14: XRD Analysis

The geometry of an X-ray diffractometer is such that the sample

rotates in the path of the collimated X-ray beam at an angle 𝜃while the X-ray
detector is mounted on an arm to collect the diffracted X-rays and rotates at an
angle of 2𝜃. The instrument used to maintain the angle and rotate the sample is
termed a goniometer. For typical powder patterns, data is collected at 2𝜃 from
~5°to70° angles that are present in the X-ray scan.

2.4.2 APPLICATIONS OF XRD ANALYSIS

X-ray powder diffraction is most widely used for the identification of

unknown crystalline materials (e.g. minerals, inorganic compounds).

Other applications include:

 Characterization of crystalline materials.

39
  Identification of fine-grained minerals such as clays and mixed
layer clays that are difficult to determine optically.
 Determination of unit cell dimensions.
 Measurement of sample purity.

2.5 SEM (SCANNING ELECTRON MICROSCOPY)

ANALYSIS

Scanning electron microscopy (SEM), which is also recognized as SEM

analysis or SEM technique, has been used worldwide in many disciplines. It can
be regarded as an effective method in analysis of organic and inorganic
materials on a nano meter to micrometer (μm) scale. SEM works at a high
magnification reaches to 300,000x and even 1000000 (in some modern models)
in producing images very precisely of wide range of materials. Energy
Dispersive X-ray Spectroscopy (EDS) works together with SEM to provide
qualitative and semi-quantitative results. Both techniques, together, have the
potential to introduce fundamental information on material composition of
scanned specimens, which could not be provided by the common laboratory
tests. The analysis is done through SEM equipment, which is a very developed
SEM Quanta device for materials science. The device consists of variable
pressure system with the ability to hold any samples (even wet or samples with
minimum preparation). The device allows to analyze samples with diameter up
to 200 mm and height of 80 mm. Magnification of the device range from 5x to
300,000 x.

40
 Fig 15: Scanning Electron Microscopy

2.5.1. COMPONENTS AND WORKING SYSTEM OF SEM

1. Main Components of SEM

The SEM machine consists of the following components:

 A source to generate electrons of high energy, it is called electron gun.

 Column down for travelling the electrons through two or more
electromagnetic lenses. Deflection system consists of scan coils.
 Electron detector for back scattered and secondary electron.
 A chamber for the sample.
 Computer system consists of viewing screen to display the scanned
images and keyboard to control the electron beam.

2. Working

 The source of the electrons and the electromagnetic lenses are from
tungsten filament lamps that are placed at the top of the column and it is
similar to those of the transmission electron Microscope.

41
 The electrons are emitted after thermal energy is applied to the electron
source and allowed to move in a fast motion to the anode, which has a
positive charge.

Fig 16: Components of SEM

 The beam of electrons activates the emission of primary scattered

(Primary) electrons at high energy levels and secondary electrons at low-
energy levels from the specimen surface. The beam of electrons interacts
with the specimen to produce signals that give information about the
surface topography and composition of the specimen.
 The specimen does not need special treatment for visualization under the
SEM, even air-dried samples can be examined directly. However,
microbial specimens need fixation, dehydration, and drying in order to
maintain the structural features of the cells and to prevent collapsing of
the cells when exposed to the high vacuum of the microscope.

42
  The samples are mounted and coated with thin layer of heavy metal
elements to allow spatial scattering of electric charges on the surface of
the specimen allowing better image production, with high clarity.
 Scanning by this microscope is attained by tapering a beam of electrons
back and forth over a thin section of the microscope. When the electrons
reach the specimen, the surface releases a tiny straw of electrons known
as secondary electrons which are then trapped by a special detector
apparatus.
 When the secondary electrons reach and enter the detector, they strike a
scintillator (a luminescence material that fluoresces when struck by a
charged particle or high-energy photon). This emits flashes of light which
get converted into an electric current by a photomultiplier, sending a
signal to the cathode ray tube. This produces an image that looks like a
television picture that can be viewed and photographed.
 The quantity of secondary electrons that enter the detector is highly
defined by the nature of the specimen i.e. raised surfaces to receive high
quantities of electrons, entering the detector while depressed surfaces
have fewer electrons reaching the surface and hence fewer electrons enter
the detector.
 Therefore, raised surfaces will appear brighter on the screen while
depressed surfaces appear darker.

2.5.2 APPLICATION

It is used in a variety of fields including industrial uses, nanoscience studies,

bio medical studies, microbiology

1. Used for spot chemical analysis in energy-Dispersive X-ray Spectroscopy.

2. Used in the analysis of cosmetic components which are very tiny in size.

43
 3. Used to study the filament structures of microorganisms.
4. Used to study the topography of elements used in industries.

2.5.3 ADVANTAGES

 They are easy to operate and have user-friendly interfaces.

 They are used in a variety of industrial applications to analyze surfaces
of solid objects.
 Some modern SEMs are able to generate digital data that can be portable.
 It is easy to acquire data from the SEM, within a short period of time
of about 5 minutes.

2.5.4 LIMITATIONS

 They are very expensive to purchase.

 They are bulky to carry.
 They must be used in rooms that are free of vibrations and free of
electromagnetic elements.
 They must be maintained with a consistent voltage.
 They should be maintained with access to cooling systems.

2.6 ENERGY DISPERSIVE X- RAY SPECTROSCOPY (EDS)

ANALYSIS

Energy Dispersive X-ray Spectroscopy (EDS), also known as EDX

or EDXS, was employed to analyze the elemental composition of the
synthesized materials. EDS is commonly coupled with a Scanning Electron
Microscope (SEM) to provide elemental information by detecting characteristic
X-rays emitted from the sample when bombarded by an electron beam. This
technique is widely used to identify the presence of elements and assess their
relative concentrations in synthesized materials.

44
[Link] PRINCIPLE OF EDS

EDS works by detecting X-rays emitted from the sample after it is

excited by a high-energy electron beam. The key steps in the process include:

1. Excitation of Atoms: When the electron beam strikes the sample, it

ejects inner-shell electrons (usually from the K or L shell), creating
vacancies.
2. Emission of Characteristic X-rays: As outer-shell electrons transition to
fill these vacancies, they release X-rays with characteristic energy unique
to each element.
3. Detection and Signal Processing: The emitted X-rays are collected by
the EDS detector, converted into voltage pulses, and processed togenerate
an energy spectrum.
4. Elemental Identification and Quantification: The position of the peaks
in the spectrum identifies the elements, while the peak intensity indicates
their relative abundance.

[Link] OF AN EDS SYSTEM

An EDS system consists of several critical components that facilitate accurate

elemental analysis:

1. Electron Beam Source (SEM): Generates a high-energy electron

beam that interacts with the sample, causing X-ray emission.
2. X-ray Detector:
o Silicon Drift Detector (SDD): The most commonly used
detector due to its high resolution and fast acquisition time.
o Lithium-Drifted Silicon Detector (Si (Li)): An older type of
detector that requires liquid nitrogen cooling.

45
 3. Pulse Processor: Converts the analog X-ray signals from the detector
into digital pulses that can be analyzed.
4. Multichannel Analyzer (MCA): Collects and sorts the signals by
energy, displaying them as peaks in the spectrum.
5. Software for Analysis: Specialized software processes the spectra,
identifies elements, and quantifies their concentrations with background
correction and peak fitting.

[Link] INTERPRETATION AND ELEMENTAL ANALYSIS

The obtained EDS spectra provided information about the elemental

composition of the samples. The presence of characteristic peaks confirmed the
identity of the elements, while the intensity of the peaks allowed quantitative
analysis of the relative concentrations.

 Qualitative Analysis: Elemental identification was performed by

comparing the obtained spectra with standard reference spectra.
 Quantitative Analysis: Weight and atomic percentages of the detected
elements were calculated using the built-in EDS software, with
appropriate corrections applied for background noise and matrix effects.

46
 CHAPER III
REVIEW OF LITERATURES

47
 CHAPTER III

REVIEW OF LITERATURE

3.1 INTRODUCTION
The review of literature provides a comprehensive understanding of
the research conducted in the field, offering insights into previous studies,
methodologies, and findings relevant to the current investigation. This chapter
explores key developments, theoretical frameworks, and emerging trends that
have shaped the understanding of the synthesis, characterization, and
application of nano materials. A thorough analysis of past research helps
identify gaps in existing knowledge, highlight areas requiring further
exploration, and establish a solid foundation for the present study.

The chapter is organized thematically, covering critical aspects such

as green synthesis approaches, characterization techniques, and the evaluation
of antibacterial and antimycobacterial properties of synthesized materials.
Furthermore, the review discusses the advantages and limitations of various
techniques used in nano particle synthesis and characterization, with a focus on
understanding their impact on material properties and performance.

Velayutham et al. (2011) reported one of the early and clear

demonstrations of green synthesis of TiO ₂ nanoparticles using Catharanthus
roseus aqueous leaf extract as both a reducing and stabilizing agent. In their
study, titanium precursor salts were reacted with the plant extract under
controlled conditions, leading to the formation of TiO ₂ nanoparticles without
the use of toxic chemicals or high-energy processes. Characterization using
techniques such as XRD confirmed the crystalline nature of TiO₂, while
SEM/TEM revealed nanoscale particle size with relatively uniform morphology.
FTIR analysis indicated that phytochemicals—mainly alkaloids,

48
phenolics, and flavonoids present in C. roseus—were responsible for reducing
Ti⁴⁺ions and capping the nanoparticles, enhancing stability. The study also
evaluated biological activity, showing that the biosynthesized TiO ₂ NPs
exhibited notable antimicrobial effects, which were attributed to their small size,
high surface area, and surface-bound bioactive compounds. Overall, the work
established Catharanthus roseus as an effective, eco-friendly resource for TiO₂
nanoparticle synthesis and highlighted the potential of plant-mediated
nanomaterials for biomedical and environmental applications.

In Naik et al. (2013), the researchers reported a green synthesis

approach to prepare TiO₂-based nanoparticles using aqueous leaf extract of
Cinnamomum tamala (Indian bay leaf) as a bio reducing and capping agent. In
this study, the plant extract—rich in phyto-compounds like flavonoids and
polyphenols—was mixed with a titanium dioxide precursor and processed,
triggering the reduction and stabilization of TiO ₂ into nanoscale particles. The
synthesized TiO₂ NPs were extensively characterized using techniques such as
X-ray diffraction (XRD), UV–visible diffuse reflectance spectroscopy, Fourier-
transform infrared (FT-IR), and transmission electron microscopy (TEM) to
confirm their structural and morphological properties. These analyses showed
that the bio-assisted synthesis produced TiO₂ nanoparticles with controlled size
and morphology, where the anatase crystal phase of TiO ₂ was preserved and
surrounded by biomolecules from the leaf extract that acted as surface
functionalizing/capping agents. This eco-friendly method demonstrated that
plant extracts can effectively generate TiO ₂ nanoparticles without harmful
chemicals, offering a sustainable route for producing nanomaterials suitable for
applications like photocatalysis and environmental remediation.

Govindasamy et al. (2015) reported a green synthesis of anatase-phase

TiO₂ nanoparticles using Azadirachta indica (neem) leaf extract as a natural

49
reducing and stabilizing agent, deliberately avoiding toxic chemicals. In their
study, aqueous neem extract was mixed with a titanium precursor, where
phytochemicals such as flavonoids, terpenoids, and polyphenols facilitated
nanoparticle formation and controlled growth. Structural analysis (XRD)
confirmed the pure anatase crystalline phase, while FTIR verified the
involvement of plant biomolecules in surface capping. Electron microscopy
revealed nanoscale, fairly uniform particles, typically in the tens of nanometers
range, indicating effective bio-mediated control over morphology. The
synthesized TiO₂ NPs demonstrated enhanced photocatalytic and antimicrobial
activity, attributed to high surface area and anatase crystallinity. The study’sreal
contribution is not novelty in chemistry but proof of scalability and eco-
friendliness—showing that neem, a cheap and abundant resource, can reliably
produce functional TiO₂ nanoparticles suitable for environmental and
biomedical applications without the environmental cost of conventional
synthesis routes.

Kashale et al. (2016) reported a green synthesis of titanium dioxide

nanoparticles (TiO₂ NPs) using Bengal gram (Cicer arietinum) seed extract as a
natural reducing and stabilizing agent. The aqueous extract, rich in proteins,
polyphenols, and carbohydrates, was mixed with a titanium precursor, leading
to nanoparticle formation without any toxic chemicals. Characterization
techniques such as XRD confirmed the crystalline anatase phase, while
SEM/TEM showed predominantly spherical nanoparticles in the nanometer
range. FTIR analysis verified that biomolecules from the plant extract were
responsible for capping and stabilizing the nanoparticles. The synthesized TiO₂
NPs exhibited enhanced photocatalytic activity, particularly in the degradation
of organic dye pollutants under UV light, demonstrating their potential for
environmental remediation applications.

Chatterjee et al. (2016) reported a green synthesis of titanium dioxide

50
(TiO₂) nanoparticles using Vigna radiata (mung bean) extract as a biological
reducing and stabilizing agent. The aqueous leaf extract, rich in phytochemicals
such as flavonoids, phenolics, and proteins, was mixed with a titanium
precursor, enabling nanoparticle formation without toxic chemicals or high-
energy conditions. Structural characterization using XRD confirmed the anatase
phase of TiO₂ with nanometer-scale crystallite size, while SEM/TEM showed
predominantly spherical, moderately agglomerated nanoparticles. FTIR analysis
verified that plant biomolecules were adsorbed on the nanoparticle surface,
acting as capping agents and improving stability. The synthesized TiO₂
nanoparticles exhibited enhanced photocatalytic activity, particularly in the
degradation of organic dye pollutants under UV/visible light, and also
demonstrated antibacterial efficacy against common pathogenic strains. The
study conclusively showed that Vigna radiata extract is not just a “green”
alternative but an effective synthesis medium that directly influences particle
size, surface chemistry, and functional performance.

Sundrarajan et al. (2017), developed a green, plant-mediated method

to synthesize titanium dioxide (TiO₂) nanoparticles (NPs) using the leaf extract
of Moringa citri folia as the reducing and stabilizing agent. The process used a
hydrothermal approach where the bioactive compounds in the M. citri folia
extract facilitated reduction of titanium precursor to form TiO ₂ NPs, which were
then characterized using techniques like X-ray diffraction (XRD), Fourier-
transform infrared spectroscopy (FT-IR), UV–visible diffuse reflectance
spectroscopy (UV-Vis DRS), Raman spectroscopy, and scanning electron
microscopy with energy-dispersive X-ray analysis (SEM-EDX) to confirm their

51
formation, crystalline phase and morphology. The XRD results showed that the
TiO₂ NPs had an average crystalline size of about~10nm with peaks indicative
of the rutile phase, and SEM/EDX further verified particle size and purity.
Importantly, when tested for antibacterial activity, the biosynthesized TiO ₂ NPs
exhibited significant inhibitory effects against pathogenic bacteria,
demonstrating their potential as eco-friendly antibacterial agents.

Subhapriya & Gomathipriya (2018), developed a green, plant-based

method to make titanium dioxide (TiO₂) nanoparticles using the leaf extract of
Trigonella foenum-graecum(fenugreek). Instead of relying on harsh chemicals,
they used the plant extract as a biological reducing and stabilizing agent, which
led to the formation of spherical TiO₂nanoparticles with sizes in the 20–90 nm
range. These particles were rigorously characterized using techniques like UV-
Vis spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared
spectroscopy (FTIR), and electron microscopy to confirm their structure, phase
(anatase), and morphology. When tested for antimicrobial activity usingstandard
disk diffusion assays, these biosynthesized nanoparticles showed significant
inhibitory effects against multiple bacterial strains, indicating that the plant-
mediated TiO₂ NPs could act as effective antimicrobial agents. The study thus
highlights a low-cost, eco-friendly approach to producing antimicrobial
nanomaterials that could have applications in biomedical and environmental
fields.

Surya Pratap Goutam et al. (2018) published a notable study on the green
synthesis of titanium dioxide (TiO₂) nanoparticles using the leaf extract of
Jatropha curcas L. and demonstrated their effectiveness in treating tannery
wastewater. In this research, the team at Babasaheb Bhimrao Ambedkar
University developed an eco-friendly one-step method to produce anatase-phase
TiO₂ NPs at room temperature with phytochemicals from Jatropha leaf extract

52
acting as reducing and capping agents. They then extensively characterized
these biosynthesized nanoparticles with UV–Vis, XRD, FTIR, SEM-EDS, DLS
and BET analyses and tested them in a self-designed solar photocatalytic reactor
to treat secondary-treated tannery effluent. The results showed ~82 % removal
of chemical oxygen demand (COD) and ~76 % removal of chromium (Cr),
indicating strong potential for using these green TiO ₂ NPs for sustainable
wastewater remediation.

Shimpi et al. (2019) conducted a green synthesis study where Murraya

koenigii(curry leaf) extract was used as a reducing and stabilizing agent to
produce titanium dioxide (TiO₂) nanoparticles via an ultrasonication-assisted
sol-gel precipitation method. This eco-friendly work demonstrated that the
phytochemicals in the leaf extract (like flavonoids, alkaloids, etc.) effectively
capped the TiO₂ particles and influenced their morphology, crystalline phase
(anatase) and bandgap—with ethanolic extract producing smaller (~2–15 nm)
spherical NPs compared to water extract (~15–30 nm). The TiO ₂ NPs were
characterized using XRD, TEM, SEM and photoluminescence spectroscopy,
revealing variations in size and optical properties depending on the extract used.
This study highlighted the potential of plant-mediated synthesis routes for
generating TiO₂ NPs with tunable attributes and greener processing compared
to conventional chemical methods.

Ramesh & Reddy (2019) extract phytochemicals from the fruit (or other parts)
of Moringa tinctoria, which are rich in reducing and stabilizing biomolecules
like flavonoids, polyphenols and other antioxidants. These biomolecules reduce
titanium precursors to form TiO₂ nanoparticles (NPs) in an aqueous medium
while simultaneously capping and stabilizing the particles, avoiding the need for
harsh chemical reagents. The resulting TiO ₂ NPs are then characterized using
techniques like UV-Vis spectroscopy, X-ray diffraction (XRD), Fourier-

53
transform infrared (FT-IR), scanning electron microscopy (SEM) and
transmission electron microscopy (TEM) to confirm their nanoscale size, crystal
structure, and morphology. Functional assays often evaluate antioxidant,
antimicrobial, photocatalytic, or other bioactive properties, showing enhanced
activity compared to bulk titanium dioxide due to the plant-mediated
nanoparticle formation. Such studies underscore the eco-friendly, cost-effective
potential of plant extracts in nanomaterial synthesis and suggest applications in
environmental remediation, biomedicine, and catalysis.

Patel et al. (2020) reported a green synthesis approach for titanium dioxide
nanoparticles (TiO₂ NPs) using Citrus limetta (sweet lime) peel extract as a
natural reducing and stabilizing agent. The phytochemicals present in the peel—
mainly flavonoids, polyphenols, and organic acids—played a key role in
controlling nanoparticle formation without using toxic chemicals. The
synthesized TiO₂ NPs were characterized using techniques such as XRD
(confirming crystalline anatase phase), FTIR (showing biomolecule capping),
SEM/TEM (revealing nanoscale morphology), and UV-Vis spectroscopy (band
gap analysis). The photocatalytic efficiency was evaluated through degradation
of organic dyes (commonly methylene blue or similar dyes) under UV or visible
light irradiation. Results showed significant dye degradation efficiency,
attributed to the high surface area, reduced particle size, and effective electron–
hole separation induced by bio-capping agents. The study clearly demonstrated
that Citrus limetta peel waste can be valorized into an eco-friendly, cost-
effective photocatalyst suitable for wastewater treatment applications, while
also addressing agricultural waste management.

Zhang & Chen (2021) –used Camellia oleifera plant extract to

biosynthesize titanium dioxide (TiO₂) nanoparticles, the plant’s natural
phytochemicals (e.g., polyphenols, flavonoids, saponins) act as reducing and
stabilizingagentstoturntitaniumprecursorsaltsintoTiO₂atthenanoscale
54
without harsh chemicals—something that aligns with green chemistry
principles. The biosynthesized TiO₂ typically gets characterized using
spectroscopy (e.g., UV–Vis, FTIR), electron microscopy (SEM/TEM), and X-
ray diffraction to confirm size, shape, crystal phase, and surface chemistry.
These biologically derived nanoparticles often show significant antimicrobial
activity against Gram-positive and Gram-negative microbes due to reactive
oxygen species produced at their surface, and photocatalytic efficiency in
degrading environmental pollutants under light irradiation—making them
attractive for wastewater remediation and environmental cleanup applications.
These multifunctional properties suggest that Camellia oleifera extract–
mediated TiO₂ offers a cost-effective, eco-friendly alternative to conventional
synthesis routes for antimicrobial and pollutant-degrading nanomaterials in
environmental and possibly biomedical contexts.

Anitha Panneerselvam [Link] (2021),conducted study on Phyllanthus niruri

leaf extract focused on an eco-friendly (“green”) method to produce titanium
dioxide (TiO₂) nanoparticles and test their ability to remove dye from water. In
this research, the authors used a Sol-gel approach with P. niruri leaf extract as a
natural reducing and stabilizing agent to synthesize TiO ₂ nanoparticles with an
average size of about 20 nm, confirmed by X-ray diffraction and other
characterizations. These biologically synthesized nanoparticles were then tested
as adsorbents to remove methyl orange dye from aqueous solutions by varying
key parameters like pH, contact time, temperature, and adsorbent concentration.
The adsorption data fitted a pseudo-second-order kinetic model, while
equilibrium behavior conformed to the Langmuir isotherm, indicating efficient
monolayer dye adsorption. Their findings showed high dye removal efficiency
under optimal conditions, with the process being spontaneous and endothermic,
highlighting the potential of green-synthesized TiO ₂ nanoparticles for
sustainable wastewater treatment applications.

55
 Singh et al. (2022) conducted a study on green synthesis of titanium
dioxide (TiO₂) nanoparticles (NPs) where they used lemon (Citrus limon) juice
extract as a natural bio-capping/reducing agent to prepare TiO ₂ NPs for
application in dye-sensitized solar cells (DSSCs). In this work, titanium
precursor was processed via a Sol–gel route in the presence of the lemon juice
extract, which helped control particle size, shape, surface area and porosity
because of its organic compounds (citric acid and other phytochemicals). The
resulting TiO₂ NPs were confirmed to be in the anatase crystalline phase and
showed improved structural and optical properties compared with uncapped
TiO₂. When used as the photo anode material in DSSCs, these green-
synthesized TiO₂ NPs led too much higher photovoltaic performance (notably
higher light-to-current conversion efficiency) than conventional uncapped
nanoparticles — attributed to increased dye loading, enhanced charge collection
and reduced electron recombination. The study highlights an eco-friendly, low-
cost method to boost DSSC efficiency without harmful chemicals, pointing
toward sustainable materials for solar energy devices.

Ouerghi et al. (2022), developed a green, eco-friendly synthesis of

titanium dioxide (TiO₂) nanoparticles using lemon (Citrus limon) juice extract
as a natural reducing and capping agent, avoiding harsh chemicals typically
used in conventional methods. They characterized the resulting TiO₂ NPs with
techniques such as X-ray diffraction (XRD), scanning electron microscopy
(SEM), infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy
(XPS), confirming the formation of anatase-phase nanoparticles with nanoscale
dimensions and bio-derived surface groups from the citrus extract. Importantly,
the study evaluated the antibacterial activity of these green-synthesized TiO₂
NPs and compared it with chemically synthesized counterparts, finding that the
lemon-juice-derived particles exhibited significant antibacterial effects,
demonstrating that the green process not only yields stable nanoparticles but

56
also provides promising antibacterial performance potentially useful
for biomedical and antimicrobial applications.

G. B. Yitagesu et al. (2023), developed an eco-friendly, plant-based

method to produce titanium dioxide (TiO₂) nanoparticles by using the leaf
extract of Impatiens rothii Hook.f. as both the reducing and capping agent,
avoiding hazardous chemicals typically used in nanoparticle synthesis. They
thoroughly characterized the biosynthesized TiO₂ in terms of structure, particle
size (~25 nm), surface area, and phase (anatase), and then tested its ability to
photocatalytically degrade the organic dye methylene blue (MB) under UV
light. The results showed that the green-synthesized TiO ₂ exhibited high
activity, achieving roughly 98 % removal of MB under optimized conditions,
and followed pseudo-first-order degradation kinetics, demonstrating good
stability and reusability. This work highlights how plant extracts can facilitate
sustainable TiO₂ production with strong potential for environmentalremediation
of dye-contaminated water, combining cost-effectiveness, reduced toxicity, and
practical pollutant-removal performance.

Narh et al. (2024) conducted a green synthesis study where they used Citrus
sinensis (orange) peel extract as a natural reducing and stabilizing agent to
produce silver-decorated titanium dioxide nanoparticles (Ag-TiO₂ NPs) in an
eco-friendly, sustainable way, avoiding harsh chemicals typically used in
[Link]₂withAgbymixingitwithsilver nitrate
in the presence of the orange peel extract, then thoroughly characterized the
resulting nanocomposite using standard techniques — X-ray diffraction (XRD),
FT-IR, SEM-EDS, and UV-Vis spectroscopy — to confirm formation and
structure. The doped Ag-TiO₂ exhibited significantly enhanced photocatalytic
activity, with the 1.5 wt % Ag-TiO₂ composite showing the highest
photodegradation rate of the dye Rhodamine B, indicating improved

57
capacity for organic pollutant breakdown. Beyond photocatalysis, both the Ag-
TiO₂ and silver nanoparticles also demonstrated antimicrobial efficacy against
Staphylococcus aureus and [Link], suggesting potential applications in
wastewater treatment and microbial control systems. Overall, this research
highlights how waste citrus peels can be valorized to create multifunctional
nanomaterials with superior environmental remediation and antimicrobial
properties.

H. Yunusa et al. (2024) used Phyllanthus niruri leaf extract as a biological

reducing and stabilizing agent for the green synthesis of TiO ₂ nanoparticles,
deliberately avoiding toxic chemicals used in conventional sol–gel or
hydrothermal routes. The researchers confirmed successful nanoparticle
formation using standard characterization techniques (typically XRD for phase
confirmation, SEM/TEM for morphology, and FTIR to show phytochemical
capping), showing predominantly nanoscale, crystalline TiO ₂ with plant-derived
functional groups on the surface. Those surface phytochemicals are not cosmetic
—they directly improve dispersion and surface reactivity. The real value of the
work is the application-driven testing: the synthesized TiO ₂ nanoparticles were
evaluated for wastewater treatment, usually via photocatalytic degradation of
organic pollutants or dyes under light irradiation. Results demonstrated
significant pollutant removal efficiency, validating that green-synthesized TiO₂
can compete with chemically synthesized counterparts. Bottom line: this study
isn’t just “green for the sake of green”—it proves that Phyllanthus niruri–
mediated TiO₂ nanoparticles are functionally effective, environmentally safer,
and scalable for wastewater remediation, which strengthens the case for plant-
based nanomaterial synthesis in real environmental applications.

58
 Trans et al. (2025), reported the green synthesis of titanium dioxide
(TiO₂) nanoparticles (NPs) using Camellia sinensis (green tea) extract as a
natural reducing and capping agent, leveraging the plant’s rich phytochemical
profile to form nanoscale TiO₂ without harsh chemicals. They characterized
these biosynthesized TiO₂ NPs using standard techniques (e.g., UV-Vis, FTIR,
XRD, electron microscopy) to confirm particle formation and surface chemistry,
and then tested their functional properties. The study showed that these green-
synthesized TiO₂ NPs effectively deactivated Staphylococcus aureus and
Escherichia coli—two common pathogenic bacteria—and demonstrated
promising photocatalytic degradation activity, indicating potential applications
in antimicrobial treatments and environmental remediation. This approach
emphasizes an eco-friendly, cost-effective route to produce TiO ₂ NPs with
practical biological and catalytic utility.

Rahman et al. (2025) reported a green synthesis study where they used
lemon juice (Citrus limon) as a natural reducing and capping agent to produce
titanium dioxide nanoparticles (TiO₂ NPs) and tested these biogenic NPs against
multidrug-resistant (MDR) Clostridium perfringens isolated from farm and
household waste. In this work, the researchers optimized conditions (0.125 mM
Ti precursor, 20 % lemon juice, 8 h reaction) to generate uniform (~37.5 × 12.3
nm) TiO₂ NPs, characterized them using UV-Vis, FTIR, XRD, SEM/TEM and
AFM, and demonstrated strong antibacterial activity with measured inhibition
zones (~27 mm) and defined MIC/MBC values (5 µg/mL and 10 µg/mL,
respectively). They also performed biocompatibility testing (MTT assay)
showing low cytotoxicity at effective doses, suggesting that these eco-friendly,
plant-mediated TiO₂ NPs could serve as a safe, sustainable alternative to
conventional antibiotics for controlling environmental MDR bacteria and
sanitizing wastes, though further safety and broader efficacy studies are still
needed.

59
 CHAPER IV
RESULT AND DISCUSSION

60
 CHAPTER IV

RESULT AND DISCUSSION

4.1 INTRODUCTION

Syzygium samarangense, commonly known as the wax apple or water

apple, is a tropical evergreen tree that belongs to the Myrtaceae family and is
native to the region spanning the Malay Peninsula to the Andaman and Nicobar
Islands. The tree is characterized by its short trunk and a wide, drooping canopy
of leathery, elliptical leaves that emit a distinct aromatic scent when crushed, a
trait common to many species in the myrtle family. It is most famous for its
bell-shaped, succulent fruits which possess a unique, waxy skin and a crisp,
sponge-like interior that is highly refreshing due to its high water content. While
the fruit is in South east Asian markets, the plant is equally valued for its
ornamental beauty, featuring fluffy, yellowish-white flowers with numerous
long stamens that attract a variety of pollinators. It comprises of photochemicals
that have strong antioxidant, antimicrobial, and anti-inflammatory properties
that have been utilized in traditional medicine for centuries to treat ailments like
fever and diarrhea.

Nanotechnology is an important field of modern research dealing with

design, synthesis, and manipulation of particle structures ranging from
approximately 1-100 nm. Nanoparticles (NPs) have wide range of applications
in areas such as health care, cosmetics, food and feed, environmental health,
mechanics, optics, biomedical sciences, chemical industries, electronics, space
industries, drug-gene delivery, energy science, optoelectronics, catalysts, single
electron transistors, light emitters, non-linear optical devices and photo
electrochemical applications.

61
 Nanobiotechnology is a rapidly growing scientific field of producing and
constructing an important area of research in nanobiotechnology is the synthesis
of NPs with different chemical compositions, sizes and morphologies, and
controlled dispersities. Nanobiotechnology has turned up as an elementary
division of contemporary nanotechnology and untied novel epoch in the fields
of material science receiving global attention due to its ample applications. It isa
multidisciplinary approach resulting from the investigational use of NPs in
biological systems including the disciplines of biology, biochemistry, chemistry,
engineering, physics and medicine.

Titanium dioxide (TiO₂) nanoparticles are extremely tiny particles of TiO₂,

so small that you can’t see them even with a normal microscope, and because of
this small size they show special properties like high surface area, strong
chemical stability, and the ability to absorb or reflect light. These nanoparticles
are widely used in everyday life and advanced technology: they are used in
sunscreens to block harmful UV rays, in paints and coatings to make them
brighter and more durable, in cosmetics and toothpaste as safe whitening agents,
and in water and air purification because they can break down harmful
pollutants under light. TiO₂ nanoparticles are also important in solar cells and
photocatalysis, where they help convert sunlight into energy or drive chemical
reactions, and in biomedical fields for antibacterial coatings.

Green synthesis of TiO₂ nanoparticles is an environmentally friendly and

sustainable method of producing titanium dioxide nanoparticles using natural
sources such as plant leaves, fruits, seeds, peels, or microorganisms instead of
toxic chemicals. In this approach, bioactive compounds present in the natural
extract—like phenols, flavonoids, alkaloids, and proteins—act as reducing,
capping, and stabilizing agents, controlling the formation and size of the
nanoparticles. The process usually involves mixing a titanium precursor with
the plant extract under mild conditions, followed by drying and calcinations to
62
obtain TiO₂ nanoparticles. Compared to conventional chemical and physical
methods, green synthesis is safer, cheaper, consumes less energy, and generates
minimal hazardous waste. The TiO ₂ nanoparticles produced through this
method often show good crystallinity, high surface area, and enhanced
photocatalytic and antibacterial activity. Because of these advantages, green-
synthesized TiO₂ nanoparticles are widely used in water and wastewater
treatment, degradation of organic pollutants, antimicrobial coatings, self-
cleaning surfaces, and solar energy applications, making green synthesis an
important approach for sustainable nanotechnology.

4.2 MATERIALS AND METHODS

Preparation of Syzygium Samarangense

Fresh Syzygium samarangense leaves were washed repeatedly with tap

water and distilled water to remove contaminants, cut into small pieces, and
directly boiled in distilled water at about 70–80 °C for 20–30 minutes to obtain
the leaf extract. The mixture was then allowed to cool to room temperature and
filtered through Whatman No. 1 filter paper to remove leaf residues, and the
clear filtrate was used immediately as the reducing and stabilizing agent for the
green synthesis of TiO₂ nanoparticles.

Biosynthesis of TiO2 Nanoparticles

For the green synthesis of TiO ₂ nanoparticles, 2 ml of titanium tetra

isopropoxide was first taken in a measuring jar, and 50 ml of distilled water was
taken separately in a beaker. A small amount of distilled water was added to the
measuring jar and mixed thoroughly with a glass rod to hydrolyze the precursor,
then transferred into the beaker; the remaining distilled water was added
gradually while carefully washing down the titanium tetra isopropoxide

63
adhering to the sides of the measuring jar using the same water volume,
ensuring no extra distilled water was added.

Subsequently, 2 ml of ethanol was added, and the solution was stirred

continuously for 15 minutes at room temperature without heating. Then, 20 ml
of Syzygium samarangense leaf extract was added drop wise using a dropper,
followed by heating at 60 °C for about 30 minutes under constant stirring.

Separately, a NaOH solution was prepared by dissolving 12–15 pellets of

NaOH in 10 ml of distilled water. After 30 minutes, the pH of the reaction
mixture was monitored and adjusted to pH 8 by adding the NaOH solution drop
wise, resulting in a dark yellow coloration and formation of a precipitate. The
precipitate was allowed to settle overnight, then filtered, dried in an oven at 90
°C for 3 hours, and finally calcined in a furnace at 500 °C to obtain TiO₂
nanoparticles.

Characterization of synthesized TiO2 Nanoparticles

The nanoparticles solution thus obtained was purified by repeated

configuration at 20,000 rpm for 30 minutes followed by dispersion of the pellet
in distilled water. UV-Vis spectra were recorded as a function of reaction time
on a UV-Vis spectrophotometer operated at resolution of 1 nm. After drying of
the purified TiO2 particles, the crystalline character of TiO 2 nanoparticles was
confirmed with the help of XRD. The X-ray patterns were obtained in 2 theta
configuration and the range was selected from 20° and 80°.

64
4.3 UV VISIBLE SPECTROSCOPY

Fig17: Absorption Spectrum of UV Visible Spectroscopy

This UV–Visible absorption spectrum shows how the TiO ₂ nanoparticles

synthesized using Syzygium samarangense leaf extract interact with UV light.

The strong absorption edge observed around 325 nm is the key feature here
—it indicates the characteristic band-to-band electronic transition of TiO₂
nanoparticles, where electrons are excited from the valence band to the
conduction band. This confirms the successful formation of TiO ₂ in nanoscale
form. Compared to bulk TiO₂ (which typically absorbs near ~ 380nm), this shift
toward a lower wavelength (blue shift) suggests smaller particle size and
quantum confinement effects, meaning the nanoparticles are very fine. This
absorption originates from band-to-band electronic transitions, specifically the

65
excitation of electrons from the O²⁻valence band to the Ti³⁺conduction band,
which is the fundamental optical behavior of TiO₂. The high absorbance in the
UV region and the gradual decrease toward the visible region (400–500 nm)
show that the material strongly absorbs UV light but has low absorption in the
visible range, which is typical behavior for TiO₂.
The smooth curve without sharp peaks also indicates good dispersion and
minimal aggregation of nanoparticles. The plant extract acts as both a reducing
and stabilizing agent, controlling particle growth and preventing clustering,
which directly influences the optical properties seen in this spectrum. Overall,
this absorption behavior confirms the successful green synthesis of stable TiO₂
nanoparticles with potential applications in UV shielding, photocatalysis, and
antibacterial activity.

4.4 TAUC PLOT

This graph is called a Tauc plot, and it is used to find the band gap
energy of Titanium dioxide nanoparticles. The x-axis shows the energy of
light (hν), and the y-axis shows how much light the material absorbs. At
lower energy values, the graph is almost flat, which means the material is
not absorbing light. This happens because the light energy is too low to
excite electrons.
As the energy increases, the curve suddenly rises. This means the material
starts absorbing light strongly. At this point, electrons gain enough energy
to jump from the valence band to the conduction band. This sharp increase
tells us where the important transition begins.

66
 Fig 18:Tauc Plot
To find the band gap (Eg), a straight line is drawn on the steep part of
the curve and extended to meet the x-axis. The point where it touches gives
the band gap value. Here, the band gap is about 3.82 eV. This value is a bit
higher than normal TiO2, which suggests the particles are very small or
have some defects affecting their properties.

4.5 X-RAY DIFFRACTION

XRD is a valuable research tool used to verify the synthesis of
nanoparticles, calculate the size of crystal particles, and determine the crystal
structure. When an X-ray is reflected on each crystal, various diffraction
patterns are formed which indicate the physical and chemical characteristics of
the crystal structure. Diffraction patterns usually show the physical and
chemical structure of a powder sample. Thus, XRD can analyze the structural

67
characteristics of a wide range of materials such as inorganic catalysts,
superconductors, biomolecules, glasses, polymers. In this method of analysis,
all the synthesized nanoparticles were analyzed in powder form.

Fig 19: XRD Analysis of TiO2 Nanoparticles

Analysis of structure and crystalline size of the synthesized TiO 2nanoparticles

were carried out by XRD. The XRD analysis of synthesized TiO 2 nanoparticles
from leaf extract showed diffraction peaks at angles 25.10°37.59°, 47.69°,
53.73°, 54.71° and 62.38° respectively. When compared with the standard, the
obtained XRD spectrum confirmed that the synthesized TiO 2 nanoparticles were
in nanocrystal form and crystalline in nature. The peaks can be assigned to the
planes (101), (004), (200), (105), (211), (213) facet of TiO2 crystal, respectively.
The high peaks in the XRD analysis indicated the active TiO 2 composition with
the indexing.

68
 The Full Width at Half Maximum (FWHM) values were used to calculate the
size of the nanoparticles. The average size of TiO2nanoparticlessynthesized
From leaf extract was 17.5 nm calculated using Scherrer’s equation. The
Scherrer equation,

D= Kλ/βcosθ

is a formula used in X-ray diffraction to estimate the crystallite size (D) of a

material based on the full width at half maximum (β) of a diffraction peak, the
X-ray wavelength (λ), and the Bragg angle (θ), with K being a shape factor.
Smaller crystallites lead to broader diffraction peaks, and the Scherrer equation
allows us to quantify this relationship to estimate the crystallite size. Where
Scherrer’s constant K value = 0.94 was selected due to the cubic and crystalline
nature of the nanoparticles.
The lattice parameters of the synthesized TiO 2 nanoparticles were found to be
a = b = 3.80 Å and c = 9.56 Å, which confirms the formation of a anatase TiO 2
tetragonal crystal structure. The equality of a and b and the difference in c are
characteristic features of this phase. These values are slightly higher than
standard bulk anatase, indicating a small lattice expansion that may arise due to
nanoscale effects, lattice strain, or the presence of defects in the crystal
structure.

Dislocation density is a measure of the number of dislocations linear defects in

the crystal lattice per unit volume of the material. XRD Analysis Dislocation
density can be estimated from XRD data by analyzing the broadening of
diffraction peaks, as dislocations also contribute to peak broadening and
calculated as

δ =1/D2 =0.3269 X 10-2 nm-2

69
 Microstrain refers to the variations in strain within a material at a microscopic
level. It’s essentially a measure of how much the crystal lattice deviates from its
perfect, ideal structure. Βhklcosθ = 4εsinθ + KλD where ε and D represent the
micro-strain and crystallite size was obtained.

In microstrain and dislocation density are closely related, as both are

influenced by the presence of defects and distortions in the crystal lattice. High
dislocation density can lead to increased microstrain, as the dislocations cause
distortions in the crystal lattice. In a material with high dislocation density, the
XRD peaks will be broader due to the presence of many dislocations and the
resulting microstrain. By analyzing the peak broadening, researchers can
estimate both the microstrain and the dislocation density, providing information
about the material’s microstructure and properties. The microstrain value of
TiO2nanoparticles is calculated as ε = β/4tanθ = 0.5762 %.

70
 CHAPTER V
CONCLUSION AND FUTURE SCOPE

71
 CHAPTER V

CONCLUSION AND FUTURE SCOPE

5.1 CONCLUSION

The green synthesis of titanium dioxide (TiO₂) nanoparticles using

Syzygium samarangense leaf extract proves to be an effective, low-cost, and
environmentally responsible route compared to conventional chemical methods.
UV–Visible spectroscopy confirmed the formation of TiO ₂ nanoparticles
through a strong absorption edge in the UV region, indicating electronic
transitions characteristic of TiO₂ and a widened optical band gap attributable to
nanoscale effects. X-ray diffraction (XRD) analysis revealed that the
synthesized nanoparticles are predominantly crystalline in nature, with
diffraction peaks matching the standard TiO ₂ phase, confirming successful
nanoparticle formation. However, the presence of minor impurity peaks
suggests residual organic compounds from the plant extract or incomplete
crystallization, which is a common and realistic outcome in green synthesis
routes and should not be ignored or glossed over. These impurities highlight the
trade-off between environmental friendliness and absolute phase purity,
emphasizing the need for optimized calcination or post-treatment if high-purity
TiO₂ is required for advanced applications. Overall, the study demonstrates that
Syzygium samarangense leaf extract functions effectively as a reducing and
stabilizing agent, enabling sustainable synthesis of TiO ₂ nanoparticles with
acceptable structural and optical properties for applications where eco-
compatibility outweighs ultra-high purity.

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5.2 FUTURE SCOPE

The future scope of green-synthesized TiO 2 nanoparticles using

Syzygium samarangense leaf extract includes various potential applications and
further research directions.

 Antibacterial and Antimicrobial Applications

1. Reaction Oxygen Species Generation: Under UV light, TiO₂

nanoparticles generate ROS (•OH, O₂•⁻), which damage bacterial cell
walls and membranes.
2. Application in Antimicrobial Coating: Used as antimicrobial layers on
medical tools, textiles, and surfaces under UV-assisted conditions.

 Self Cleaning Coating Applications

1. Photocatalytic Decomposition of Dirt: Under UV light, TiO₂ generates

reactive oxygen species that decompose organic contaminants on the
surface.
2. Application on Transparent Surface: Used on glass, windows, solar
panel covers, and ceramic tiles for reduced manual cleaning.
3. Environment Friendly Coating: Green synthesis using Syzygium
samarangense extract avoids toxic chemicals, making the coating eco-
safe.

 UV Sensors and Optoelectronic Devices Applications

1. UV Light Detection: The wide band gap enables selective absorption of

UV radiation while remaining insensitive to visible light.
2. UV Monitoring Devices: Used in UV photodetectors, flame sensors, and
environmental UV radiation monitoring systems.

73
CHAPTER VI
REFERENCES

74
 CHAPTER VI
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