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 Green Synthesis of Copper Oxide
Nanoparticles and Applications

By:
Shakeel Ahmad Khan
PhD Scholar Chemistry (Shakilahmad56@gmail.com)
Hallym University South Korea
Dr. Sammia Shahid
PhD Chemistry, Associate Professor, Chairman
Department of Chemistry, University of Management
and Technology Lahore, Pakistan
Faheem Ijaz
PhD Scholar Chemistry
Department of Chemistry, University of Management
and Technology Lahore, Pakistan

1

 DECLARATION
We, Shakeel Ahmad Khan, Sammia Shahid and Faheem
hereby declare thDWWKHPDWWHUSULQWHGLQWKHWLWOHG*UHHQ
Synthesis of Copper Oxide Nanoparticles and
$SSOLFDWLRQVLVWKHLURZQZRUNDQGKDVQRWEHHQSULQWHG
published and submitted as research work, thesis or
publication in any form in any University, Research
institution etc. in Pakistan or Abroad.

2

 ACKNOWLEDGEMENT

All Praises for ALLAH ALMIGHTY, the Most

Merciful and Most compassionate, the creator of the
universe, who guides us to research what is mysterious in
this universe and helps in difficulties, enabled me to
complete this research work successfully. I offer my
humblest and sincerest words of thanks to Holy prophet
HAZRAT MUHAMMAD (PBUH) who is forever a torch
of guidance and knowledge of humanity.
It is a matter of great pleasure and honor for me
to express my deep sense of gratitude to my kind and
respected Professor, Dr. Sammia Shahid, Associate
Professor, Chairperson Department of Chemistry,
University of Management and technology Lahore, for
her esteemed Supervision, patients, motivation, incessant
support and constructive criticism throughout my
research.
My sincere thanks go to all the worthy Professors,
especially Dr. Khurram Shahzad Munwar, Dr. Sohail
Nadeem and Dr. Ayesha Muhy-ud-Din. I would also

4

express my sincere thanks to Dr. Ghulam Hussain for
his valuable guidance and suggestion in this research
work.
Last but not the least; I would like to thank my
parents (Atta Muhammad Sajid and Jamila Akhter),
brothers (Muhammad Rizwan Sajid and Khaleel
Ahmad Khan), sister and Uncle Dr Abd-ur-Razaq Khan
Rashid for supporting me throughout my life and praying
for my success. They continuously encouraged me during
the hard time of my studies.

5

ABSTRACT
The study reports a superficial method for the green
synthesis of copper oxide nanoparticles by a solution
combustion method using Abutilon indicum water
extract. The Copper oxide Nanoparticles were
characterized by XRD, SEM, TEM and UVVisible
studies. XRD data indicates the formation of pure
monoclinic crystallite structures of CuO Nanoparticles.
SEM images showed that the particles have sponge like
structure with large surface area and the average
crystallite sizes were found to be 60120 nm. These
observations were confirmed by TEM analysis.
Photocatalytic activity studies of CuO Nanoparticles
revealed that they act as very good catalyst for the
effective degradation of acid black 210 in the presence of
Sunlight. The CuO Nanoparticles found to inhibit the
activity of 1,1-Diphenyl-2 picrylhydrazyl (DPPH) free
radicals effectively. CuO Nanoparticles exhibited
significant bactericidal activity against
E.coli,Staphyloccus aureus, Klebsiell and Bacillus
subtilis. This research revealed a simple, ecofriendly and

6

robust method for the synthesis of multifunctional CuO
nanoparticles employing under-utilized medicinal plants.

7

 TABLE OF CONTENTS

TITLE PAGE 1
DECLARATION 2
DEDICATION 3
ACKNOWLEDGEMETN 4-5
ABRACT 6-7
TABLE OF CONTENTS 8
Chapter 1 Introduction 9-45
Chapter 2 Literature Review 46-77
Chapter 3 Materials and Methods 48-92
Chapter 4 Result and Discussion 93-113
Chapter 5 Conclusion 114- 115
Chapter 6 References 116- 124

8

Chapter 1 INTRODUCTION
1.1 Nanoscience
During the last few decades, the interests of scientists are
increasing continuously day by day in the field of science
and technology at the nanometer range. Institutions of
Government, some research centers of public, different
type of Government and private universities and some
firms have investigated these significant resources
throughout the world. This technology of nanoscience is
still at an early stage of progress. Future scientific and
technological results are not easy to estimate and chase.
It has been agreed the extensive scope of the pasture
concerned and the possible scientific progression. In this
perspective, forethought methods can play a vital role by
individuating, surrounded by this wide choice of
nanoscience. It is most promising meadow of research
and handling for people, organizations, laboratories and
research centers. Though it is generally acknowledged
that growing technologies like biotechnology,
nanotechnology etc. will have greater than ever socio-
economic impacts. There are major boundaries in terms

9

of accessible financial resources, public and supporting
accountability.
This has managed to the inevitability of scenery examine
significances not only at a macro-altitude of study but
also at a micro level research. Forethought crams to
identify research priorities at a macro-level are
extensively subtle. In most of the cases it leads to alike
end in terms of macro-fields. Almost each country of the
world has shown a great curiosity to empower
considerably in the area of nanoscience. The conclusion
of this era is only a preliminary step, agreed that more or
less any scientific research can be studied at the
nanometer range with some new and very attractive
upshots. ,QIDFWWKHWHUPQDQRWHFKQRORJ\LQFOXGHVVXFK
wide variety of tools, procedure and promising
applications. The definite length scale namely nanometer
is the billionth part of one meter which brings human
thoughtful capability to that attitude where all branches
in natural sciences unite, i.e. the atomic and molecular
aspects of study.

10

If nerve cells, filaments of DNA, copper particles or
allotropic form of carbon i.e. buck balls are studied, we
conclude that all these living as well as nonliving things
consists of atoms and molecules. There is nothing any
conceptual difference between these biotic and abiotic
things. Nano-science is a major branch of science which
provides the prospects to recognize and wangle at atomic
or molecular level. Nano-science and nanotechnology is
supposed to wrap all fields of science and technology
because any material particle is made up of atoms and
molecules. It is the branch of science and technology
which confronts to the atomic and molecular structure of
microscopic particles of matter. But scientists and
researchers are not being in agreement on defining this
branch as above. A supplementary pragmatic definition
of nanoscience can be attained by focused on the basic
goods of the nanoscale materials and the opportunity of
using, operating and organizing them into assembly in
order to carry out definite functions. These
impersonations can be better studied in the frame of a
discussion on efficiency (Balzani et al., 2005). In nano-

11

chemistry, small structures or small sized materials are
studied. The dimension spans of nanotechnology range is
from sub-nanometer to hundreds nanometers. This
technology is assumed to understand as a technology of
devise, production and applications of very small
structures and materials. Physical properties of materials
and amount of are also some basic understandings of
nano-chemistry (Guozhong et al., 2003).
In science and technology, was considered well-
progressed interdisciplinary field of research in last few
year. Particularly metal oxide nanoparticles have
extensive well-known their application of in many fields
of science including chemical industry, electronics,
biomedical sciences, biosensor, molecular catalysts,
magnetic materials and drug gene delivery due to their
physical and chemical properties different from those in
the bulk material (Bednorz et al., 1986). Main focus of
Nanoscience and nanotechnology is synthesis of new
nanoparticles with various sizes and new morphology.
Due to change in morphology strongly effects on their
broadly varying properties. Much attention attracted by

12

nanoparticles on account of their unique properties and
energetic application which are strongly influenced by
their size, morphology and structure. Cupric oxide
nanomaterials have attracted much attention on account
of their distinctive properties among other metal oxides
nanomaterials (Ng et al., 2006).
In Nanotechnology materials are fabricated at atomic
scale to achieve unique properties which suitably
employed to attain for the preferred applications. Mostly
natural process also occurs in the nanometer scale.
Nanotechnology and biology deals various biomedical
problem and modernized the field of health and medicine
(Curtis et al., 2001). For human benefits, controlled
disparity, chemical composition, morphology and
different sizes nanoparticles are basic purpose of
nanotechnology. Due to high specific surface area metal
nanoparticles have been considered broadly.
Nanoparticles show specific features, such as catalyst,
optical, antimicrobial (Elumalai et al., 2010). The study
and applications in Nanoscience and technology are
deviating and extending form conventional devices of

13

physics to self-assembled molecular devices. Many new
materials are being made which have nanoscale
dimensions and controlling matter at atomic level.
Nanotechnology may progress more new devices with
innovative applications in evolving field such as
electronics, biosciences, medicines and energy
production. As a new technology, it has some severe
issues and shocking one is issue of toxicity and influence
of nanoparticles on environment. Nanotechnology has
prospective effect on the economy of world. Finally,
nanotechnology can be used as technology for fabrication
of nanomaterials and their application in innumerable
fields (Cristina et al., 2007). In recent years due to the
distinguishing features in physical and chemical
application of nanomaterials, they have received much
greater attention. A significant numbers of experimental
approach have been utilized to manufacture nanocrystals.
Particle size of the final products exquisitely depends
upon the experimental conditions applied (Ball et al.,
1992).

14

Nanostructures provided unique combination of small
size with larger surface area. Bulk materials lack similar
properties demonstrated by nanoparticles. They have
significant applications in biological sensors, optical,
photovoltaic cells, conductive materials and coating
formulations. Control of the size and shape of the
particles and their stability are quite challenging and
similar is the case when attempting to uniformly
dispersed them in a matrix and trying to optimize their
release rate (Anyaogu et al., 2008). An enormous amount
of interest has developed in scientific circles due to
incomparable, unusual and captivating biological,
physical, and chemical properties of nanometer-sized
materials. A substantial change has been noticed after the
particles were down sized to nanometer range. Massive
attempts have been made to express and characterize the
physical and chemical properties of metal oxide
nanoparticles. These have significant application in
numerous technological fields (Ferreira et al., 1996).
As in nanomaterials, dots are decreased from a micro-
meter to a 10-9m size; the resulting properties are altered

15

intensely e.g. chemical reactivity, electrical conductivity,
biological activity, hardness and active surface area are
all known to be changed. As result both their size and
high surface to volume ratio, the bactericidal
achievement of metal nanoparticles has been
recommended. Such features should permit them to
combine with membranes of bacteria, rather than the
effect being exclusively due to the discharge of metal
ions (Morones et al., 2005). The expansion of reliable
and prompt processes for the synthesis of nano-ranged
metal dots has fascinated important consideration due to
their uncommon size dependent optical and electronic
properties. Now, to synthesize various types of
nanoparticles, a large number of biological physical and
chemical methods were available. Though, it is a fact that
stability and reproducibility of the nanomaterials with
controlled size are very difficult to attain by common
chemical reduction methods. Manufacture of
nanoparticles by using extracts of plant is quite novel
method and it leads to true green chemistry at a very
inexpensive cost (Mohanpuria et al., 2008). The unit of

16

length nanometer is very significant because it shows as a
connection for smallest devices to be manufacture and
the world of macro objects in this universe. The
structures and system having one or more dimensions in
nanometer scale are the possible smallest things which
we have produced up till now. The modern research and
technologies are able to produce solid materials in
nanometer range or so smaller than this particular range.
As a result, the specific properties of material change in a
very peculiar way and are completely different from their
counterpart in macro form. This unexpected change is
due to change in size of particles of nanomaterials
(Eychamuller et al., 2000).
Sometimes nanoparticles show properties related to size
and sometimes do not show, but these properties vary
considerably from the properties of the same material in
bulk. Molecules having size in the range of 2500nm and
10000nm still are not measured as nanoparticles. The
particles in the range of 1-10nm having narrowed size
distribution and with at least one dimension are called
nano clusters. When these nano clusters or nanoparticles

17

form agglomerates these are known as nanopowders. The
single crystals having their size in nanometer range
called nanocrystals (Fahlman et al., 2007).
1.2 Copper Oxide Nanoparticles
Cupric oxide has shown wide applications in the fields
which range from energy conversion and storage,
environmental science electronics, and sensor. Cupric
oxide is a p-type semiconductor material which has a
narrow band gap of 1.2 eV. In the last few years, they are
getting much attention for their unique properties and
potential applications as heterogeneous catalysts, lithium
ion batteries, gas sensors, antibacterial agents and solar
cells. In addition, CuO Nps are robust, stable and their
shelf life is longer as compared to organic antimicrobial
agents (Ren et al., 2009).
Colloidal metal and metal oxide nanoparticles of Silver,
Gold and cupric oxide have been used extensively in
optoelectronic, industrial and biomedical applications.
CuO nanoparticles have monoclinic crystallite structures
and due to their versatile physical and chemical
properties, they have been used in numerous technical

18

and scientific applications. They have been utilized as
antibacterial agent, antioxidant agent, drug delivery
agent, and imaging agent in field of biomedicine. In
industrial field, they have been used widely as p-type
semiconductor materials, transistors, in the construction
of batteries, gas sensors, and field emitters. The
applications of nanoparticles can be changed depending
on the shape and size, which affects physical and
chemical properties of cupric oxide nanoparticles
directly. In plant extract mediated synthesis method,
some experimental parameters i.e mass ratio between
Copper salt and extract, nature of plant extract,
temperature and reaction time, have shown direct
influence on the morphology of Cupric oxide
nanoparticles (Sankar et al., 2014; Yallappa et al., 2013).
Cupric oxide nanoparticles have got great consideration
because it is the simplest member of the family of copper
salt and shows a range of possibly useful physical
properties such as electron correlation effects, spin
dynamics and high temperature superconductivity. In the
field of energy saving, by using copper oxide

19

nanoparticles in energy transferring fluids, the viscosity
of fluid improves and thus boosts thermal conductivity
(Kwak et al., 2005).
The information related to antimicrobial activity of CuO
nanoparticles is very limited. Copper oxide is
inexpensive than silver and gold. It can be mixed easily
with polymers and is relatively stable in terms of both
physical and chemical properties. Cupric oxide
nanoparticles are highly ionic with respect to other metal
oxides nanoparticles and may be predominantly valuable
antimicrobial agents as they can be synthesized with
extremely unusual crystallite morphologies and high
surface areas (Stoimenov et al., 2002).
1.3 Green synthesis of Nanoparticles
Plant extracts use is gradually evolving research area
known as green synthesis of nanoparticles (NPs). Green
synthesis is potentially advantageous over chemical or
microbial approach as it simplifies the process and also
results in production at larger scale (Das et al., 2013).
Finding suitable, nontoxic and natural product to prepare
metal nanoparticles in an aqueous environment was quite

20

an effort. Several naturally existing materials were
utilized to construct nanoparticles but apparently plants
seem to outrun the other contenders. Plant origin
nanoparticles are much durable, offer various sizes and
shapes and moreover their production can be at much
faster pace (Iravani et al., 2011).

Figure 1: Green synthesis of CuO nanoparticles

Green chemistry is about biosynthesis of metal or metal
oxide nanoparticles using environment friendly methods.
This concept entails avoid using harsh & toxic reducing
agents (e.g. hydrazine hydrate, sodium borohydride,

21

dimethyl formamide (DMF), ethylene glycol, and so on),
and expensive chemicals. Many of these reducing agents
have been declared major culprits notoriously known as
biological hazards and lead to environmental toxicity.
For the synthesis of nanomaterials, with growing interest
in the reduction or total removal of waste and the
implement of appropriate procedures and processes. In
these processes, we used major true principles of green
chemistry to grow of natural and biomimetic procedures
(Raveendran et al., 2003).
In the medical application when we have used chemical
synthesis methods then these chemical adsorbed on the
material surface which have great impact on our lives. In
synthesis of nanomaterials different factors effect in the
production of high quality nanomaterials with respect to
chemical purity, crystallinity, phase selectivity, and
homogeneity in dot size and shape with controlled state
of accumulation in inexpensive method is still a task to
material chemists. Due to increase awareness towards
green chemistry and other biological processes, the desire
to grow green approach for the preparation of

22

nanoparticles has increased. From plant extract
nanoparticles were synthesized which provide
environmental friendly atmosphere that deal several
benefits of eco-friendliness and compatibility for
pharmaceutical material and other biomedical
application. They do not utilize poisonous chemicals for
the manufacture procedure (Parashar et al., 2009).
Recently, for the development of novel routes to
synthesize nanoparticles, the material scientists and
researchers have focused on green routes. Greener
procedures are eco-friendly, inexpensive, very cheap and
without use of harsh chemicals (Sankar et al., 2013). For
the synthesis of nanoparticles, Solution Combustion
method is clear, smooth, and simpler and consumes short
energy and time. Significant features of greener
procedures include less or zero pollution to the
atmosphere, advantageous products can be prepared
easily and environmental friendly. This green
methodology based on biomaterial to remove toxic
chemical materials. Normally, the waste products from

23

the plants are frequently easily available in the
environment at low cost.
During solution combustion synthesis method, for
uniform distribution the reactants and combustible fuel
are mixed. In this way pure product material was
obtained in fine power form. Plant extract which act as
Combustible fuel plays a key role in the release of energy
and during combustion reduced size particles were
obtained. During solution combustion synthesis method,
appropriate plant extract (combustible fuel) which
behave as a reducing in an aqueous solution containing
metal salts particularly metal nitrates. When reaction take
place between reactants a great amount of energy release
so it is called exothermic reaction (Nagappa et al., 2007).
Based on applied synthesis protocols, in distinctive plant
extract facilitated nanoparticles synthesis, the plant
extract is mixed with a certain mass ratio metal salts.
Nanoparticles are formed and different ranges of
temperatures form room temperature to hundred degree
centigrade. Nanoparticles are formed at different time
period that from minutes to hours. Usually, in plant

24

extract great amount metabolites are present such as
terpenoids, tannins, flavonoids and proteins that behave
reducing and stabilizing agents (Sankar et al., 2014;
Schrofel et al., 2014).
1.4 Application of CuO Nps
Cupric oxide nanoparticles fabricated three-
dimensional orientation pattern which particularly
used in gas sensors. Copper oxide has adequate
surface area and interspaces which have ability to
absorb gas .So they behave as good gas sensors.
Energy transmitting fluids filled with cupric oxide
nanoparticles can increase liquid viscosity and
improve thermal conductivity. Copper oxide
nanoparticles have narrowband gap and gives
photocatalytic properties as well as photoconductive
characteristics.
Cupric oxide nanostructured belongs to transition
metals and shows reversible ability of two to three
times greater than the graphite as negative electrode
materials for lithium ion batteries. It is experimentally
showed that the particle structure has a significant

25

 impact on cupric oxide nanoparticles electrochemical
behavior towards lithium.
Copper is broadly used material because it is much
cheaper and shows high conductivity. It is highly
reactive for the creation of conductive structures for
printing in ink-jet.
Copper oxide has attracted great attention because it
acted as p-type semiconductor. Cupric oxide is
obtained from copper salts and this simplest
compound showed broad useful properties such as
high temperature superconductivity, solar cells,
electron correlation and spin dynamics.
1.5 Antioxidant activity
Antioxidant agents are important for food storage. These
agents suppress the oxidative rancidity in food by
controlling the free radicals production. Many biological
systems have not the ability to overcome the oxidative
abilities of free radicals which are generated during some
metabolic processes. As a result these species and
systems come under the oxidative stress. Oxidative stress
has its role in many minor and major diseases such as

26

aging process, heart diseases, and neurodegenerative
diseases and in cancer (Astley et al., 2003).
It is naturally that living cells have antioxidant protective
system in them. This system makes them able to
inactivate as well as prevents the excessive formation of
ROS. When an imbalance takes place between the
formations of reactive oxygen species (ROS) and body
antioxidant defense then antioxidants protect from
damaging potentials of oxidative stress. Antioxidants
now found many applications in food industry. These
agents prevent flavor losses, food deterioration and
nutritional losses in many commercial food items. Both
natural and synthetic antioxidants are available. Natural
antioxidants are more preferred over synthetic because of
safety and health point of view (Gorinstein et al., 2003).
The most common reactive oxygen species in humans
which are produced during human metabolism are O2-,
H2O2 and OHx . If the equilibrium between reactive
oxygen species and antioxidants is disturbed then ROS
cause many irregularities in the human body. These are
incessantly generated within the human body. These

27

ROS damage different bimolecular species like DNA,
lipids, lipoproteins and proteins. When such bimolecular
species are damaged, they cause human diseases such as
arthritis, diabetes mellitus, neurodegenerative diseases,
cancer, and atherosclerosis etc. A lot of work is going on
in the field of free radical biology to avoid and overcome
the chronic human diseases (Elmastas et al., 2006).
Like food products polymers are also oxidized by
atmospheric oxygen, especially at high temperatures.
Oxidation of polymers causes cracks and polymer
backbone breakdown. Oxidation process also influences
the durability and mechanical properties of rubber and
plastic. Synthetic antioxidants such as n-propyl gallate
(NPG), butylated hydroxyanisole (BHA) and butylated
hydroxytoluene (BHT) are very effective but due to
carcinogenicity these have limited uses (Ahamed et al.,
2008). 1RZ D GD\V GLIIHUHQW W\SHV RI QDQRSDUWLFOHV DUH
being synthesized which show good antioxidant
properties. For the treatment of cancer and cardiovascular
diseases CuO is used as a powerful antioxidant. In the

28

present study CuO show good antioxidant activity by
potentially scavenging DPPH radicals.
1.6 Photocatalytic degradation dye
7RGD\VZRUOGLVGHQVHO\LQGXVWULDOL]HG7KHUHVXOWRIWKLV
industrialization is the production of unlimited and
uncontrolled pollution in different forms. This pollution
is causing many health and environmental issues. A large
part of this pollution comes from textile and paper
industries in the form of carcinogenic and non-
degradable dye effluents. It is common practice of most
of the industries that they use color dyes and discharge
waste without any proper treatment into the natural
ecosystem (Robinson et al., 2001). These dye materials
are so stable that they are not degraded by applying UV
radiation and hydrogen peroxide as oxidizing agents. As
a result scientists developed advance method to
overcome this issue. At present photocatalytic method for
the degradation of dyes has got great importance due to
its simplicity and effectiveness (Nishio et al., 2006).
Photocatalytic reactions involve the generation of free
electrons and whole due to the absorption of light energy

29

and these electrons then cause oxidation of the target dye.
Similarly the synthesized metal nanoparticles also show
good degradation of the color dyes. Various methods are
available for the synthesis of metal nanoparticles. But the
green synthesis is most preferred because it is more
economical when compared with chemical and physical
method. Among the synthesized metal nanoparticles,
copper oxide nanoparticles are very important
(Krishnaraj et al., 2010). These are extensively used as
catalysts, and especially in environmental catalysis. For
example, carbon monoxide which is a dangerous gas can
be oxidized using CuO catalysts. Its catalytic activity is
more efficient than noble metal catalysts (Jernigan et al.,
1994). It is important note when colored dye went in
aquatic ecosystem where life exits these life effected
from these effluent. Largest synthetic dye are called azo
dyes in which nitrogen attach to other nitrogen with
double covalent bonds. In largest scale, azo dyes are now
utilized in textile industry, paper industry, food industry,
additives, cosmetics industry, xerography industry, laser
materials and laser printing industry (Bafana et al.,

30

2011). CuO is an important catalysts being utilized to
abolish industrial pollutants present in the atmosphere. It
is obvious from the former studies that the catalytic
reaction of CuO is a structure responsive process and the
oxygen surface lattice of CuO is drawn in in the reaction.
Morphology and the crystal planes of the particles are
mainly based on the catalytic reactivity of CuO
nanoparticles. So the controlled shape synthesis of the
particles is an imperative factor in improving the
catalytic performance (Zaman et al., 2012; Nezamzadeh-
Ejhieh et al., 2010).
CuO is a not expensive and environmental friendly
catalyst used for degrading pollutants. The morphology
of CuO plays the main role in degrading a pollutant. So it
is the synthesis of new CuO nanostructures with the
desired shape and improved catalytic activity for the
degradation of dyes as well as organic pollutants is the
need of time (Narayanan et al., 2004). In the recent
research work by the scientists in different regions of the
world it is preferred to use the environmental friendly as

31

well as cheap methods for the synthesis of the metal
nanoparticles.
1.7Antimicrobial activity
The present study reveals that nanoparticles having
smaller size and nanoparticles that are synthesized at low
temperature showed a great antimicrobial activity. It was
reported that effect of temperature had a great effect on
the antimicrobial activity of nanoparticles. At a range of
400 to 700C there is an enhanced effect of antibacterial
behavior on both Gram-positive and Gram-negative
bacteria as well as zones of inhibitions (Azam et al.,
2012). Surface area is another factor by which
antimicrobial activity of the microbe can be observed. So
it was seen that nanoparticles having large surface area
showed more reactions with the surface of bacteria
(Holister et al., 2003).
Now a day, regular growth of many antibiotic bacterial
strains has nowadays made it imperious to invent some
new drugs or wide spectrum of operative antimicrobial
effect. The present study on nanoparticles reveals that
many metals and metallic oxides showed great potential

32

effects for killing the harmful bacteria. Nanoparticles
with greater surface to volume ratio and structure of their
crystal generate biotic responses. These are very different
from previous forms of metals. Nanoparticles having
small range of particles showed many times less toxic
effect to the cells of mammals. These show protracted
results in organisms as a source (Rakhmetova et al.,
2010).
Some factors provide greater retention time for the
interaction of bacterium nanoparticles. These factors
include size of nanoparticles, stability of nanoparticles
and growth medium concentration. The size of bacteria
is in a range of micrometer. But the pores in the cell
membrane of bacteria range in nanometer. In general,
bacterial cells are in the micron-sized range. So,
nanoparticles are prepared in such a way that they must
cross the pores of the cell membrane (Azam et al., 2012).
In aqueous medium, the large amount of Copper oxide
particles is insoluble. Therefore has limited
bioavailability as a beneficial antimicrobial agent. In
comparison, Cupric oxide nanoparticles are soluble in

33

water and produce colloidal solution. Furthermore, in
biological organizations, high surface area and
morphology of crystal provide greater reactivity and
provide grounds of their target specificity (Lemire et al.,
2013).
1.8 Characterization techniques
In the present age of science, it is believed that
nanotechnology and Nanoscience are most important and
most growing technologies in the globe. In recent years,
nanoparticles show substantial properties. In determining
the chemical, biological and physical properties, these
materials show abrupt behavior. These properties lead to
the scientist interest at a peak due to its size and
improvement in behavior. This technique provides a vital
role in all disciplines of science i.e. electronics,
mechanics, space science, polymer chemistry, nuclear
chemistry, optical science and information technology.
This technology is proving itself an emergent field of
science.
The nanotechnology is based on the fact that physical and
chemical characteristics of the nanomaterials varies

34

strongly when the size of the material particle changes.
Properties differ greatly when material is wrinkled form
heavy particles to nano-sized particles. But to measure
the size of a nanoparticle is a difficult task. Researchers
are trying to prepare different methods of synthesizing
nanoparticle. For synthesis of nanoparticles, scientists are
trying to improve new techniques of characterization of
equipment.
Thus in the growth of this field, researchers are trying to
in the nanotechnology and Nanoscience has encouraged
the researcher and scientists to develop and devise some
new sophisticated and prcised equipment. This
equipment will change their way of thinking and
modified their expressions for the betterment of this field.
They shall be able to control many parameters like
morphological parameters etc. so these researches shall
be helpful for the betterment of mankind in the next few
years.
A number of new devices have been constructed by a
number of scientists which are very helpful in controlling
the composition and morphology of nanoparticles.

35

Various techniques which are important in determining
the nanoparticle structure, morphology, type of elements
presents, dielectric constant analysis, magnetic
properties, trace elements and impurities are being used
in this field of chemistry. These devise are very helpful,
precise and accurate in their quality control. The
techniques include:
X-RAY DIFFRACTION
SCANNING ELECTRONS MICROSCOPY
TRANSMISSION ELECTRON MICROSCOPY
PHOTOLUMINISENSES
ENERGY DISPERSIVE X-RAY ANALYSIS
UV-VISIBLE ABSORPTION SPECTROSCOPY
1.8.1 X-ray diffraction (XRD)
X-ray diffraction plays a key role at the pitch of
nanoparticles research and Nano chemistry. This
application is being used It is used to study precarious
structures such crystal size, strain of crystal and crystal
structure. The formula of Scherer is used for calculating
the crystal size. The formula is:
' &RV

36

Where
D = diffraction in the size of crystal,
 ZDYHOHQJWKRIWKHEHDP
&RV %UDJJVDQJOHRIURWDWLRQ
 IXOOEUHDGWKKDOIPD[LPXP
In this type of diffraction analysis, many different
patterns of diffraction are prepared randomly. After
solving these patterns of diffraction, some very useful
information is obtained about the structure and size of
nanoparticles. The sample to be determined is present in
powdered form or in the form of a single crystal which
tells us about the orientation of different particles in
sample. This chemical characterization technique tells us
some important information about the structure of
particles. The only condition for obtaining better results
of scattered pattern of diffraction is that, the sample
VKRXOGVDWLVI\%UDJJVHTXDWLon i.e.
GVLQ Q

37

1.8.2 SCANNING ELECTRON MICROSCOPY
(SEM)
During SEM analysis of given sample a high energy and
highly focused being of electron instead of ordinary light
is used irradiate the sample under examination. It uses
focus beam of high energy electrons scanning over the
surface of thick or thin specimen to produce images.
SEM magnifications can go to more than 300000X and
resolution 10nm. For producing the SEM images, the
area of sample under inspection is irradiated with highly
energetic electron beam. As result signals are produced
due to scattering of electrons from sample surface, the
produced signals are amplified using amplifying part of
instrument, then these amplified signals are analyzed and
data obtained is converted into images. The final results
from SEM analysis are obtained in the form of highly
resolved images. SEM can be used to study the
topography, morphology, chemistry and crystallography
of the sample being inspected. The diameter and length
of the synthesized particles is also calculated.

38

The SEM analysis is carried out scanning the sample
under high energy focused beam of electron. During
scanning when these electron beams collide with sample
being observed, a number of interactions take place due
to absorption of the high energy electrons. As a result
primary electrons, secondary electrons, back scattered
and X-ray are produced .these all type of radiations
contain important characterization information about the
sample. These are then monitored and detected. The
detector of the instrument converts these radiative signals
into images after some complicated processes.
1.8.3 Transmission Electron Microscopy (TEM)
TEM examinations of the samples provide high
resolution images. From these images important
information like the size, surface features, shape of the
particles, the size distribution and morphology of the
nanoparticles is obtained. During routine transmission
electron microscope analysis, a uniform and thin sample
is prepared for examination. Then this specimen is
irradiated with uniform current density electron beam.
The electron guns are used as source of electron beam.

39

Type of gun depends upon the sample, instrument and
analysis requirement. When these high energy electrons
collide with sample, these do not scatter back but most
them transmit through the sample.
These transmitted electrons provide required information
about the sample. These transmitted electrons are
amplified and magnified with help of 3-4 lens system
present in the instrument. Objective lens converts these
electronic signals into images. The images can be
obtained directly with the help of a digital CCD camera
or conventionally on a photographic plate. Normally
TEM is operated at voltage 120 to 200 KV. When
working voltage is 200-500 KV it is called medium
voltage microscopy which provides high resolution and
better transmission. When TEM uses acceleration voltage
in the range 500 KV to 3MV then it is called as high
voltage electron microscopy. High resolution-TEM
provides better characterization of the particles and also
shows the arrangement the atoms, so it can be used to
study defects and surface atomic arrangement of the

40

particles. It becomes more efficient when used in
combination with diffraction studies of nanoparticles.
1.8.4 Energy Dispersive X-Ray analysis (EDX)
Energy dispersive X-ray spectroscopy sometimes called
as energy dispersive X-ray analysis or energy dispersive
X-ray microanalysis is an analytical technique used for
the elemental analysis or chemical characterization of a
sample. The EDX analysis is performed with the help of
SEM. EDX analysis involves the bombardment of
focused and high beam of electrons on a solid sample and
resulting X-ray spectrum is evaluated to obtain results. It
is used both for qualitative and quantitative analysis. Its
basic principle is to use some X-ray source to excite
sample. Then these excited samples emit characteristic
wavelength radiation in the form of spectrum depending
upon the elemental composition. As each element present
in the sample emits specific wavelength X-rays which are
observed with the help of energy dispersive spectrometer.
In this way the constituent elements and their
composition is estimated.

41

1.8.5 Fourier Transform Infrared Spectroscopy
(FTIR Spectroscopy)
FTIR Spectroscopy is the technique very common in
analysis of samples in any state of matter. This technique
provides us information about the investigation sample in
the form of the IR spectra. The spectrum is obtained
either in absorption, emission, photoconductivity mode
or in Raman scattering. The spectrometer used for this
purpose is named as Fourier transform spectrometer.
When the sample is injected to take Infrared spectrum, a
large no. of absorbance or transmittance bands are
appeared. The possibility for a large number of
compounds to have the same IR spectrum is ruled out.
Therefore in comparison with standard spectra, unknown
compounds are identified. From this information,
bonding type and functional groups in the sample are
identified.
The size and shape of nanoparticles are best studied using
FTIR Spectroscopy. When compared with bulk form,
Nanoparticles have very large surface. The properties of
the nanoparticles changes when the size changes.

42

Particles formation, adsorbents on the surface of
nanoparticles is measured accurately using FTIR
Spectroscopy. It obeys the bonding and type of bonding
between polymers and nanoparticles. It also conform the
elements present in the sample at Nano scale.
1.8.6 UltravioletVisible Spectroscopy (UV-Vis.
Spectroscopy)
The electronic transitions and electronic structure of the
sample is correctly measured using UV-visible
spectroscopy. A sample in solution form is prepared and
is introduced in the UV-visible spectrophotometer. A
given range of wavelength is measured under ultraviolet
visible region of the spectrum. Sometimes both sample
under observation and reference sample are run at the
VDPHWLPH max is determined correctly through canning
process. This spectrophotometer functions on the
principle of Beer-lambert law. Mathematically, this law
can be explained as:
A=log I/Io or A = cx
Where x = path length of the sample, C = concentration.
= constant of molar concentration

43

7KLV ODZ LV GHILQHG WKDW WKH DEVRUSWLRQ LV GLUHFWO\
proportional to the molar concentration if intensities of
incident and transmitted light are I and Io 7KLV
technique of analysis is very helpful in chemical analysis
and research work because it is utilized for qualitative
and as well as quantitative analysis. This technique is
used to measure the chemical characterization of
nanoparticles. The particles size of the nanoparticle is
estimated correctly by using this technique. Mono
dispersive or poly dispersive particles are estimated using
this technique.
1.9 Abutilon indicum (the plant used in present study)
An erect, woody and shrubby plant, named Abutilon
indicum (local name; peely booti or karandi) extensively
spread in the tropical countries like Pakistan. The aim of
the present study was to collect the extract of leaves and
to manufacture CuO nanoparticles. After the preperatiton
of nanoparticles, biomedical uses of CuO nanoparticles
are determined. The plant used in the study i.e. Abutilon
indicum is classified in the plant family malvaceae. From
the plant Abutilon indicum, Alkaloids, flavonoids,

44

steroids, terpenoids, glycosides, saponins and many more
compounds have been prepared. The extract of this plant
is very useful for the treatment of ulcers, leprosy,
jaundice, hypoglycemia etc. a number of diseases
mentioned above are treated using this plant extract. The
treatment of cough and piles is treated by using the seeds
of this plant. For the treatment of diuretic, anthelmintic,
pulmonary sedative, haematuria and fever, its bark is
used which plays a vital role against these diseases.
When Abutilon indicum is used externally, swelling as
well as pains is relieved. A large number of medicines
are prepared from the seeds and roots of this plant.

45

Chapter 2
Summary for the Green synthesis of CuO
nanoparticles
Ijaz et al., [2017] described the synthesis of copper oxide
(CuO) nanoparticles using an ecofriendly technique and
were evaluated their antimicrobial, antioxidant and
photo-catalytic dye degradation potentials. A superficial
method (solution combustion method) was employed for
the synthesis of copper oxide nanoparticles from an
aqueous extract of Abutilon indicum. The CuO
nanoparticles were characterized using x-ray diffraction
(XRD), energy-dispersive x-ray spectroscopy (EDX),
scanning electron microscope (SEM) and ultraviolet-
visible (UV-Vis) spectroscopic techniques. The
antimicrobial activity of the CuO nanoparticles was
determined by agar well diffusion method, while their
antioxidant properties were assessed by DPPH radical
scavenging, ferric reducing antioxidant power (FRAP),
total antioxidant, ferric thiocyanate (FTC) and total
phenolic content (TPC) assays. The photo-catalytic

46

degradation activity of synthesized CuO nanoparticles
was assessed by the degradation of Acid Black 210 (AB)
dye under sunlight irradiation. XRD, EDX and SEM
results confirmed the successful synthesis of CuO
nanoparticles of 16.78 nm sizes, with hexagonal, wurtzite
and sponge crystal structure. Photo-catalytic data
revealed that the nanoparticles are a good catalyst for
effective degradation of Acid Black 210. The
nanoparticles also exhibited remarkable antioxidant
activity, with IC50 and FRAP values ranging from 40
0.23 to 84 0.32 g/ml, and 0.65 0.01 to 9.10 0.21
Trolox equivalent/mL, respectively. Significant
bactericidal activity was manifested by the CuO
nanoparticles against Klebsiella and Bacillus subtilis with
zone of inhibition of 14 0.05 and 15 0.11 mm,
respectively. The synthesized CuO nanoparticles exhibit
antibacterial and antioxidant potential, indicating that
they are good candidates for future therapeutic
applications.

47

 Figure 2: Green synthesis of CuO NAPs using Abutilon
indicum through combustion method.
Fatih et al., [2016] investigated that when extract of
medicinal plant (Matricaria chamomilla) was used, the
fabrication of copper oxide nanoparticles (CuO NPs)
showed both reducing and capping agent. They also
investigated the antioxidant properties of the
nanoparticles and their interface with DNA plasmid. For
the determination of chemical characterization and

48

morphology of nanoparticles, different techniques were
used e.g. UV-Vis spectroscopy, FTIR, DLS, XRD, EDX,
SEM etc. The CuO NPs exhibited diameter of 140 nm
size. The shape of such type of particles is spherical or
somewhat mono-distributed.

Figure 3: Microwave assisted green fabrication of CuO

NPs using M. chamomilla
Gowdhami et al., [2016] studied the antibacterial activity
of greenly synthesized CuO nanoparticles by the stem

49

extract of plant Cissus quadrangularis. The
characterization of nanoparticles were synthesized by
UV-Vis spectroscopy, SEM, XRD, FTIR and further
subjected for antibacterial activity against the pathogens
S. aureus, E.coli, K. pneumoniae, P. aeruginosa, S. typhi,
S. dysenteries and V. cholera. The synthesized particles
had the hexagonal and cubic in shape. The diameter of
these particles was about 58 nm according to their SEM
photograph. The product obtained existed in pure phase
as it is confirmed from XRD results. Intensive peaks are
seen with blue shifts in them when the spectrum was
observed using FTIR, which indicated structure
crystalline and shorten the distance of lattice. They
deduced that these Nps showed a great resistance against
all the pathogen tested The nanoparticles used in
pharmaceutical industry as bactericide, disinfectant and
germ killer, and make this method of green synthesis are
reliable method for large scale synthesis of inorganic
materials.

50

 Figure 4: Microwave irradiated green synthesis of CuO
NAPs using Cissus quadrangularis
Naika et al., [2015] explained the solution combustion
method. This is a greener process because Gloriosa
superba L. extract used as fuel to synthesize cupric oxide
nanoparticles. Monoclinic nature of copper oxide
nanoparticles were analyzed by XRD. Blue shift value

51

cupric oxide nanoparticles were measured from UV-
Visible absorption spectrum. CuO nanoparticles are
spherical in nature was confirmed with the help of SEM
images. Transmission electron microscope characterized
the size of copper oxide nanoparticles in the range of 5-
10 nm. Cupric oxide nanoparticles were efficiently
showed antimicrobial application against pathogenic
microbial strains namely K. aerogenes, P. desmolyticum
and E.coli are Gram negative and S. aureus are Gram
positive bacteria.

52

 Figure 5: Muffle furnace irradiated green synthesis of
CuO NAPs using Gloriosa superba
Jayakumarai et al., [2015] used the leaf extract of Albizia
lebbeck for the green synthesis of copper oxide
nanoparticles which was very simple, effective and rapid
research. The synthesized CuO NPs were characterized
by using SEM, EDS, TEM, XRD and UV-Vis
spectroscopy. The size of copper oxide nanoparticles is
less than 100 nm and they all are spherical in shape. It

53

was proved by them that the extract of A. lebbeck leaves
can be used as a cheap and effective reducing agent for
the bulk production of CuO NPs.

Figure 6: Green synthesis of CuO NAPs using Albizia

lebbeck
Kumar et al., [2015] reported green approach to
synthesize of cupric oxide nanoparticles. This is
biological and eco-friendly method. They fabricated them
using harmless Aloe vera leaf extract. (PXRD) and TEM

54

were used for the characterization. These fabricated
particles were found in monoclinic phase with average
particle size of 20 nm. They also showed the enhanced
antibacterial activity of these nanoparticles against some
bacteria which cause serious infections in fish e.g. A.
hydrophila, P. fluorescens and F. branchiophilum even at
lower concentrations.

Figure 7: Green synthesis of CuO NAPs using Aloe vera

55

The method of solution combustion was studied by
Lingaraju et al., [2015] for the synthesis of Copper oxide
nanoparticles. The extract of aqueous leaf of Rauwolfia
serpentina was used as fuel for combustion. PXRD,
SEM, UVvisible, and TEM were used for
characterization and morphology of the nanoparticles.
They produced monoclinic crystals as revealed by PXRD
patterns. Large surface area of sponge-like image was
shown by SEM. Agar well method of diffusion was used
for the antibacterial properties of CuO nanoparticles
against Gram negative E. coli (NCIM-5051) and P.
desmolyticum (NCIM-2028) and Gram positive bacteria
S. aureus (NCIM- 5022).

56

 Figure 8: Green synthesis of CuO NAPs using
Rauwolfia serpentina
Udayabhanu et al., [2015] also reported solution
combustion method. This green approach is very
important for the synthesis of cupric oxide nanoparticles
(CuO NAPs). Tinospora cordifolia water extract was

57

used in this method. They used XRD, SEM, TEM and
UVvisible for morphology and characterization of these
nanoparticles. The prepared nanoparticles were
monoclinic crystallite structures as shown by XRD.
Sponge like structure and large surface area was
determined from SEM images. Average crystalline sizes
were characterized in TEM which were found to be 68
nm. CuO Nps showed potential pathogenic antibacterial
activity against K. aerogenes, P. aeruginosa, E. coli and
S. aureus. This method was eco-friendly and nontoxic for
the preparation of versatile cupric oxide nanoparticles
applying under-utilized remedial plants.

58

Figure 9: Green synthesis of CuO NAPs using Tinospora
cordifolia
Awwad et al., [2015] reported synthesis of CuO
nanoparticles by a facile and green method using Malva
sylvestris leaf extract. The copper oxide nanoparticles
were analyzed to characterize from these techniques FT-
IR, SEM, XRD and UV-vis spectroscopy. The average

59

size of crystallites was 14 nm. Important feature of this
fabrication was the tunability of the structural features of
the cupric oxide nanoparticles by controlling the ratio of
the amount of Malva sylvestris leaf extract to copper
ions. The synthesized CuO NPs exhibited important
antimicrobial activity against pathogenic strain both
Gram-positive and Gram-negative microbes.
Brajesh et al., [2015] reported biologically fabricated
copper oxide nanoparticles (CuO-NPs) of a desired size.
In their study, 10 mM solution of copper nitrate with
plant extract Andean blackberry fruit (ABF) and leaf
(ABL) was used to prepare cupric oxide nanoparticle.
They also assessed its antioxidant activity. CuO-NPs
were characterized with the help of UVvisible, TEM
and XRD analysis. Antioxidant activities were also
reported in their studies.
Reyad-ul-ferdou et al., [2015] explained that Abutilon
indicum is largely grown in South Asia. A.indicum plant
contain large amount of chemical constituents which was
utilized in folk medicine. Golden yellow flowers and
hairy stem of A. indicum mostly found in Pakistan and

60

India. Abutilon indicum plant leaves were used to cure
different diseases such pulmonary, erotic and sedative.
Plant bark, seeds, roots and oil were employed in several
diseases like astringent diuretic, expectorant, demulcent,
anti-inflammatory and anthelmintic. In pile complaints,
plant leaves were used for treatment. Flowers of these
plant contained significant constituents which enhanced
semen in men. Alkaloid, Saponins, Amino acid,
Flavonoids, Glycosides and steroids was present in plant
which was measured in phytochemical analysis. This
plant exhibits several potential pharmacological
activities.
Acharyulu et al., [2014] reported the biological method
for green fabrication of CuO nanoparticles they used
extract of Phyllanthus amarus leaf which was eco-
friendly and non-toxic. Powder XRD analysis showed
that CuO nanoparticles had an average size are in 20 nm
while their shape was found to be monoclinic. These
particles also proved to have antibacterial properties
against Gram +ve (B. subtilis and S. aureus) and Gram -
ve (E.coli and P. aeruginosa). Antibacterial action was

61

reportedly much stronger against B. subtilis followed by
S. aureus, P. aeruginosa and E.coli.
Abboud et al., [2014] also reported biologically oriented
fabrication of nanoparticles. They have claimed to have
adapted a cheaper, cleaner and efficient technique for
fabrication of these nanoparticles. They reported the first
time use brown alga (Bifurcaria bifurcata) for the
fabrication of copper oxide nanoparticles of dimensions
5-45 nm. They used UVvisible and FT-IR spectrum
analysis for the characterization of these nanoparticles.
X-ray diffraction was used to characterize the
crystallinity of copper oxide nanoparticles. They also
reported the antibacterial action of these nanoparticles
against gram +ve and gram -ve bacteria.
Rajeshwari et al., [2014] explained an eco-friendly
method. In this study, Indian medicinal plant
Tabernaemontana divaricate extract was used to prepare
cupric oxide nanoparticles which are biosynthesis
method. 50% concentration of leaf extract was used to
synthesize highly stable and spherical in shape cupric
oxide nanoparticles. UV-Vis, XRD, FT-IR, SEM, EDX

62

and TEM these techniques were used to characterize
nanoparticles. The average sizes 484 nm was
synthesized. In this research, they observed antimicrobial
activity of cupric oxide nanoparticles against urinary tract
organisms.

Figure 10: Green synthesis of CuO NAPs using

Tabernaemontana divaricate
Ali et al., [2014] reported a new simple and cost-
effective chemical reduction process. In this method
these chemicals CuSO4, NaOH and NH3 were used in situ
to synthesize cuprous oxide nanoparticles on cotton
fabric. In reduction process copper sulphate reduce in the

63

presence of cotton fabric which act as reducing agent and
help to prepare Cu2O in nanometer range as a stabilizer.
These techniques such as XRD, SEM and EDX were
subjected to characterize synthesized cotton/nano
cuprous oxide composite. To observe the alter functional
group of cotton in addition with cuprous oxide
interaction with cotton fabric were studied using Fourier
transforms infrared spectroscopy. Fabric nanocomposites
were used against pathogen like S. aureus and E.coli in
antibacterial activity. Moreover, the treated fabric
developed into highly hydrophobic and sensed.

64

 Figure 11: Synthesis of cuprous oxide nanoparticles on
cotton fabric
Prasanta et al., [2014] reported green approach toward
synthesis of cupric oxide nanoparticles. In this green
approach, coffee powder and tea leaves were used as
extract for the synthesis of CuO under microwave
irradiations. By using microwave at 540W, cupric oxide
nanoparticles were synthesized to irradiate metal salt and
extracts of coffee and tea in 3:1 ratio for 7-8min. SEM,

65

XRD, FT-IR and UV-Vis were used to characterize the
prepared particles. To check out the activity of these
particles against six human pathogenic organisms. These
particles showed excellent activity against these
pathogenic bacteria. These CuO nanoparticles offered
several benefits of eco-friendliness and compatibility for
pharmaceutical and other biomedical applications.
Renu et al., [2014] studied green process in the formation
of cupric oxide nanoparticles. 5 mM CuSO4 treated with
plant leaves extract (Carica papaya).UV-Vis
spectrophotometer was used to measure the kinetics of
the reaction. Characterization techniques such as SEM
and DLS showed rod shape cupric oxide nanoparticles
have average particle size 140 nm. Copper ions reduced
in the presence of bioactive functional groups which
were examined through FT-IR spectrometer. Crystallite
nature of cupric oxide nanoparticles were confirmed from
XRD spectra. Cupric oxide nanoparticles have catalytic
behavior. Coomassie brilliant blue R-250 dye effectively
degraded by cupric oxide nanoparticles in the beneath of
sunlight.

66

 Figure 12: Green synthesis of CuO NAPs using Carica
papaya
Henam et al., [2014] reported use of Centella asiatica
leaves extracts for the preparation of copper oxide
nanoparticles at room temperature. This method is
completely a green and environmental benign method.
No toxic and harmful solvent was used in this process.
Morphology and size were characterized using SEM,
UVVisible, IR and EDX. CuO NAPs prepared by this
method were suggested to be used for the photo catalytic

67

degradation of methyl orange. This catalytic effect of
copper oxide nanoparticles can be contributed to its small
size. Nanoparticles have many active sites as compared
to the bulk materials because of its greater S/ V ratio.

Figure 13: Green synthesis of CuO NAPs using Centella

asiatica
Ipsa et al., [2013] studied a novel biological method. In
this method, they were synthesized Copper nanoparticles

68

using clove. In the presence of clove extract, copper
sulphate was reduced to copper nanoparticles. Crystallite
structure of cupper nanoparticles was confirmed with
XRD. TEM revealed copper nanoparticles size in the
range of 5-40nm. Spherical and granular nature of copper
nanoparticles was investigated from SEM. Copper
nanoparticles peak was characterized to use UV-Visible
absorption spectroscope.

Figure 14: Green synthesis of CuO NAPs using

Syzygium aromaticum (Clove)

69

Vinod et al., [2013] reported that Copper oxide (CuO)
nanoparticles have catalytic, antibacterial, photonic,
electric, and optical and nanofluid activities and these
properties, as all other nano materials depend on aspect
ratio and shape of these particles. They showed the
fabrication of these nanoparticles by green technology
using a natural nontoxic hydrocolloid, Gum karaya, and
investigated the antibacterial properties of CuO
nanoparticles.
Gopalakrishnana et al., [2012] reported the synthesis of
cuprous oxide nanoparticlHV IURP )HKOLQJV VROXWLRQ
using Tridax procumbens leaf extract. By using green
technology, they synthesized nanoparticles which is not
involved any toxic chemicals. Plant shoot and leaves
contained water soluble carbohydrates which played an
important role to copper ion in nano-size cuprous oxide
particles. Cuprous oxide nanoparticles surface coated
with polyaniline by using H2O2 as oxidizing agent which
create resistance against oxidation of cupric oxide.
Synthesize cuprous oxide nanoparticles and PANI coated
cuprous oxide were characterized at different analytical

70

techniques such as XRD, SEM, UV-Vis and FT-IR
spectroscopy. These particles were evaluated against a
variety of surplus infections and toxins in human like E.
coli by using disc diffusion method.
Soheyla et al., [2012] reported biosynthesis of copper
oxide nanoparticles using microorganisms which is an
emerging eco-friendly method of fabrication. They
synthesized copper oxide NPs having well-defined sizes
and shapes. They used green process for the production
of nanoparticles. And stabilization was done using P.
aurantiogriseum, P. citrinum and P. waksmanii. These
stabilizing species were extracted from soil. The NPs
fabricated this way had uniform size with spherical
shape. They characterized the shape and size by using
SEM. DLS was applied to determine the polydispersity
of NPs. The outcomes originate by SEM and DLS was
further tested by UV-VIS and fluorescence spectrum.
Jiao et al., [2012] reported metal hyper-accumulator plant
process. From metal hyper-accumulator plant process,
CuO/ZnO nanoparticles were synthesized. This method
commonly used to clean soil polluted toxic and heavy

71

metals. In this study, they suggested that toxic metals
were obtained from plant shoots and leaves. These toxic
metals transferred from plants to animal and to human
beings. So hyper-accumulator plant is better way to
prepare nanoparticles. Near copper mine they collected
Brassica juncea leaves which utilize as raw chemical to
synthesize Cu/ZnO nanoparticles. For extraction, ethanol
was plant leaves and extracted Copper and Zinc reacted
to HNO3 to from Cu/Zn (NO3)2. Sodium hydroxide and
ethanol was used to precipitate the Cu/Zn chlorophyll.
About 97nm diameter of synthesize nanoparticles.
Panneerselvam et al., [2011] reported the production of
CuO/ZnO nano-photocatalyst light assisted degradation
of textile dye in an aqueous solution by the wet
impregnation technique. The CuO/ZnO samples were
annealed at the 550 C for 5 hours were changed from the
purple to grey color, which indicates that CuO clusters on
the ZnO may not be metallic Cu but it may be the CuO-
ZnO. Characterization was done by TEM, DRS and XPS
analysis which confirms that only CuO phase is present
as the prepared sample.

72

Nekrasova et al., [2011] investigated the effects of
copper oxide nanoparticles and copper ions on the
activities of antioxidant enzymes, lipid peroxidation rate
and photosynthesis. They reported that NPs were
accumulated much actively by the plants as compared to
the larger particles of same substance. They also showed
that the lipids can be oxidized by both copper ions and
NPs. Catalase activities were increased by factor of 1.5 to
2.0 with NPs. Rate of photosynthesis was depressed in
the presence of concentrated copper ions and in the
presence of NPs, whereas in the presence of NPs, same
depression was observed at 1.0 mg/l.
Rajendra et al., [2011] nanostructured crystalline copper
oxide were prepared by the assistance of
alcohol/nonionic polymeric surfactant. For
characterization TEM, SEM, XRD, FTIR and UV-Vis
were used. It was observed that surface area of metal
nanoparticles depend upon the ratio of alcohol to anionic
surfactant. Greatest surface area was obtained by using
methanol in the synthesis process. Catalytic activity was
checked for freshly prepared CuO nanoparticles.

73

Kooti et al., [2010] suggested a simplistic method for
preparation of Cu2O NPs by reduction of the Fehling's
solution, using glucose as the reducing agent. Method
was very simple and facile. They used copper sulfate as a
starting material and added potassium sodium tartrate in
alkaline media for the production of Fehling's solution.
Triton-X 100 was used as the surfactants to control the
production of Cu2O NPs from Fehling solution. The
resulting Cu2O NPs were characterized by XRD, SEM,
EDX, TEM and FTIR spectroscopy.
Abdul et al., [2009] stated a novel suitable method to
synthesize of cupric oxide nanoparticles using
Phormidium cyanobacterium. They suggested that
synthesis of copper oxide nanoparticles occurs by
extracellular hydrolysis of the copper ions by certain
anionic reductase produces by bacteria at ambient
conditions and aerobic environment. Proteins reduce Cu
(II) into copper oxide nanoparticles and help stabilization
of formed nanoparticles at room temperature. These
biologically synthesized NPs were characterized by
TEM, SEM, XRD and FTIR analysis.

74

Guogang et al., [2009] attempted successfully the
synthesis and characterization of copper oxide (CuO)
nanoparticles with respect to the antimicrobial
applications. They showed the presence of pure Cu and
CuO nanoparticles in nanoscaled CuO, produced by
thermal plasma technique. Size of particles was found in
the range 20 to 95 nm by using the TEM. The
nanoparticles of CuO were tested against a range of the
bacterial pathogens, including S. aureus and E. coli and
were found effective with minimum bactericidal
concentrations ranging from the 100g/mL to
5000g/mL. The antibacterial activity of CuO NAPs to
decrease bacterial population up to zero was possible in
the presence silver nanoparticles.
Fei et al., [2008] reported synthesis of CuO particles by
hydrothermal method. They investigated the influence of
the hydrothermal temperature and time on growth of
NAPs. XRD, SEM, HRTEM, TEM, and ED were used
for the characterization. The flower-like structures
showed high chemiluminescence intensities and the
reactivates for the CO oxidation.

75

Dongyun et al., [2008] studied TritonX-100 based water-
in-oil reverse micelles system. In this research, cupric
oxide nanoparticles were synthesized using TritonX-100.
Different techniques were employed to characterize
nanoparticles such as XRD and TEM. The ratio of water
to surfactant played an important role in synthesis of
grain size, morphology and distribution. The control
effect of water content dissolved in reverse micelles was
associated to the improvement of the exchange rate of
reactants among micelles. This control effect also
depends upon the relevant change in the interfacial film
flexibility of reverse micelles with the increasing amount
of water in the micro emulsion.
Chun-Hong et al., [2007] reported the synthesis CuO
NAPs with the estimated average sizes of 40 to 420
nanometers. They adapted seed-mediated method in the
aqueous solution. After 2 hours, nanocubes are
synthesized at room. Well-defined Cu2O nanocubes were
synthesized to react CuSO4 with sodium dodecyl sulfate
reaction mixture. The nanocubes most probably, formed
by the controlled aggregation of the Cu2O particles. The

76

surface rebuilding of nanocubes influenced through SDS
surfactant and the sulfate ions. Optical characters showed
nanocubes are smaller than the 100 nm absorbs at 490
nanometer. Absorption band at the 515525 nm showed
that the size nanocubes were larger than 200 nm.

77

Chapter 3 Materials and Methods
3.1 Chemicals
Cupric Nitrate Trihydrate
Acid Black 210
Copper Oxide Nps
Nutrient ager
Nutrient Broth
Methanol
DPPH
BHT
Distill water
3.2 Glassware
Glass Conical Flasks
Glass Beakers
Test tubes and stand
Glass Petri plate
Glass Measuring Flasks
Pipettes
Micropipette
Cork Borer
Sprit Lamp

78

 Quartz Cuvettes
China dish (100ml)
Steel spatula
3.3 Instrument
Incubator Shaker
Incubator (Oven)
Spectrophotometer
Autoclave
Electronic Water Bath
Ultrasonic Shaker Bath
Laminar flow
Electronic milligram balance
3.4.1 Collection of Plants Materials
Fresh leaves of Abutilon indicum were collected
from Christain graveyard Jallo and near ring road
interchange Barki, District Lahore, Pakistan. The leaves
of Abutilon indicum were identified and authenticated by
Taxonomist, Department of Botany, G.C University,
Lahore, Pakistan.
3.4.2 Extract Preparation

79

 Fresh leaves of Abutilon indicum were taken from
the Jallo and washed gently for the removal of dust
particles. After washing the leaves from tap water, these
leaves were dried in a shady place at the temperature near
to room temperature. After drying, the leaves were
ground to powdered form using mixer grinder and sieved
from 200 mesh. Powdered plant material was poured in
Soxhlet apparatus for continual solvent extractions. The
temperature of the apparatus was set at 90-950C and it
takes approximately 3 hours for completion. After
experimentation, the solution was filtered using cintered
glass crucible and further concentrated. For
concentration, rotatory flash evaporator was used and
vacuum was created for this purpose. Furthermore, the
solution was poured in water bath for drying of solution.
The crude plant extract was taken in an air-tight bottle
and stored in refrigerator.
3.4.3 Nano particles synthesis
CuO nano-particles synthesized by eco-friendly
green combustion route using Abutilon indicum plant leaf
extract as fuel. The Cu(NO3)2.3H2O was taken from lab.

80

1.205g of Cu (NO3)2.3H2O was mixed with 0.3g Abutilon
indicum extract in 20ml of double distilled water. The
solution was homogenized for 2-5min with constant
stirring. The combustion of mixture was done in muffle
furnace at 400 5C. The combustion was completed
within 2-3 min. Fine material of CuO nanoparticle was
obtained. Then resultant solution is filtered. Continuous
washed this solution with distilled water and methanol to
remove impurities. The obtained Cupric Oxide
nanoparticles were calcinate for 2h to attain pure form.
Black fine CuO nanoparticles obtained. These particles
stored in airtight container.
3.5 Characterization
The synthesized CuO nanoparticles were
characterized by using Energy Dispersive X-ray to
analyze composition. The absorption spectrum of the
sample was analyzed on UVvisible spectrophotometer.
Morphological features of prepare nanoparticles were
determined using Scanning Electron Microscopy (SEM).
3.6 Photocatalytic Degradation of Dye

81

 The activity of CuO nanoparticles were estimated
by the degradation of Acid Black 210 (AB) dye under
sunlight irradiation following the method described by
Khan et al., [2018]. Take 0.005g of synthesized CuO
nanoparticles with 5 mL distilled water in test tube and
ultra-sonicated to disperse particles in aqueous solution
for activity. 10ml of (13 mM) Acid Black 210 dye
solution was added in test tube with 5ml Cupric Oxide
nanoparticles solution. To obtain adsorption-desorption
stage, prepared solution was kept in the dark for 30
minutes. CuO nanoparticles suspension with acid black
210 was exposed for different time intervals (20, 40, and
60 minutes).This experiment was performed (between
11am to 1pm). Then the obtaining suspensions were
centrifuged for two minutes. The cons. of the Acid Black
210 in the resultant solution was monitored by UV-
visible spectrophotometer in the wavelength range of
200800 nm. The distill water is used as reference. To
measure molarity given formula was used.

82

 
Molarity = x




3.7 Different Antioxidant Activities

3.7.1 Radical of 2, 2-diphenyl-1-picrylhydrazyl
Scavenging Assay
By using Khan et al., [2013] method, the known
antioxidant value of butylated hydroxytoulene was
compared with unknown values of given sample of 2, 2-
diphenyl-1-picrylhydrazyl. 3ml of methanolic solution of
DPPH (0.1mM) was mixed with different concentration
of samples. At room temperature, the mixture containing
components was continuously mixed and after it rest for
one an hour. The absorbance of given solution was
measured at 517 nm wavelength against methanol as
blank in the spectrophotometer. Greater concentration of
free radical scavenging activity was examined at lower
DEVRUEDQFH LQ VSHFWURSKRWRPHWHU ,Q WKLV IRUPXOD F
VWDQGIRUFRQWUROOHGDQG6VWDQGIRUVDPSOH
Antioxidant activity = AC AS /AC 100
Involved calculation:-

83

 1. DPPH 0.1mM (0.04g/L)
2. Sample solution 1mg/ml (1g/L)
Further dilution i.e 0.5 mg/ml, 0.25 mg/ml, 0.125 g/ml,
0.06 mg/ml are made from stock solution.
3.7.2 Total Antioxident Activity by
Phosphomolybdenum
By using Khan et al., [2016] and Ahmad et al.,
[2017] Phosphomolybdenum complex formation method,
the total antioxidant activities of samples are evaluated.
4ml of reagent solution (0.6M sulphuric acid, 28mM
sodium phosphate and 4mM ammonium molybdate) was
added with 0.5mg/ml of the sample in test tube. The
blank solution contains 4ml of reagent solution. The test
tubes are caped and incubated in water bath at 95C for
ninety minutes. The incubated samples were cooled at
room temperature and the absorbance was measured at
695nm against blank. The antioxidant activity is
expressed relative to that of butylated hydroxytoulene
(BHT).
Involved calculation:-
1. 28mM sodium phosphate [10.64g/L]

84

 2. 4mM ammonium molybdate [4.94g/L]
3. 0.6M sulphuric acid [33.4 ml/L]
10.64g of Sodium phosphate, 4.94g of ammonium
molybdate and 33.4ml of sulphuric acid are dissolved in
one Littre distilled water.
3.4.3 Farric Reducing Antioxidant Power (FRAP)
By using Khan et al., [2017], the FRAP assay is
done to evaluate sample activity. The stock solution are
synthesized by mixing 10mM TPTZ (2,4,6-Tripyridyl-s-
triazine), 300mM acetate buffer (3.1g sodium acetate
trihydrate and 16ml acetic acid ) ;and 20mM ferric
chloride hexahydrate with pH 3.6 solutions in 40mM HCl
solvent. Before using, 25ml acetate buffer, 2.5ml TPTZ
solution in 2.5ml ferric chloride hexahydrate solution
was prepared and then warmed at 37C for working.
Trolox and sample solutions are synthesized in methanol
(1mg/ml). 10 L of each of sample and BHT solution are
taken in separate test tubes and 2990 L of FRAP
solution are added in it to make total volume up to 3ml.
For 30 minutes FRAP solution mixed with sample for
reaction in dark. Reading was measured of colored

85

product (ferrous tripridyltriazine complex) at 593nm. The
FRAP values are determined as micromole of Trolox
equivalent per mL of sample by computing with standard
calibration curve constructed for different cons. of
Trolox. Results are expressed in TE M/mL.
Involved calculation:-
1. TPTZ 10mM [3.12g/L]
2. HCl 40mM [3600 L/L]
3. Fecl3.6H2O 20mM [5.4g/L]
4. Trolox 1M [250g/L]
3.7.4 Total Phenolic Content (TPC)
By using Khan et al., [2017] method, total
phenolic of sample are examined. 0.1ml (0.5mg/ml) of
sample was added with 0.1ml of 2N Folin-Ciocalteu
reagent and 2.8ml of 10% Na2CO3.From UV-visible
spectrophotometer absorbance was measured after 40min
at 725nm. Total phenolic are examined as milligrams of
Gallic acid equivalents per gram of sample by computing
with standard calibration curve constructed for different
concentration of Gallic acid. The standard curve is linear

86

between 0.05mg/ml to 0.4mg/ml of Gallic acid. Results
are expressed in GAE g/mL.
Involved calculation:-
1. F.C Reagent 2N
2. Na2CO3 10% [100g/L]
3.7.5 Ferric Thiocyanate (FTC) Assay by Linoleic
Acid
By using Khan et al., [2017] thiocyanate method,
the antioxidant activity of sample on inhibition of linoleic
acid peroxidation is studied. The 2.5ml of linoleic acid
emulsion (0.02M,pH 7), 2.0ml of phosphate buffer
(0.02M,pH 7) and 0.1ml of sample solution (0.5mg/mL)
are combined.0.28g of linoleic acid,0.28g Tween-20 as
emulsifier and 50.0mL of phosphate buffer are mixed
together to prepare linoleic acid emulsion. For 5 days,
this reaction mixture was heated using incubator at 400C.
For controlling the solution, the mixture without sample
was used. In a beaker, 5ml of 75% alcohol, 0.5ml of 30%
ammonium thiocynate and 0.1ml of 20mM ferrous
chloride (in 3.5% HCl) was mixed and 0.5ml of mixture
was mixed in it and left for some time (about three

87

minutes) at room temperature. After adding FeCl2 to
reaction mixture, absorbance was recorded using 592nm
wavelength. The antioxidant activity was expressed as
percentage inhibition of peroxidation (IP%).
IP% = [1-(abs. of sample)/(abs. of control)] 100
Involved calculation:-
1. Disodium hydrogen phosphate
0.02M [2.84g/L]
2. Ferrous chloride
20mM [3.99g/L]
3. Hydrochloric acid
3.5% [94.6mL/L]
4. Ethanol
75% [3L/L]
5. Ammonium thiocyanate
30% [3g/30mL]
3.8 Antibacterial Activity
The bacteria E.coli, B.subtilis, Staphyloccus
aureus and Klebsiella was used in the present research
for the antibacterial activity of CuO nanoparticles. These
classes of bacteria were used due to following reasons.

88

The class of bacteria E.coli is a type of Gram negative
bacteria. These bacteria cause different type of infections
such as urinary tract infections, food poisoning, neonatal
meningitis etc. [Khan et al., 2017; Khan et al., 2016].
The class of bacteria i.e. B. subtilis, Staphyloccus
aureus, Klebsiella are a type of Gram positive bacteria
creates infections like pneumonia, impetigo, sepsis,
urinary tract infections, septicemia, meningitis, cellulitis,
diarrhea, tissue infections, Boils, food poisoning and
toxic shock syndrome etc. in living organisms. In this
activity Agar well diffusion method was employed to
check the retorted growth of antimicrobial activity of
cupric oxide nanoparticles in living organisms. For
positive control, bacteriological ampicillin was taken as
Standard antibiotic while for negative control of all the
species, only distilled water was used.
3.8.1 Sample Preparation
The Ultra sonication method was used for solution
preparation of CuO nanoparticles. And this solution was
prepared in distilled water. Two different concentrations
of nanoparticles solutions were prepared i.e. 3mg and 5

89

mg. 1 mg concentration of the reference antibiotic was
used for the present study.
3.8.2 Preparation of Nutrient Broth
In a beaker, 0.8g of nutrient broth was weighed
and 100ml of distilled water was poured in it for the
preparation of nutrient broth solution [Hussain et al.,
2017]. When the solute dissolved completely in the
water, it was heated for short duration to obtain the clear
solution. The solution was buffered at a pH of 7.4. The
autoclave was used for the sterilization of sample at
1210C for 15min.
3.8.3 Preparation of Inoculum
For the preparation of inoculum, firstly laminar
was rinsed with methanol for sterilization and disinfected
by UV radiation for ten minutes further to kill the germs
as much as possible. The sample of broth was subjected
in laminar for slow cooling. The bacteria culture slant
was taken from the refrigerator and poured in laminar
flow. A chrome wire was taken and subjected to spirit
lamp. When red hot, dipped in slant and then transmitted
in nutrient broth. Now the nutrient broth prepared, was

90

introduced in shaker incubator for 24 hours. The
temperature of the culture was maintained 37C. Now
this medium was used as inoculum.
3.8.4 Preparation of Nutrient Agar
100 ml of distilled water was taken in a conical
flask. 0.8 g of nutrient broth and 1.3g of nutrient agar
was drizzled in it and stirred well and heated gently until
a clear solution was obtained. Again the pH was adjusted
at 7.4 for this purpose. The mixture was put in autoclave
for sterilization for 15 min. The temperature of the
medium was set at 121C.
3.8.5 Preparation for petri dishes
Took two petri dishes and sterilized in autoclave
for 15 min and placed in laminar flow. Nutrient agar was
taken and transferred in laminar flow for some time. Now
20 ml of agar was poured in petri dishes. Inoculum was
taken from shaker incubator and poured 1 ml in petri
dishes. Shake well and left for solidification for some
time. Cork borer was sterilized and for holes at peripheral
positions were made in solidified medium. The
concentrations of CuO were subjected into two of these

91

holes with the help of micro pipette. In other two holes,
water was introduced in one hole for negative control,
and standard ampicillin was poured in the other hole for
positive control. The plates prepared as above were
subjected into incubator. The temperature of the culture
was maintained at 37C. After 24 hours, the petri dishes
were taken and zone diameters around the wells were
examined and recorded.

92

Chapter 4 Results and Discussion
4.1 Scanning Electron Microscope
The morphological and structure properties of CuO
nanoparticles were observed using scanning electron
microscopy (SEM).The images taken by SEM of the
synthesize particles are shown in figure. The SEM
images show that the synthesized particles have size
which is not in nanometer range and their structures are
not homogenous. From figures 15, 16, 17 it is simply
experimental that the particles prepared are in nanometer
range. From these images shown below indicate that only
few particles which spherical shape has been synthesized.
Some nanoparticles are well separated from each other
while most are present in agglomerated form. Thus, these
SEM results confirmed the nanostructure behavior of the
synthesized particles.

93

 Figure 15 SEM images of Copper Oxide
Nanoparticles

94

 Figure 16 SEM images of Copper Oxide
Nanoparticles

95

 Figure 17 SEM images of Copper Oxide
Nanoparticles
4.2 Energy Dispersive X-ray
EDX studies were carried out in order to confirm
copper oxide nanoparticles and also to examine the
chemical formation and composition of the prepared
sample. The EDX spectra for CuO nanoparticles were
recorded which confirm the presence of all constituent
elements in the investigated sample and are represented
in table. The EDX spectra for the synthesized

96

nanoparticles are shown in figure 18. The EDX spectra
confirm the presence of CuO in the prepared sample. It is
clear from the figure.
The EDX pattern of the CuO Nps synthesized using
Abutilon indicum leaf. The EDX peak positions were
consistent with the Copper oxide and sharp peaks of
EDX indicate the crystalline structure. Other Peaks were
observed due to impurities Diffraction Standards. The
strong intensity and narrow width of CuO diffraction
peaks indicate that the resulting products were of highly
crystalline in nature. Hence we can conclude that fuel has
played profound role in controlling particle size.

97

 
Figure 18 EDX spectra of Copper Oxide
nanoparticles

98

 Table 1. Constituent Element and their percentage
4.3 Photocatalytic dye degradation
In the present study, aqueous solution of 13 M ACID
BLACK 210 dye was used which was catalyzed with
CuO nanoparticle as photo-catalyst. For different time
intervals, it was introduced in UV-visible absorption
spectrophotometer as shown in figure 19. The sunlight
exposure time was monitored at 610 nm for ACID
BLACK 210 and a characteristic absorption peak was
obtained. The degradation of the dye was continuously
monitored at different time intervals. It was examined
that after one hour only, this dye had been degraded
completely.
The disintegration of organic dye takes place as follows.
&XSULFR[LGHOLJKWHOHFWURQKROH
(OHFWURQR[\JHQPROHFXOHVXSHr oxide free radical
+ROH+\GUR[\OLRQ+\GUR[\OIUHHUDGLFDO
+ROH:DWHU3URWRQ+\GUR[\OIUHHUDGLFDO
+\GUR[\OIUHHUDGLFDO$FLG%ODFN3URGXFWRI
Degradation

99

 2[\JHQPROHFXOHIUHHUDGLFDO$FLG%ODFN
product of Degradation
In the first step, cupric oxide nanostructure comes in
contact with light. It creates a photo-generated electron
and a hole. This photo-generated electron reacts with
oxygen molecule to form super oxide free radical in the
second step. In the third step, hole reacts with water and
hydroxyl ions to produce highly mercurial hydroxyl
radicals. These superoxide free radicals and hydroxyl
free radicals reacts violently with the acid black 210
organic dye and decompose/ decolorize it in the next
step. The degradation/decomposition rate of the organic
dye totally depends upon morphological and crystal
structure of photochemical catalyst.
Active sites of photocatalyst can be increased by
increasing the surface area and crystallinity which in turn
increase the effectiveness of photocatalytic reactions by
separating electron- hole pairs.

100

 These results showed that water used in the
experimental work as negative control, did not show any
antibacterial activity. Antibiotic Ampicillin which was
used as a reference is definitely much more effective for
anti-microbial activity. And nanoparticles of cupric oxide
are also very effective for affirmative control of
antibacterial activity. These particles killed/inhibited a
wide array of bacterial evolution. The anti-microbial
activity of four types of bacterial strains with zones of
inhibitions is pasted for reference in figure no 20.
From the above mentioned results, it is clear that
the concentration of 5mg CuO nanoparticle is much
effective for S. aureus, Klebsiella and B. subtilis having
ZOI 10, 14 and 15 respectively. While the concentration
of 3mg is effective for Klebsiella and B. subtilis only viz.
12 and 14. As compared with standard, it is clear that the
concentration of 5mg is much more effective for
klebsiella and B. subtilis in the range of 14 and 15
respectively. While ZOI range of standard is 8 and 12mm
in standard ampicillin.

102

 Actually, the copper ions released form CuO
nanoparticles get entered into the cell membrane of
bacteria. Here, the ions break the structure of cell
membrane by attaching with cell wall which is negatively
charged. These ions involved in cross-linkage of nucleic
acid strands by binding them with DNA molecule of
bacteria. It forms a disordered helical structure of DNA
molecule here which causes denaturation of proteins and
some other biochemical processes in the cell. Hence the
cell of bacteria completely destroys and dies in the end.
Various factors that affect the sensitivity of
bacteria towards cupric oxide nanoparticle are size of
particles, synthesis temperature of particles, structure of
bacterial cell wall, and nanoparticle contact degree with
germs. Our present study agrees that very small size of
nanoparticle is much more effective for antibacterial
activity.

103

 Figure 20 Zone of inhibition of antibacterial activity
of CuO nanoparticles

104

 Table 2. Zone of Inhibition of antibacterial activity of
CuO nanoparticles
Bacillus
Escherichia Staphyloccus Klebsiella
subtilis
Concentrations coli aureus ZOI
ZOI
ZOI (mm) ZOI (mm) (mm)
(mm)
Negative
Control
Nil Nil Nil Nil
(Water)
Nil
Positive
Control
10 12 8 12
(Ampicillin)
1mg
CuO Nps
7 10 14 15
5mg
CuO Nps 6
6 12 14
3mg

105

4.5 Antioxidant Activities
4.5.1 DPPH Free Radical Scavenging Assay
A very stable deep violet colored organic free
radical named 2, 2-diphenyl-1-picrylhydrazyl radical
(DPPH) scavenging assay contains different dilutions of
solvent Peucedanumgraveolens extracts and acts as an
antioxidant. The absorption peak of DPPH is at 515-528
nm. It change its colour from deep violet to light yellow
when it receives photons from donor atom i.e. hydrogen.
At this stage it loses its chromospheres. It is well
thought-out that the capacity of any extract of DPPH free
radical scavenging is owing to the existence of phenolic
compounds in it. As it is clear from the above discussion
that when the amount of phenolic compounds or
hydroxylation degree of such compounds increases, the
antioxidant activity of DPPH radical scavenging activity
also increases.

106

 DPPH Free Radical Scavenging Assay
100

80
IC50 (microgram/ml)

60

40

20

0
1000g 500g 250g 125g 60g BHT
Concentration of Copper oxide nanoparticles

Figure 21 DPPH Free Radial Scavenging activities

against copper oxide nanoparticles
Using DPPH assay, different concentrations of CuO
nanoparticles were observed. And results are mentioned
in the Figure 21 in the form of their IC50 values. As it is
cleared from the above mentioned fig. that the highest
value is 1000 g / mL and the lowest value is 60 g / ml.
The graph showed that the effect of Free radical
scavenging increases when the concentration increases
and more will be antioxidant activity. The IC50 values of
CuO nanoparticles increases in the following order,

107

 1000 g < 500 g< 250 g< 125 g< 60 g
In the present research, the ability of extract of DPPH
assay was determined to act as an electron or hydrogen
atom donor which is able to convert DPPH (violet color)
into reduced form i.e. DPPH-H (yellow color).
4.5.2 Total Phenolic Contents (TPC)
The total Phenolic substance of the methanol was
removed and different concentrations of CuO
nanoparticles were run ranging from 0.86 g to 19.70g per
hundred grams GAE. Most high percentage of TPC
esteem was noticed in 1000g of the methanolic
concentrate. While minimum TPC esteem was observed
in the weakening of 60g of methanolic remove in
addition CuO nanoparticles. These results are mentioned
in the following figure 22 in the form of graph. The TPC
activity of cupric oxide nanoparticles increases in the
following order.

60g < 125g < 250g < 500g <1000g

108

 Total phenolic content activity
25.00
percentage of GAE/100g

19.70
20.00

15.00
10.05
10.00
4.98
5.00 1.82 0.86
0.00
1000 g 500 g 250 g 125 g 60 g

Different concentrations of Cupric Oxide

nanoparticles

Figure 22 Total phenolic content activities of Copper

Oxide nanoparticles
4.5.3 Antioxidant Activity Determination in Linoleic
Acid System
Antioxidant activity of Different concentrations of
CuO nanoparticles was determined using the inhibition of
linoleic acid oxidation. The thiocynate method was used
for its determination. The peroxidation percentage
inhibition was found to be 28% (maximum) starting from
17% (minimum). The 28% inhibition revealed by the
1000g concentration of the nanoparticle. and minimum

109

inhibition (17%) was showed by 60g solution of the
CuO nanoparticle. These results are shown in the form of
graph in figure 23. All the extracts show signs of
significant inhibition of peroxidation and were compared
with BHT having 81% inhibition of peroxidation. All the
extracts showed lower peroxidation when compared to
BHT.

Ferric reducing activity

30.0
%AGE inhibition of peroxidation

28.0 27.0
25.0
25.0

20.0
20.0
17.0
15.0

10.0

5.0

0.0
1000g 500g 250g 125g 60g

Different concentrations of Copper Oxide nanoparticles

Figure 23 Antioxidant activity determinations in

Linoleic acid system of CuO nanoparticles

110

4.5.4 Total Antioxidant activity by
Phosphomolybdenate
Copper oxide nanoparticles antioxidant activity
was checked by phosphomolybdenum. The range of the
absorbance was found to be 0.6920, 0.3730, 0.1897,
0.1197 and 0.1049. While the value of standard butylated
hydroxytoulene (BHT) ranges from 0.68 to 0.1 for this
activity. As it is cleared that this activity reveals in close
conformity with the standard employed such as BHT.
The results of antioxidant activity by
Phosphomolebdenate by Copper Oxide nanoparticles are
represented in the figure 24 in graphical form.

111

 Total Antioxidant activity of
Phosphomolybdenum


Absorbance

Absorbence of CuO Nps Blank BHT

Figure 24 Total antioxidant activities by

Phosphomolybdenate of CuO nanoparticles
4.5.5 FRAP Assay
An intense blue color is appeared with an
adsorption maximum at 593 nm at low pH. At this stage,
ferric-tripyridyltriazine (FeIII-TPTZ) complex is reduced
to the ferrous (FeII) form. Any half-reaction will reduce
the FeIII-TPTZ complex. This reaction is non-specific.
The conditions for the test to occur favor the reduction of
TPTZ complex and, thereby, color development. But the
only condition is that the reductant (antioxidant) must be

112

Chapter 5 Conclusion
By using solution combustion method, copper oxide
nanoparticles were synthesized to use Abutilon indicum
leave extract as fuel. EDX, SEM and UVVisible
techniques were utilized to characterize the as formed
nanoparticles. EDX data suggests crystallite composition
of CuO nanoparticles were formed. SEM images showed
that nanoparticles possess sponge like structures with
large surface area. The average crystallite sizes were
found to be 60120 nm. The carcinogenic dye such as
acid black 210 degraded very effectively by CuO
nanoparticles in the presence of Sunlight. The dyes which
contain Monoazo dye degraded easily while antraquinon
structural dyes degraded hardly. Methyl and chloro
groups present in structure which degraded slightly as
compared the nitro group. Alkyl groups less soluble in
water, so disfavor degradation process. The dyes
containing more sulfonic substituents are less reactive in
the photocatalytic process, while hydroxyl group
intensifies the electron resonance in the molecule and the
degradation rate of the dye. The plant extract contained

114

alkaloids, glycosides, tannins, and flavonoids. These
constituent are phytochemicals. Phytochemicals are used
in different diseases like astringent diuretic, expectorant,
demulcent, anti-inflammatory and anthelmintic. These
nanoparticles showed potential antioxidant activity.
Synthesized copper oxide nanoparticles used in
pharmaceutical and medical industries as an antioxidant.
The CuO NPs inhibit the scavenging of DPPH free
radicals effectively with IC50 value of 1000 g/ml. Using
ager well diffusion method, copper oxide Nps exhibited
significant antimicrobial activity against both classes of
bacterial strains. Natural occurring plants acted as both
reducing and stabilizing agent for the synthesis of Nps.
This green method of synthesizing CuO nanoparticles
could also be extended to fabricate other industrially
important metal oxides.

115

Chapter 6 References
Abboud,Y.; Saffaj, T.; Chagraoui, A.; El Bouari, A.; Brouzi, K.; Tanane,
O.; Ihssane, B.; Biosynthesis, characterization and antimicrobial activity
of copper oxide nanoparticles (CONPs) produced using brown alga
extract (Bifurcaria bifurcata). Applied Nanoscience. 2014; 4(5): 571-576.
Abdul, R.; Amri, I.; Desi, J.; Stella, M.; Hanggara, S.; Synthesis Of
Copper Oxide Nano Particles By Using Phormidium Cyanobacterium.
Indonesian Journal of Chemistry. 2009; 9(3): 355-360.
Acharyulu, N. P. S.; Dubey, R. S.; Swaminadham, V.; Kalyani, R. L.;
Pratap, K.; Pammi, S. V. N.; Green Synthesis of CuO Nanoparticles
using Phyllanthus Amarus Leaf Extract and their Antibacterial Activity
Against Multidrug Resistance Bacteria. International Journal of
Engineering Research & Technology. 2014; 3(4): 639- 641.
Ahmad W, Khan SA, Munawar KS, Khalid A. Kawanl S. Synthesis,
characterization and pharmacological evaluation of mixed ligand-metal
complexes containing omeprazole and 8-hydroxyquinoline. Tropical
Journal of Pharmaceutical Research. 2017; 16(5): 1137-1146.
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Antifungal and GC-MS Analysis of Seeds of Mustard Brown.
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of Taibah University for Science. 2015; 9: 712.
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evaluation of stable copper nanoparticles using T. arjuna bark extract.

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2013; 110: 108-115.
Khan, S. A.; Shahid, S.; Kanwal, S.; Hussain, G. Synthesis
characterization and antibacterial activity of Cr (III), Co (III), Fe (II), Cu
(II), Ni (III) complexes of 4-(2-(((2-hydroxy-5-nitrophenyl) diazenyl)
(phenyl) methylene) hydrazinyl) benzene sulfonic acid based formazan
dyes and their applications on leather, Dyes and Pigments,
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characterization of Cu-doped ZnO nanoparticles and their antioxidant,
antibacterial, antifungal and photocatalytic dye degradation activities,
Dig. J. Nanomater. Bios, 2017; 12(3): 877- 889.

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