Chapter 2

Materials and methods

This chapter presents materials and methods of synthesis of metal

nanoparticles by herbometallic method. Silver nanoparticles,silver

nanoflowers and silver nanodendrites by green methodand silver

nanorods by single replacement reaction and characterization

techniques

70
 2. MATERIALS AND METHODS

Metal nanoparticles are playing vital role in research and

industry. By the use of metal nanoparticles tremendous applications

are noticed from house hold to industrial. So the production of metal

nanoparticles must be in the large scale to reach the industrial

necessities. In the survey many physical and chemical methods have

been utilizing,but the hazardous chemicals in the preparation method

lead to environment pollution which causes serious diseases not only

for human beings but also for animals. So there is a rising need to use

nature friendly techniques for the synthesis of metal nanoparticles.

Because of this reason the synthesis of metal nanoparticles (MNPs)

using nature is attracted by many researchers. Metal nanoparticles

can be developed without using any toxic chemicals with naturally

available things such as plant extracts. So eco friendly green synthesis

does not require more effort of preparation, and it involves less

manufacturing cost, very less equipment and minimum usage of

chemicals.

Metal nanoparticles can be generally fabricated in following ways

1. (Top down)Bulk materials converted into nanosize materials.

2. (Bottom up) Nanoparticles are formed by building atom by atom.

To prepare metal nanoparticles various physical and chemical

methods are available, various techniques are prescribed in literature.

Few of them are ball milling, chemical vapour deposition, physical

vapour deposition, Solgel technique, laser synthesis, inert gas

71
condensation, electro deposition and plasma arching, polyol synthesis

etc.

2.1 Various processes of preparation of nanomaterials

2.1.1 Ball milling or mechanical crushing

This method is preferred mainly in the preparation of metal

nanoparticles. In this method small balls are allowed to rotate inside a

drum and are made to fall on a solid with high gravitational force,

which crushes the solid into nano-crystals.

High Energy Ball Milling can produce:

 Metal NPs (Fe, CO),

 Inter-metallic NPs (FeCO, CeCO5 , Nd2 Fe14B) ,

 Single Crystal NPs (Sm2CO17) (Metal Oxides/Ferrites/

Carbonates NPs are (ZnO, Fe3O4 , CoFe2O4 , LiNbO3, CaCO3),

 Carbon NPs (Graphite, Pure Carbon)

Disadvantages of Ball milling process:

 Basic material is contaminated.

 Contamination is a result of wear and tear, this happens mainly

from the balls and slightly from the casing.

 It causes noise pollution, in case of the hollow cylinder.

 With more of metal machine noise level are extremely high.

 Elongated preparation time, since large quantities are prepared,

takes longer time.

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  Cleaning is hard after utilization, dismantling the machinery

and erecting it back is difficult.

 Difficult to prepare smaller quantities and hence wastage is

more [257].

2.1.2. Physical vapour deposition (PVD)

By using this deposition technique, we can manufacture thin

films, thin films of various metals deposited on different surfaces. The

source material can be evaporated by using electron beams, thermal

energy, sputtering technique, and molecular beam epitaxy and

cathode arc plasma.

This technique involves condensation from the vapour phase as

follows.

1. By the sublimation of evaporating of a material.

2. Transportation of the material to the substrate from source.

3. Formation of the thin film and particle by nucleation and

growth.

PVD is one of the methods used to coat [Link]

PVD is still one of the most effective methods in the case of improving

a surface‟s strength and durability.

PVD involves different methods:

 Evaporation: Material is transformed to a gas phase by heating,

thendiffuses through a vacuum to the substrate.

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  Sputtering: Plasma is generated first; this plasma contains

argon ions and electrons. Next, atoms from the target are ejected

after being struck by argon ions. The atoms from the target then

travel through the plasma and form a layer on the substrate.

 Molecular beam epitaxy: The substrate is cleaned and loaded

into a chamber that is evacuated and heated to drive off surface

contaminants and to roughen the surface of the substrate. The

molecular beams emit a small amount of source material

through a shutter, which then collects on the substrate [258].

Pulsed Laser Method: This method is capable of high rate of

production of 3g/min. This method is mainly used in the synthesis of

silver nanoparticles. Silver nitrate solution and reducing agent are

allowed to follow into a blender like device. This device consists of a

solid disc and this disc rotates with solution. Hot spots are created on

the surface of the disc by subjecting it to pulses from a laser beam.

Silver nitrate reacts with reducing agent at these hot spots and as a

result, small silver particles are formed. These particles can be

separated by centrifuge. The size of the particles is controlled by the

energy of the laser and angular velocity of the disc.

Using this Physical Vapour Deposition nanoparticles can be prepared

are,

MNPs are (Au,Ag,Co, Pt, Fe, Y),Metal Oxide (TiO2,ZnO),

Semiconducting (Ge),

Magnetic (Fe-Pt), Hybrid (ZnS:Mn), Quantum Dots (Ge)

74
 Sputtering Electron Beam Evaporation:

Metal NPs (Au, Ag, Pt),

Inter-Metallic (Co-Pt),

Composites (Pt-MWCNT, CoPt-Graphene ).

Laser Ablation/Pulse Laser Deposition:

Metal NPs (Ag, Fe, Ni, Se), Magnetic NPs (FeCo, FePt),

Metal Sulphide (PbS), Metal Oxides(ZnO).

Disadvantages:

This process is more costly because of the special requirement

for cleaning and also difficult machines, require machinist to operate

it, this is slow process and ecologically sound method.

[Link] vapour deposition (CVD)

A precursor is introduced into a reaction chamber and is

controlled by balanced flow regulators and control valves. Precursor

molecules pass by the substrate, are drawn into the boundary layer,

and are deposited on the surface of the substrate[258].

This process is mostly used in the synthesis of pure metal

nanoparticles and semiconductor industry for depositing thin films of

various materials. This is mainly chemical process. This method can

also be used to grow surfaces and produce the desired deposit. If an

object has to be coated with nanopowders, the substrate is exposed to

precursors and then the object has to be introduced inside the

75
chemical vapour deposition area at a high temperature so that the

nanopowders can be adsorbed by all over the object. The reaction of

adsorbed molecules takes place to produce crystals.

There are three main steps in CVD process.

1. Initially material is heated to form a gas.

2. Precursor molecules pass by the substrate are drawn into

theboundary Layer deposit on a solid surface under

vacuumcondition.

3. By products formed by the gas phase reaction has to be removed

from the surface. Homogeneous nucleation takes place in

gasphase. Heterogeneous nucleation takes place on

substrate[259] .

Chemical Vapour Synthesis can yield the nanoparticles like

Metal Oxide NPs (CoO, SiO2, ZnO, Fe2O3), WS2 NPs,

CuSi/SiO2NPs, NPs of Al doped ZnO, fluorine doped tin oxide

and Cr doped ZnO.

Disadvantages:

 It requires high temperatures.

 The material which cannot hold high temperatures can be

physical vapour deposited.

 Usage of more toxic chemicals in the technique.

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2.1.4. Sol-gel technique

This technique is used to generate nanoparticles and

nanopowders. Sol-gel technique is based on the different combinations

of precursors and on their mechanical and physiochemical properties;

various sol-gels can be formed. Nanoparticles can be generated by sol-

gels using the process of precipitation, gelation, hydrothermal

treatment, hydro-dynamic cavitation.

Among all ofthis hydro dynamic cavitation technique is

oftenused, inwhich nanoparticles can be generated through

creationand release of gas bubbles inside the sol-gel solution.

The sol-gel solution is taken in a drying chamber and thoroughly

mixed by applying enormous pressure and high temperature also

further exposing it to cavitational disturbances.

This process creates hydrodynamic bubbles in the sol-gel. These

bubbles will undergo nucleation, growth and quenches to form

nanoparticles.

Sol-Gel Synthesis method can produce the

 Metal oxide NPs (ZnO, SnO2 , Fe2O3 , Ta2O5 ),

 Ceramic NPs (TiO2 , SiO2 ),

 NPs of CdSe, ZnB2 , GdVO4

 Magnetic NPs (Fe-Co)

 Metal Aluminates

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 The advantages and disadvantages of sol gel method are in the

below table2.1[260].

[Link] Solgel Advantages Disadvantages

matrices
1 Inorganic Goodoptical transparency, Brittleness,
Chemical robustness Low porosity

2 Organic Good tunable porocity and Fragile,limited

Electrochemical activity optical
Transparency
3 Hybrid Flexible rigidity, Poor optical
controlled porosity,balance transparency
hydrophobicity and and structural
hydrophilicity collapse on
(repel and attractive to drying
water(hydrozenbond))

Table 2.1: Advantages and disadvantages of sol-gel preparation

2.1.5. Polyol synthesis

Since the first description by Fievet, Lagier and Figlarz in 1989,

synthesis of nanoparticles in polyol (high boiling, multivalent alcohols)

process have been developed into a widely applied method, and

nowadays the standard repertoire for preparing high quality

nanomaterials is required. The polyols take advantage of several features

such as:

 Water comparable solubility of simple metal salt precursors.

 High boiling points (up to 320 °C).

 Reducing properties for the instantaneous synthesis of metals.

 Coordinating properties for surface functional and colloidal

stabilization of nanoparticles.

78
  Wide adaptability of the polyols ranging from low weight ethylene

glycol (EG) to high-weight polyethylene glycols (PEGs).

Metal nanoparticles, metal oxidesnanoparticles, metal chalcogenides,

and non-metal nanoparticles can be prepared via the polyol

[Link] method is more useful to synthesize

silvernanoparticles,silver nanorods and silver nanowires,

Polyol Synthesis Metal NPs (Ag, Pt, Cu, Pd, Pr), metal oxide NPs

(ZnO, Indium-tin-oxide, Gd2O3, Cu2O, Pr6O11), magnetic NPs (Fe3O4)

and metal hybrid NPs (CoSb3, FeCo). The result of polyol synthesis can

produce pure crystalline state metal tungstates, stabilisation of low-

melting elements and controlled thermal decomposition of polyols to

obtain high quality, lanthanide-modified carbon dots (C-dots).

Disadvantages:

High temperature and hazardous chemicals were used.

2.2. Adopted synthesis processes for NPs preparation

Present work focuses on green preparation of metal

nanoparticles. In this work we have opted two synthesis processes first

one is Herbo metallic method and second one is simple Green

synthesis with different herbs. These two methods are natural

methods and they are non polluting. In these synthesis techniques

there is no requirement of high chemical processing and top end

equipment.

79
 In this work natural herbs playing main role as a reduction

agents, and only single chemical required in green synthesis, where as

in Herbo metallic method medicinal herbs and metals were used for

the synthesis. Both processes are non toxic and least cost.

Herbo metallic
method

SYNTHESIS

Green synthesis

Figure 2.1:Synthesis methods

2.2.1Herbo metallic method

Herbo metallic method is a top down approach. This is a three

step iterative process where bulk form of metal is converted to

nanoparticles. This oldest Indian Ayurvedic system of preparation of

medicine is gaining lots of importance these days because of the

material produced were in the range of nanoscale. This method

prescribed in Ayurveda is in utilization long before the word

nanotechnology is coined. With less equipment like house hold things

one can prepare metal nanoparticles. In this method the materials we

used are naturally available. In this way we present the preparation of

gold, silver, ZnO, SnO2, ZnPbSn, Pb and Hg nanoparticles,which are

mentioned in the following Figure2.2.

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 Gold
nanopart
zinc lead icles
Tinoxide
tin nanopart
nanopart icles
icles

Lead MNPs Zinc

Oxide
nanopart
nanopart
icles
icles

Mercury Silver
nanopart nanopart
icles icles

Figure 2.2: Metal nanoparticles prepared by Herbo metallic method

The final product of Herbo metallic preparation is called

Bhasma. There are many Bhasmas and various preparations in the

Indian Traditional Medicine system (Ayurveda). Different Bhasmas

contain different metalnanoparticles and are prepared with different

herbs.

In Herbo metallic method metals were processed with herbal

juices and modification of base metals into nanoparticles by the use of

plant extracts to eradicate the toxic effects of metal.

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 Figure 2.3: Herbo metallic method

Herbo metallic method has three mandatory processes which include

purification, grinding and calcinations.

 In Purification process, the molten or red hot metal quenched

in specific liquids number of times.

 Grindingprocess breaks solid materials into smaller pieces.

 In Calcination heating a substance to a high temperature, to

bring aboutthermal decomposition.

These three steps were repeated several times to get the proper

physic chemical properties.

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2.2.1.1Purification

In purification the molten metal (which are at higher

temperatures) is poured in specific liquids, which are relatively cool

(nearly room temperature) and this sudden cooling is called

quenching. There are two types of purification, normal purification and

special purification.

Normal purification:

In this process, molten metal is quenched with rice gruel,

sesame (tiltil) oil and cow urine. In the following figure 2.4 shows the

normal and special purification process.

Ricegruel:

A cup of plain ricegruel prepared with rice and water, and then

filtered after cooking 20minutes. Riceguel have about 138 calories, 1.3

g of fat, 14.2g of sodium, 48.4g of potassium, 28 g of carbohydrates

and 1.3 g of fiber.

Sesame oil:

Sesame (Til) oil is having vitamins like: B1(Thiamine), B2

(Riboflavin), B3 (Niacin), B5 (Pantothenic Acid), B6 (Pyridoxine), B9

(Folate), A (Retinol), E (Tocoferol) and Minerals like: Calcuim,

Magnesium, Sodium, Potassium, Phosporous, Iron.

The health benefits of sesame oil include its ability to improve

hair and skinhealth, and stimulate strong bone growth. It has ability

to reduce blood pressure, and can also maintain good heart health.

This can control anxiety and depression. This oil protects infant

83
health, and also cures the dental problems, prevent from cancer,

improve the digestive process, and lower the inflammation.

Figure 2.4: Normal purification and special purification

Cow urine:

In Ayurveda, Gomutra(Cow urine) is claimed to be helpful in the

treatment of leprosy, abdominal colic pain [261], bloating, and cancer,

anaemia, fever, epilepsy [262]. In a study by Mandsaur has claimed

that it may also benefit for cancer patients[263].It also cures liver

ailments and asthma[264].It is a bioenhancer and it has enhancing

anti microbial activity of antibiotic and antifungal agents

[265,266,267].The cow urine investigation shows that it contain

84
nitrogen, sulphur, phosphate, sodium, manganese, carbolic acid, iron,

silicon, chlorine, magnesium, maleic, citric, tartaric, succinic acids

and calcium salts, vitamin A, B, C, D, E, minerals, lactose, enzymes,

creatinine, hormones and gold acids[263].

Special purification:

In special purification, molten metal is quenched with lime

juice several times. The metal nanoparticles prepared by citric fruit as

a reduction agent will have less or non toxic as compared with other

reduction agents. When we quench the material several times in lime

juice hazardous bulk metal became non toxic because of the chemical

constituents present in the lime juice. These chemicals every time

induced in the metal when they dipped in lime juice so the

deformation of the metal takes place and also the tough bulk metal

reduced to soft and smooth.

Lime juice:

Lime juice main components are citral, limonene, β-pinene and

fenchone (up to 15%). The beautiful aroma compounds that present in

limejuice are terpineol, bisabolene and other terpenoids. The fresh

juice of acid limes averages approximately 7.7% citric acid and 0.3%

invert [Link] reports from Diaz et al. found Iron absorption

efficiency is increased in women (suffering from iron deficiency) by

adding lime juice to meals [268].

85
The chemical constituents of lime are as shown infollowing figure2.5.

Citric acid C6H8O7 L-Ascorbic Acid C6H8O6

PhlorinC12H16O8 Eriocitrin C27H32O15

Hesperidin C28H34O15 Narirutin C27H32O14

Neoeriocitrin C27H32O15 Neohesperidin C28H34O15

Diosmin C28H32O15 Luteolin 7-O rutinoside

C27H30O15

Figure 2.5:Chemical constituents of lemon juice

86
 Lime juice is more useful in reducing nonalcoholic fatty liver

disease (competitively inhibiting CYP 3A4 monooxygenase activities)

[269,270]. Rodrigues [271,272] results showed that lime juice have

antibacterial and anticholera [Link] lime juice

composition[273]shownin the below table2.2.

[Link] Compound Value

1. Ph 2.81
2. Acidity(g100g-1 6.05
citric acid)

3. Soluble solids 8.42

4. VitaminC(mg.100 22.86
mL-1)
5. Fructose(g.100 g-1) 0.98
6. Glucose(g.100 g-1) 0.89
7. Sucrose(g.100 g-1) 0.07
8. K(µg. g-1) 376.79
9. Ca(µg. g-1) 23.24
10. Mn(µg. g-1) 0.08
11. Fe(µg. g-1) 1.71
12. Cu(µg. g-1) 0.35
13. Zn(µg. g-1) 0.29

Table 2.2:Compounds of lime juice

In the revolutionary work on the citrate reduction method,

Turkevich in 1951and Frens in 1973 detailed the description about

the basic experimental method and they have also observed that the

variation of temperature and concentration of citric reduction agent

have remarkable effect on the metal nanoparticles size and size

distribution[30]. In recent times, the crucial role of citrate reduction

87
agent on the nanoparticle size was demonstrated based on

experimental and theoretical modelling results [274]. The below

flowchart figure 2.6 reveals the transformation of metal by the effect of

temperature and abrupt cooling during the purification process.

Figure 2.6:Flow chart of results of quenching

This process detoxifies the bulk metal. By the Iterative

purifications metal becomes highly crystalline and also phase

transition of metal is occurred. Quenching prevents undesired

lowtemperature processes, such as gradual phase transformations. It

does this by reducing the window of time during which these

undesired reactions are both thermodynamically favourable, and

88
kinetically accessible. For instance, purification can reduce the crystal

grain size of metallic materials, decreasing their hardness.

[Link] Grinding

Figure 2.7:(a).Aloe-vera plant (b). mortar and pestle

Grinding (milling) is a process that breaks solid materials into

smaller pieces by mortar and pestle. In this process of breaking,

separating, sizing, or classifying aggregate material take place. The

grinding of solid matters with Aloe vera juice occurs under exposure of

mechanical forces that trench the structure by overcoming of the

interior bonding forces. After grinding, the state of the solid is

changed. The grain size disposition and the grain shape are also

changed. Grinding serves the purpose of increasing surface area of a

metal.

Aloe vera:

Aloe vera contains antioxidant vitamins A, C, E, vitamin

B12, folic acid and choline. The hormones called auxins and

gibberellins both help in healing [Link] has anti-inflammatory

property. It provides 12 anthraquinines and eight enzymes. These

areacting as analgesics, antibacterials and antivirals. Minerals such as

89
calcium, copper, selenium, chromium, manganese, magnesium,

potassium, sodium and zinc are present in aloe vera. Four fatty

acids are present, including cholesterol, campesterol, beta-sisosterol

and lupeol all providing anti-inflammatory results.

[Link] Calcination

Figure 2.8: Electrical muffle furnace

Calcination is the process of applying regular heat to the sample.

In the calcination processone can reach to high temperatures that are

capable of making desired changes in the material at the molecular

level. Basically a sample first undergos purification then the sample

was grinded using mortar and pestle by adding Aloevera juice as

supporting liquid. This wet grinded sample was prepared as small

pellets using pelletizer. The sample in the form of pellets was dried

under sunlight and then dried substance kept in muffle

[Link] using muffle furnace surve the following purposes:

90
 Then furnace is used for converting the sample into absolute

ashes, but without fusion.

 This ash is used for performing the analysis of different attributes

of the material.

 Calcination is the best process to study the chemical (like

reactivity, solubility, chemical constituents of sample) and physical

(like particle size and structure)properties of the material.

 In this process a material is heated to a temperature below its

melting point to effect a thermal decomposition or a phase

transition other than melting.

Figure 2.9: Herbo metallic synthesis of metal nanoparticles

The figure 2.9 shows Herbo metallic method of metal nanoparticles.

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 Then the metal nanoparticles were characterized by XRD(X-ray

diffraction) for the evaluation crystalline nature, crystal

structure,lattice parameters and particle size. SEM(Scanning Electron

Microscope) micrographs reveals surface analysis of the

material,morphology and grainsize. EDS(Energy Dispersive

Spectroscopy) peaks from various elements reveal chemical

constituents and stiochiometry of the sample. Particle size and zeta

potential measurements were done using Horiba SZ-100 nanoparticles

analyser. Particle Analyser estimates their particle size distribution,

Zeta Potential confirm stability of particles.

2.2.2. Green synthesis of Silver nanoparticles

The green method of synthesis of nanoparticles is easy, efficient,

and eco-friendly in comparison to chemical, physical and microbe

mediated synthesis methods.

As compared with the chemical and physical synthesis the green

method of preparation require less lab equipment. This processnot

requires any toxic solvents, high pressure, energy and high

temperature conversion. It requiresnatural leaf extracts. The microbe

mediated synthesis is also biological method other than green

synthesis. But the microbe involved synthesis is not feasible

industrially, due to its lab maintenance. Hence, green synthesis is the

best option to choose for the synthesis of nanoparticles.

Silver was of particular interest due to its distinctive physical and

chemical properties. Nowadays silver nanoparticles are the most

92
common nanoparticles useful for house hold things to industrial

purposes. Therefore the nanoparticles were synthesized by using

extracts of Tulasi and piper and metal ions (such as silver nitrate).

Also a simple single displacement reaction is used to prepare silver

nanoparticles. Tulasi and piper leaf extracts were selected as they are

of high medicinal value and the process do not require length

procedures to prepare the sample.

Figure 2.10: Silver nanoparticles prepared by green synthesis

AgNPs were formed using three reduction agents [Link]

tenuiflorum plant (Tulasi) [Link] betel leaf [Link] rod.

Figure 2.11 (a). Ocimum tenuiflorum plant (Tulasi) (b). Piper betel

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Importance of Ocimum tenuiflorum plant (Tulasi):

Ocimum tenuiflorum plant(Tulasi) is the holy Indian medicinal

plant which is used in regular chronic ailments like cold, cough and

throat infection and pimple mark reduction. Every part of the plant is

useful. Among the plant parts stem, leaves, bark and seeds, leaves are

most medicinally valued. Therefore we have used Tulasi leaves for the

reduction of silver nanoparticles. Using these leaves we have

synthesised silver nanoparticles(Ag NPs) and nanoflowers(Ag NFs). In

this process Ag NFs and Ag NPs were obtained by changing synthesis

parameters like temperature, stirring time. We have observed the

morphology difference between the Ag NPs prepared using decoction of

Tulasi leaves and Tulasi fresh juice.

Tulasi extract have wide applications in different fields because

of its chemical constituents (oleanolic acid, ursolic acid, rosmaric acid,

Methyl Eugenol, charvicol-Caryophyllene), so it is regarded as a

phytomedicine. Ocimum tenuiflorum have several therapeutic

properties like in cardiac activity, antidiabatic, anticancer activity.

Tulasi leaves are known to have more antioxidant activity than Tulasi

stem. It is also used in deflouridation of water[275].

Importance of Piper betel leaf

In India, particularly in south India the piper betel plant leaves

are taken orally (with nut powder, tinge or trace of calcium carbonate)

after meals. In Thailand leaves are used to prevent oral malodor.

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Medicinal values

Piper betel leaf is a antioxidant, antifungal, antibacterial, anti-

inflammatory, bioprotective, antidiabetic agent, wind-cold cough,

bronchial asthma, relieve throat pain, carminative, fever and fatigue,

rheumatism, stomachalgia, and pregnancy edema , halitosis, mouth

freshener,wound healing, a digestive and pancreatic lipase stimulant,

control excessive bleeding during menstruation.

We synthesised silver nanoparticles using betel leaf. As a result

we obtained silver nanodendrites(AgNDs)using Piper betel leaf extract

as reduction agent.

Basically green method is a twostep method. In the synthesis of

silver nanoparticles using leaves(Tulasi and piper betel) involved two

steps, in the first step 1mM AgNO3 solution has been prepared. Second

step the solution then mixed with the broth of plant leaves under

suitable conditions as specified below. Nanoparticles formation is

considered as a result of change in the colour of the solution. Then

silver nanoparticles were collected from the solution for further

characterization.

Fig 2.12 Flow chart of green synthesis

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2.2.2.1Synthesis of Ag NPs using Tulasi leaves

Preparation of Tulasi decoction:

Fresh Tulasi leaves were collected from JNTUH temple premises.

Tulasi leaves washed thrice with DDW(double distilled water) and then

wiped clean. Next 3g of leaves were weighed and added to 100ml of

DDW, this mixture is heated while stirring for about 10minutes using

magnetic heater and stirrer. Then finally the pale yellow colour

product was filtered and stored properly for further application.

Figure 2.13:(a). Tulasi leaves (b). Tulasi decoction

Preparation of Tulasi juice:

In Tulasi juice preparation, fresh Tulasi of 15g leaves were

washed with DDW water then air dried for [Link] leaves are

grinded using mortar and pestle and the green juice out of leaves is

extracted and filtered.

Figure 2.14:(a). Fresh Tulasi juice prepared by mortar and pestle (b).AgNPs

using Tulasi juice

96
Different deposition condition using Tulasi extract

Ag NPs were prepared using Tulasi decoction and fresh Tulasi

juice at the following different deposition conditions they are

 Tulasi decoction at room temperature

 Tulasi decoction at 80ºC temperature

 Fresh Tulasi juice at increased (2hrs) stirring time.

The colour of the Tulasi decoction was reddish brown and AgNO3

solution colour was white. After adding Tulasi decoction to 1mM

AgNO3 solution the colour of mixture was observed in the following

figure.

Figure 2.15:(a). AgNO3 and Tulasi decoction (b). AgNPs formed at room
temperature (c). reaction at 80ºC

Figure 2.16: Images of Ag NPs using Tulasi leaves extract under the
specified three conditions (TDN&TD Ag NPs at room and increased
temperature, TR –using Tulasi juice )

97
 The flower like structure of Ag NPs was observed when Tulasi

decoction was used for the synthesis. Spherical Ag NPs formed when

Tulasi juice was used instead.

2.2.2.2Synthesis of Ag NDs usingPiper betel leaf

The first step of green method is preparation of leaf broth. Betel

leaves collected from [Link] were washed thrice with DDW.

Leaves were wiped and air [Link] leaves were cut into fine peaces

and then added to 50ml of DDW. Next the solution was heated up to

boiling temperature then solution was filtered using filtering papers.

The solution have beautiful [Link] solution was pale yellowcolour.

Figure 2.17:(a). piper betel leaf (b). piper betel decoction

For the second step1mM AgNO3 solution has prepared and then

mixed with the extract of piper betel leaves. Then basically light yellow

colour of the solution changed to white, we were waiting for the

solution to change its colour to reddish brown as a result of silver

nanoparticles formation. But there was no change in colour then next

98
day when we took it to SEM analysis then we came to know that as a

result of silver nanodendritesformation the colour did not change.

Figure 2.18:(a).AgNO3 and piper betel leaf broth (b). after 1hr (c). after 1day

solution.

[Link] Single displacement/replacement reaction

Single replacement reactions provide an elegant way of

transforming solid nanoparticles into complex morphologies. When

the metals are arranged in the order of increasing reduction

potentials, which are determined with respect to one molar solution of

their ions and measured on the hydrogen scale, a long series or list

called electrochemical series or galvanic series is formed. That is

presented in the table2.3.

A system with high reduction potential has a great tendency to

undergo reduction. The higher a metal in the series, the greater is its

tendency to oxidise,and the metals top in the series are strong

reducing agents and their ions are stable. The bottom elements of the

series are inactive, stable metals and their ions are easily reduced to

metals. The metal in top of the series can reduce especially the ions

present lower in the series.

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 Standard oxidation potentials at 25ºC

[Link] Element Electrode reaction Eº volts

1 Sodium Na→Na+ + ℮ -2.712

2 Magnesium Mg→ Mg+++ 2℮ -2. 34

3 Beryllium Be → Be+++ 2℮ -1. 70

4 Aluminium Al→ Al+++ + 3℮ -1.67 A

5 Manganese Mn→ Mn++ + 2℮ -1.05 N

6 Zinc Zn→ Zn++ + 2℮ -0.762 O

7 Chromium Cr→ Cr+++ + 3℮ -0.71 D

8 Iron Fe→ Fe+++ + 3℮ -0.44 E

9 Cadmium Cd→ Cd++ + 2℮ -0.402

10 Cobalt Co→ Co++ + 2℮ -0.277

11 Nikel Ni→ Ni++ + 2℮ -2.250

12 Tin Sn→ Sn++ + 2℮ -0.136

13 Lead Pb→ Pb++ + 2℮ -0.126

14 Hydrozen H→ 2H++ + 2℮ 0.000 C

15 Copper Cu→ Cu++ +2℮ +0.345 A

16 Copper Cu→ Cu+ + ℮ +0.522 T

17 Silver Ag→ Ag+ + ℮ +0.800 H

18 Platinum Pt→ Pt++ + 2℮ +1.2 O

19 Gold Au→ Au+++ + 3℮ +1.42 D

Table2.3: Galvanic series

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 This series give more information about the elements having

lower reduction potential can displace another metal having higher

reduction potential from its salt solution spontaneously.

Copper metal dissolves in a silver nitrate solution. The balanced

equation for the reaction is:

Cu(s) + 2AgNO3(aq) -------> Cu(NO3)2(aq) + 2Ag(s).

Copper (Cu) oxidised by the loss of two electrons and silver (Ag)

reduced by gaining two [Link] is oxidizing agent and Copper is

acting as a reducing agent.

2.3 Characterization techniques

Metal nanoparticles prepared in herbometallic method were

characterizedusing techniques XRD(X-ray diffraction), SEM (Scanning

Electron Microscope), EDS(Energy dispersive X-ray spectroscopy),

Particle analyser and zetapotential. Silver nanoparticles prepared by

Green synthesis were characterized by techniques, SEM(Scanning

Electron Microscope), EDS Energy dispersive X-ray spectroscopy),

TEM(Transmission Electron Microscope), SAED and Uv-Vis

spectroscopy.

2.3.1 XRD(X-ray diffraction)

X-ray powder diffraction (XRD) patterns were scanned in the

Bragg angle range of 0o to 80o with a Philips X-ray Diffractometer

coupledwith graphite monochromator using CuKα(λ=1.5406Å) with an

accelerating voltage of 40 KV. In the Bragg‟s angle region (2θ)from

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0o to 80o at a speed of 0.25o per minute, the scanning was done.

Crystallite size was calculated using Scherrer formula.

Figure 2.19: Philips X-ray diffraction instrument

Applications

X-ray powder diffraction is most widely used for the

identificationof unknowncrystalline materials (e.g. minerals, inorganic

compounds). Determination of unknown solids is critical to study in

material science, engineering and biology.

Other applications include:

 Characterization of crystalline materials.

 Identification of fine-grained minerals such as clays and mixed

layer clays that are difficult to determine optically.

 Determination of unit cell dimensions.

 Measurement of sample purity.

[Link] Analysis (PSA)

Horiba-sz particle size analysis instruments provide the ability

to measure and report the particle size distribution of the sample.

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Almost all real world samples exist as a distribution of particle sizes

and it is recommended to report the width of the distribution for any

sample analyzed.

Particle Size Analysis (PSA) determines the size range, and/or

the average, or mean size of the particles in a powder or liquid sample.

PSA is a part of particle science, and it has importance in a number of

industries like the chemical, mining, food, agriculture, forestry etc.

Figure 2.20: Horiba particle analyser

2.3.3. Zeta potential

Zeta potential describes the electrostatic interactions of cells

and particles in a fluid environment. The liquid layer surrounding the

particles exists as two parts as shown in the figure2.21; an inner

region(stern layer) where the ions are strongly bound and an

outer(diffuse) region where they are less firmly associated. Within the

diffuse layer there is a notional boundary inside which the ions and

particles form a stable entity. When a particle moves, ions within the

boundary moveit. Those ions beyond the boundary stay with the bulk

dispersant. The potential at this boundary is the zetapotential.

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 Figure 2.21: Zeta potential

Zeta potential is the electrical potential at the hydrodynamic plane of

shear. Zetapotential depends on following:

 Particle surface properties

 Nature of solution

 Ionic strength

 pH value

Used to predict the mono dispersity or agglomeration of the sample

as mentioned in the below table2.4.

[Link] Zetapotential Value Particle dispersion

1 High >30 mV (+ve or Mono dispersity
-ve)
2 Low <5Mv Agglomeration

Table 2.4: Zetapotential value and particle dispersion

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 Figure 2.22: Graph of Zeta potential Vs pH

From the above graph Zeta potential Vs pH it is clear that

Zetapotenial value is <-30 or > +30 Nanoparticles repel each other and

they are in stable state.

2.3.4 UV–Visible Absorption Spectroscopy

UV–Visible Absorption Spectroscopy is generally used for

characterizing nanoparticles, especially for those Plasmon resonances,

such as gold, silver and copper. It is used to determine the electrons

in high energy orbitals having wavelength range of 100-700 nm. The

UV absorption spectra come from electron transitions from lower

energy level to a higher energy level in a molecule. Since orbitals have

quantized energy, only certain transitions can occur in the Uv-Vis

energy range. The differences in the incident and transmitted beam

give us information about the frequencies which are absorbed by the

sample molecules. The chemical structure of the sample can be

analysed depending upon the absorbance data.

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 Figure 2.23: UV-visible spectrophotometer

UV-Vis analyses in the present work were done using the 80µL

of solution in the range of 200-800 nm with 1 nm resolution. All

spectra were corrected against the background spectrum of water as

reference.

2.3.5. SCANNING ELECTRON MICROSCOPE (SEM)

Scanning Electron Microscope is an improved model of an

electron [Link] is used to study the three dimensional

image of the specimen.

Principle:

When the accelerated primary electrons strike the sample, it

produces secondary electrons. These secondary are collected by a

position charged electron detector which in turn gives a 3-dimensional

image of the sample.

Construction:

It consists of an electron gun to produce high energy electron

beam. A magnetic condensing lens is used to condense the electron

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beam and a scanning coil is arranged in between magnetic condensing

lens and the sample.

Figure 2.24: SEM working and SEM analysis with EDS Instrument

The electron detector (Scintillator) is used to collect the

secondary electrons and can be converted into electrical signal. These

signals can be fed into CRO through video amplifier as shown in figure

2.24.

Working:

Stream of electrons are produced by the electron gun and these

primary electrons are accelerated by the grid and anode. These

accelerated primary electrons are made to incident on the sample

through condensing lenses and scanning coil.

These high speed primary electrons on falling over the sample

produce low energy secondary electrons. The collection of secondary

electrons is very difficult because of their low energy. Therefore, to

collect these secondary electrons, a very high voltage is applied to the

collector.

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 These collected electrons produce scintillations on photo-

multiplier tube (detector) and are converted into electrical signals.

These signals are amplified by the video amplifier and are fed to the

CRO.

By similar procedure the electron beam scans the sample from

left to right and again from left to right etc., similar to we read a book

(figure 2.24), and the whole picture of the sample is obtained in the

CRO screen.

Advantages:

 It can be used to examine specimens of large thickness.

 It has large depth of focus.

 It can be used to get a three dimensional image of the object.

 Since the image can be directly viewed in the screen, structural

details can be resolved in a precise manner.

 The magnification may be upto 3,00,000 times greater than that

of the size of the object.

Disadvantages

The resolution of the image is limited to about 10-20 nm, hence

it is very poor.

Applications

 It is used to examine the structure of very large specimensin a

three dimensional view.

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  Similar to the application of electron microscope this scanning

electron microscope also has applications over various fields

such as biology, industries, engineering, physics, chemistry, etc.

2.3.6 Energy-dispersive X-ray spectroscopy (EDS or EDAX)

Energy dispersiveX-ray

spectroscopy (EDS, EDX,EDXSorXEDS).Sometimes called energy

dispersive X-rayanalysis (EDXA) or energydispersive X-ray

microanalysis (EDXMA),is an analytical technique used for the

elemental analysis or chemical characterization of a sample. EDAX

develops the best solutions for micro and nano characterization,

where elemental and/or structural information is required, making

analysis easier and more accurate.

 It relies on an interaction of material and X-ray excitation.

 Its characterization potential is depend on atomic structure of

each element. It permits an exclusive set of peaks on its

electromagnetic emission spectrum.

 To stimulate the emission of characteristic X-rays from a

specimen, a high-energy beam of charged particles such

as electrons or protons, or a beam of X-rays, is focused onto the

sample being studied.

 At rest, an atom within the sample contains ground state (or

unexcited) electrons in discrete energy levels or electron

shells bound to the nucleus.

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  The incident beam may excite an electron in an inner shell,

ejecting it from the shell while creating a hole where the electron

was.

 An electron from an outer, higher-energy shell then fills the

hole, and the difference in energy between the higher-energy

shell and the lower energy shell may be released in the form of

an X-ray.

 The number and energy of the X-rays emitted from a specimen

can be measured by an energy-dispersive spectrometer.

 As the energies of the X-rays are characteristic of the difference

in energy between the two shells and of the atomic structure of

the emitting element, EDS allows the elemental composition of

the specimen to be measured.

2.3.7. TRANSMISSION ELECTRON MICROSCOPE (TEM)

We know in scanning electron microscope the resolution of the

image is limited only upto 10-20 nm. This will not be useful to view

the internal features of an atom (or) the morphology of a sample of

size say 0.2 nm.

Thus to examine the sample of size up to 0.2 nm, the

transmission electron microscope can be used. In this microscope the

image is obtained by transmitting the electrons through the specimen.

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Principle:

Electrons are made to pass through the specimen and the image

is formed in the fluorescent screen, either by using transmitting beam

(Bright field image) on by using diffracted beam (Dark field image).

Construction:

It consists of an electron gun to produce electrons. Magnetic

condensing lens is used to condense the electrons and is also used to

adjust the size of the electron beam that falls onto the specimen. The

specimen is placed in between the condensing lens and objective lens

as shown in figure 2.25. The magnetic objective lens is used to block

the high angle diffracted beamsand the aperture is used to eliminate

the diffracted beam (if any) and in turn it increases the contrast of the

image.

The magnetic projector lens is placed above the fluorescent

screen inorder to achieve higher magnification. The image can be

recorded by using a fluorescent (phosphor) screen or CCD (charged

coupled device).

Working:

Stream of electrons are produced by the electron gun and is

made to fall over the specimen using the magnetic condensing lens.

111
 Figure 2.25: TEM working instrument

Based on the angle of incidence, the beam is partly transmitted

and partly diffracted, as shown in fig 2.25. Both the transmitted beam

and the diffracted beams are recombined at the E-wald sphere (sphere

of reflection which encloses all possible reflections from the

crystal/specimen, satisfying Bragg‟s law), to form the image as shown

in fig 2.25. The combined image is called the phase contrast image.

In order to increase the intensity and the contrast of the image,

an amplitude contrast image has to be obtained. This can be achieved

only by using the transmitting beam, and thus the diffracted beam

has to be eliminated.

Now in order to eliminate the diffracted beam, the resultant

beam is passed through the magnetic objective lens and the aperture

as shown in figure 2.25. The aperture is adjusted in such a way that

the diffracted image is eliminated. Thus, the final image obtained due

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to the transmitted beam alone is passed through the projector lens for

further magnification.

The magnified image is recorded in the fluorescent screen (or)

CCD. This high contrast image is called BRIGHT FIELD IMAGE.

Also, it has to be noted that the bright field image obtained is

purely due to the elastic scattering (no energy change) i.e., due to

transmitted beam alone.

Advantages:

 It can be used to examine the specimen of size upto 0.2 nm.

 The magnification is 1,000,000 times greater than the size of

the object.

 It has high resolution.

 The resolving power is 1 A° to 2 A°.

 We can get high contrast image due to both transmitted beams

(bright field) and diffracted beam (dark field).

Disadvantages:

 The specimen should be very thin.

 It is not suitable for thick samples.

 There are changes for the structural change, during sample

preparation.

 3-dimensional image cannot be obtained.

 In case of biological samples, the electrons may interact with

the samples, which may even damage the sample.

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Applications:

 The main application of TEM is in nano-sciences (nano-tubes,

micro machines etc), used to find the internal structures of

nano-materials.

 It is used to find the 2-dimensional image of very small biological

cells, virus, bacteria etc.

 It is used in thin-film technology, metallurgy, bio-chemistry,

micro-biology etc.

 It is used to study the compositions of paints, papers, fibers,

composite materials, alloys etc.

2.3.8. SAED (Selected Area Electron Diffraction)

Selected area diffraction (SAED) is a TEM technique to obtain

diffraction patterns that result from the electron beam scattered by

the sample lattice. Obeying the Bragg‟s law, the electrons are

scattered elastically by the lattice, therefore, we can index the

diffraction spots in the pattern and identify the phases in the sample

and study their structures.

Figure 2.26: SAED working

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 In the imaging system of a TEM, an SAED aperture is inserted

in the image plane of the low object lens to limit the sample volume

that contributes to the diffraction pattern. In the above figure 2.26 we

can find the SAED aperture on the image plane is equivalent to a

virtual aperture close to the specimen which functions as a diffraction

area selector.

In SAED, a parallel beam (plane wave travelling in one direction)

interacts with sample. Typically, the area size of the sample been

selected by SAED aperture is 0.5-1 μm, so the microstructure of the

sample could be fine selected and tested. An aperture is used to define

the area from which the diffraction pattern is to be recorded from a

thin sample. This aperture is typically located in the first image plane

below the sample. Typical size of an area studied by SAED is a few

hundred of nanometers. SAED diffraction patterns are either simple

spot patterns corresponding to single-crystal diffraction or ring

patterns corresponding to powder diffraction from multiple crystals

with a variable orientation.

SAED is commonly used for phase identification, structural

information, determination of structural intergrowth, determination of

growth directions etc., crystalline symmetry, unit cell parameter and

space group etc. Lattice parameters from SAED have high accuracy,

phase separation, texture and precipitation.

115