Chapter 4 Results and Discussion

Introduction Nanotechnology refers to the branch of science and engineering dedicated to materials of nano scale, having size in order of 100th of the nm or less. It has been reported that currently more than 400000 researchers are working in the field of nanoscience and technology in the world which is supposed to be up to 6 million in 2020.Various examples of nanomaterials could be observed in everyday life; smoke, volcanic ash, sea spray, welding fumes are nanosized. Human DNA strand is approximately 2.5nm in diameter, a paper sheet is almost 100000 nm thick, single gold atom is about 0.33nm, haemoglobin in our blood is 5.5 nm in diameter and one nanometer is about as long as our finger nail grows in 1 second. Nanomaterials exhibit unique properties due to their size. As we know there is increase in urbanization along with that industries are moving on and on, causing exhaust of hazardous and excessive gases along with toxic chemicals as waste products. Along with this we have to know the benefits in the secrets of natural compounds that are helpful in the fabrication of nano particles because nano particles are being used in every field for the benefit of mankind. Because of the absolute properties of biological molecules for nanoscience, these are widely used in the synthesis of nano materials. Biological molecules have reducing and capping agents that is a solid reason for their use in synthesis. These are found to be cheap and non-hazardous source. Nanoscience is the advance technology in the modern research. It discussed advanced properties with wide range of applications of nano-materials.Due to the property of wide surface area many other exclusive properties including electric, magnetic, antibacterial and optical properties these become a point of affection for chemists. Various features of synthesized nanoparticles are given in figure 1.

Figure 1.Various features of fabricated nanomaterials In the making of aircraft bodies and other aerospace constituents nanoparticles are extensively used. It increases the durability of aerospace components by 300 percent.Nanoparticles are substantially low-density materials and reduce the fuel requirements. They are also very effective for space vehicles that face intense conditions, especially heat. Nanoparticles provide human beings with this future opportunity to make materials implanted with nanosensors that can keep track of the condition of the framework of aerospace vehicles.Nanoparticles have found its way in the automobile industry as well like every other field of life. Various parts of automotives, like ball bearings, springs, etc, are being made using nanocrystalline silicon salts. These nano components increase the durability and life span of these parts of automobiles [5]. The nanoparticles could be used for making antifogging mirrors and wind shields which improves the visibility which ensures safety during travelling.

Nanomaterials are also being applied in batteries, fuel cell technology and other energy storage devices and they have improved the performance and storage capabilities of such devices. The aerogels used in the separator plates of batteries are embedded with nanomaterials. The advantage of such plates is the increased energy-storage capacity. In future, such technology could be utilized in making inexpensive hydrogen sensors for fuel cells and also in creating batteries with improved energy density and recharge timings.Building materials are very essential part of everyday life. Many types of building materials and ceramics materials have been down sized to nano to improve the durability of building materials. With the help of nano technology, smart windows are being manufactured which become dark in bright light and translucent in subdued light. Nano particles are also used in making insulating materials. The implication of nano technology in making various kinds of building materials could help create lighter and low-cost construction stuff. Materials implanted with nanoparticles are easily biodegraded so they are considered environment-friendly materials.

Nano particles are also being used in the synthesis of nanocrystalline metal carbides that are extensively utilized in drilling and cutting. Better ceramics are also being manufactured by nanomatrials. In future, materials embedded with nanoparticles may be used semiconductors and robotic machinery. Moreover, very hard materials could be drilled and cut with the aid of such materials.

Nanoparticles are now essential component of different electronic devices such as LCDs, LEDs, Smart Phones, Calculators and Screens of computers and televisions. The use of different semiconductor nanoparticles in cathode-ray tubes has resulted in better resolution of televisions and monitors. It has also helped in making flexible displays. Nano technology used in field-emission displays could be applied in X-rays, lithography and lighting systems. These FEDs could also be utilized in large television displays. Silicon has been the basic material for electronic industry but many other materials are also making their way to this field like graphene. The most fascinating applications of nanomaterials could be found in the field of biomedical science. Gels designed from nanomaterials are used as substitute of lost tissue or can be used for the reformation of natural tissues. These days nanoparticles are being used in the replacement of bones and in the re-growth of nerves. Nanoparticles can be made about the size of natural human cell and can grow with that easily hence are better alternatives. It might become possible in near future to engineer heart valves and artificial kidneys with the help of nanotechnology.Magnetic Resonance Imaging (MRI) is considered to be the one of the most important tool in disease diagnostic. Magnetic nanoparticles have used in MRI as contrasting agents. Nanorobots, made from carbon based nanomaterials, are being used for target drug delivery in lungs and heart diseases. Nanomaterials are also being used as drug carriers in many anti-cancer drugs .Environmental pollution is the most concerned area in modern world. Nanoparticles also playing their part to remove pollutants from environment. A large number of nanomaterials and their combinations are being used in treatment of water and countermeasure of dyes, heavy metals, pesticides, insecticides and other pollutants from in soil, air and water.Nanomaterials have been used for degradation of various dyes such as methyl orange and methylene blue. Kinetics information from final results shows that methylene blue and methylene orange abide by second order and first order kinetics. Altogether elimination of dyes from water can be carried out by use of variety of concentrations i.e 10 to 200 mg/L.Catalysis is one of the most important area in which nanomaterials are successfully being used. Nanocatalysts have increased the rate of reactions and the performance of reactions which has resulted in better yield of products. In shortest periods of time, better quality of products is being achieved in no time.Nanoparticles are being used in sunscreens and cosmetics industry. With the development of nanopolymers, life of pipelines has been improved. Eye glasses made of nanomaterials have the quality of better cleaning and scratch resistance. Nanoparticles are also being used in textile and pigment industry. One important application of nanomaterials is separation science; chromatography and spectroscopy. Use of nanomaterials also have improved the separation efficiency in chromatography and sample preparation with the help of nanoparticles have yields better results as well.Different methods have been used for the fabrication of nanoparticles. Few factors must be taken into account before selection of a method for nanoparticle synthesis such as reproducibility of the method, controlled particle size, cost effective and environmental friendly. Conventionally, two approaches are most commonly being used for the fabrication of nanomaterials; Top down approach and bottom up approach.The general mechanism for both of these methods is shown in figure 2.

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Figure 2. General scheme applied for Top down and Bottom up approaches.Top down approaches is the break-down of bulk material into tiny particles which could be done by attrition or ball milling. There are few other size reduction methods which could be applied as well. Pyrolysis under severe conditions is the other method in which high pressure is applied on gas or liquid materials. These methods have advantage of production of nanomaterials in large quantity as well as have controlled particle size. But this approach has some drawbacks as well; polydispersity, cluster formation, destruction of crystallographic structure, surface dislocation due to high pressure and high cost. To overcome these draw backs, bottom up approach has been extensively being used in current day research world. Materials used in this method are initially at atomic or molecular level which is combined to nanomaterials. Prime benefits of this approach are; produced nanostructured materials have fewer defects, homogenous particles are produced, size and morphology could be adjusted.Various methods have been developed and efficiently used in the research world which includes; Sol-gel, micro-emulsion, hydrothermal method, sonochemical method, vapour phase method, supercritical fluid method and co-precipitation method which are briefly discussed below.One of the most crucial processes for the synthesis of nanoparticles is the sol-gel method. Wet chemical technique is used during this process. Nanomaterials, like polymers and metal oxides, have been synthesized with the help of sol-gel method. This process begins with hydrolysis after which polycondesation is executed. As a result, a bond is formed, in the heart of which metal is present. On any one side of this metal either a Hydroxo- group or oxo-group is present. The word sol has been inferred from solution and gel is a colloid which is neither solid nor liquid. The first step involved in the sol-gel method is the selection of reactants with a specific stoichiometric quantity. Then these reactants are combined to form a homogenous mixture. The next step involves the addition of a chelating agent. It could either be citric acid or ethylene glycol. Excess of chelating agent is removed by heating, the resultant of which is a clear solution names as sol. Addition of an acidic chelating agent increases the acidity of the solution. To counterbalance this acidity, ammonia solution is added drop by drop. This solution is heated at 80-90 C and vigorously stirred. Ammonia evaporates as a result of heating and a semi solid solution is obtained which is called gel. Heating this at a high temperature, it takes the form of an ash like material. Final steps involve its annealing and packing.

The size of the nanoparticles formed during this process depends on temperature, pH and composition of the solution. An advantage of the sol-gel process is that it needs annealing temperature to a lesser extent. But a disadvantage is that the drying process takes a lot of time.Aerosol/Vapor-Phase Method: Another name of the aero-sol or vapour phase method is spray pyrolysis. During this process a substrate material which is made of an organic solution containing ferric salts and a reducing agent is vaporized into droplets. These fine droplets are metamorphosed into nanomaterials. The size of the manufactured nanomaterials depends on the size of the droplet. An alternate approach to this method is by using a laser to heat the organic solvent. Therefore this method is also called laser pyrolysis. Fabrication of nanomaterials through this technique could have better results if this process is executed in generators.Another method which is sonolysis method, used in the fabrication of controlled sized nanoparticles which occur by using organometallic forerunners. By this technique FeO nanomaterials are being synthesized that have particle size near to that of organomettalic forerunners. Nanomaterials of about 10nm were fabricated from Fe(II) acetate forerunners. These nanomaterials are obtained from elevation of chemical in the presence of low wavelengths radiations at 1.5 atmospheric pressure for three hours at moderate temperature. Material size was guarded by altering the metal forerunner that has been used for the fabrication of nano materials. It may be guarded by altering the temperature of the chemical reaction. Hypnotic nanomaterials fabrication by using Fe acetate forerunners at high temperature lead to thermolysis.While supercritical fluid method is being used in the fabrication of FeO nanomaterials with required cytology and figures. Supercritical fluids persist in one phase form above critical conditions. Among many other components CO2 is mostly utilized in the fabrication of nano materials because of their inflammable, non-hazardous nature and being commonly used in medication .Water is also being utilized in fabrication of alpha Fe2O3 and Fe3O4 nanomaterials at 22.11 atmospheric pressure and 374.3 C temperature. Mainly this method is used to avoid the organic solvents which can be toxic in nature and have harmful effect on environment. So, this approach can possibly regard as a green chemistry approach. The advantage of this method is that I can properly control the size of the particles.

In micro emulsion method two different mixtures i.e, water and oil, are used to make a thermodynamically stable clear solution. Moreover, co-surfactants are utilized for more strength in few facts. In this process many salts and inorganic materials in aqueous media although hydrocarbon amalgam require non aqueous phase.The main asset of this method is fabrication criterion that is used for the stabilization and maintenance of size and other assets. Choosing of proper binding agents is the main issue. Many types of nanomaterials are synthesized by using micro emulsion method.Thermal decomposition method is used for the fabrication of many semiconductor as well as oxide nano materials. By using reducing and capping agents this method is useful in the fabrication of controlled sized and limited sized magnetic nanoparticles. Particularly for the fabrication of nanomaterials and surfactants, organomatellic forerunner is utilized. Surfactants and solvents that are used are responsible for the size and morphology. Size and morphology depends upon reactants which include organomettalic precursors, surfactant and solvent system. Necessary parameters for controlling size and morphology are, time of reaction, developing time and temperature.Hydrothermal methodology is famous and common method for the fabrication of nanomaterials. This method requires high pressure and temp. First of all the reacting components mixed and NaOH is poured into it after dissolving in some suitable solvent. The resultant mixture is then allowed to reaction chamber and then exposed to high temperature and after completion of reaction, mixture is allowed to cool at room temperature. Then the pH of sample is maintained after filtration and washing and allowed for calcinations providing high temperature in the presence of air. This methodology is not detailed till now but has been helping in the fabrication of necessary materials. By this method necessary morphology and size can be gained by controlling certain parameter like temperature and pressure.Co-precipitation methodology is simple way for the fabrication of nanomaterials. This method is applicable for the components that are solved in aqueous or organic media. It helps in the subsequent growth of nuclei of the components that are involved. Expansion of nuclei should be frequent for the fabrication of nanomaterials. Precipitation occurs in the form of thick cloudy form. Many parameters like pH, concentration and temperature are involved in the fabrication of desired morphology and size. Starting materials are metal salts and alloys and the precipitation promoter that are utilized are hydro-oxides of sodium and ammonia. Main assistance of this method is that, it can be regained by providing similar condition. Along with the assessment, here is a drawback of this method that it consumes large amount of samples and give less production.Metal, metal oxide nanoparticles along with super paramagnetic nanomaterials are being synthesized by this method. It is considered that aqueous medium is more efficient for the synthesis of super paramagnetic nanomaterials.Nano-toxicity and environmental contamination of nano-materials is an area of concern for nanotechnology research world. For the solution of these issues eco-friendly green method of the synthesis of nano particles and chemical methods for the synthesis of nano particles are being introduced. Many books based on general information about green chemistry are published and their approaches in different fields along with various methods of synthesis. Many applications in various fields are revolutionized regarding eco-friendly method of synthesis. For the need of synthesizing less harmful and non-toxic nano particles chemists got a lot of guidance by the rules revolutionized by green chemistry. Many chemical and technical methods are successfully being used in the synthesis of nano particles but these methods are considered as expensive and toxic so there was a need to synthesize nano particles by cheap and non-toxic ecofriendly method. Although a lot of progress gained in nano sciences but still there are some bad effects that are completely unknown. So there is need for the synthesis of nano materials by that processes which are non-toxic, cheap and eco-friendly. For this purpose low temperature, inert eco-friendly method and non-hazardous solvent (water) may be adopted now a day.Green methods used for the synthesis of nano particles including extracts of plants, micro-organisms etc. are considered as a source of reducing agents. These methods have revolutionized the area of synthesis of nano particles. Plants are considered as the best source for green fabrication and are being successfully used in a variety of synthesis of nano-materials. Moreover plants are rich source of poly-phenolic compounds and these poly-phenolic compounds are being extracted from different parts of plants. Eco-friendly route have been superior to other routes because of its low cost, non-hazardous, and reproducible ability. Consequently we have been used green method for the synthesis of fine, cheap, eco-friendly and non-hazardous nanoparticles. Punica granatum peels contains various polyphenol which may serve as long lasting and environmentally benign reservoirs for production of metallic nanoparticles. To the best of our knowledge this is the first report describing the fabrication of nanoparticles (copper, cobalt and zero-valent iron) in which Punica granatums peels extact act as reducing and stabilizing agent. OBJECTIVESIn current study we developed economical, versatile and reproducible method for the synthesis of metal nanostructure including copper, cobalt and zero-valent iron by reduction process using peels extract of Pomegranate. Punica granatums peels extract has been used along with respective metal salts to produce desired nanoparticles. Metals salts are the sources of metal ion while Punica granatums peels extract provides the reducing agents for metal reduction and stabilizing agents of the nanoparticles. Synthesized nanoparticles have been characterized by various techniques like FTIR, SEM, EDX and AFM. All the results confirm the successful formation of nanoparticles. REVIEW OF LITERATUREIn the recent past, immense number of studies on the synthesis and characterization of nano-materials by various methods has been carried out. Brief information reported in the literature regarding the synthesis and characterization of nano-particle has been summarized below.Various chemical methods have been used for the fabrication of nanoparticles but due to the elimination of hazardous and expensive chemicals, green method of fabrication of nanoparticles has become more applicable and advantageous. By using greener route synthesis of metals their oxides and salts are being synthesized. This method has revolutionized the nanoscience and technology in just few years. In greener route waste materials are non-hazardous and can be wasted easily because these are of plant infusions parts. Major dedication of greener eco-friendly method is the fabrication of silver nano materials and many other metals along with their salts and alloys have been reported due to the non-hazardous and easy method. Green methods of the fabrication of nano particles are being preceded by utilizing infusions of different plants which are the source of reducing and stabilizing agents. The method that is being applied for the fabrication of any metal and its oxide can also be applicable for another metal and its oxides. It has been reported that the synthesis is being proceed at small scale in laboratories only. Although it has been expected investigation of the reaction mechanism that is involved in the fabrication of nanoparticles are needed to be known with more confirmed conclusions.

In recent research era, infusions of plants and many of species are being used in the fabrication of iron nanoparticles. Synthesis proceeds at room temperature and the infusions used were purely aqueous. After fabrication, nanoparticles were exposed for characterization. Characterizations involved were UV-visible spectrophotometry, and SEM. This method was thought to be eco-friendly and cheap method and can be used for the fabrication of iron nanoparticles at large scale.

Now-a-days iron nanoparticles caste impressive role in the screening tools for the environment. Fe nanoparticles were fabricated by greener method of fabrication by using plants infusions including Euphorbia milii, Datura innoxia, lemon grass tree etc. Iron nanoparticles were fabricated by the interaction of plant infusion and the solution of FeCl3. Infusions of plants are rich source for the reducing and capping agents. These help in the fabrication and stabilization of iron nanoparticles. Characterization of iron nanoparticles was done by UV-Visible spectrophotometry, TEM and DLS, DLS was used for measuring the size of iron nanoparticles. Iron nanoparticles fabricated from the infusions of plants stem and flowers were considered to be the source of very small size and the size of iron nanoparticles synthesized by usingEuphorbia milii was noted from 13 to 21 nm.

Different routes have been used for the fabrication of nanoparticles of metal, metal oxides and their salts. These routes include chemical, physical and biological methods but due to some drawbacks along with limitations, there is a need for a method that may be harmless, non-toxic, eco-friendly and cost effective. Various studies have been made for the fabrication of metal and metal oxide nanoparticles by using biosynthetic routes and their scope in future.Fe Nanoparticles were synthesized by green route by taking 0.1 molar solution of FeCl3, and reacting it with the infusion of leaves of eucalyptus plant. Diffraction peak showed that the nanoparticles of Fe were of amorphous nature. Adsorption capacity for dye was tested for iron nanoparticles and it was noted that there exist high capability of absorption of dye at room temperature. Almost 1.6 gram of acid black 194 dye was absorbed by only 1 gram of Fe nanoparticles. Covering agents of nanoparticles were used for purifying water and in ground water remedy.Green method of fabrication is also used for the fabrication of silver nanoparticles by using silver nitrate solution. Silver nitrate aqueous solution was treated with the infusion of various species of plants and its parts, fungi and other microbial components for the purpose of fabrication.

By studying the structure of Elettaria Cardamomum it has been observed that many functional groups were present in the structure having the property of reduction. So, for the green route for fabrication of gold nano-particles, Elettaria Cardamomum (elaichi) has been used. The infusions of plant were used of different concentrations. Aqueous solution of Tetrachloroauric acid was treated with the extract of elaichi at moderate conditions. Characterization techniques involved were UV-V spectroscopy, XRD, FTIR and laser diffraction particle size analyser. The size of Au nanoparticles was noted to be 432.3 nm.

Biological experimental inspiration revolutionized a valuable branch of nanoscience. Use of leaf infusions revolutionized an eco-friendly and emerging access. 1mm solution of AgNO3 was prepared and allowed to react with the leaf infusion of pomegranate fruit. Poly phenolic compounds present in the infusion of leaf were the source of reducing and stabilizing agents for the silver nanoparticles. Pomegranate leaf infusion was screened out and then used for the fabrication of silver nanoparticles of different morphology and sizes. Silver nanoparticles were characterized by different techniques to check absorbance with respect to time and sizes. Characterization was done by UV-visible spectrophotometer and TEM. TEM showed the average size for silver nanoparticles about 10 to 15 nm. Their structure was exposed to be spherical. These nanoparticles were considered as highly toxic for drug resistant antibodies for human.

Recent investigation shows that by using Lycopersicon esculentum callus and leaf infusions, Ag and Au nanoparticles were fabricated. It realized that callus tissue culture and infusion of leaf were capable for the fabrication of silver and gold nanoparticles. The synthesis of Ag and Au nanoparticles was confirmed by colour change and spectrophotometry. The colour change to brown indicated the fabrication of silver nanoparticles and colour change to ruby confirmed the fabrication of gold nanoparticles in the reacting solution. Infusion from callus was found to be more frequent in the fabrication of silver and gold nanoparticles, in comparison with the leaf infusion. Morphology of both metal nanoparticles was appeared to be spherical and the size was noted about 30-40 nm. Size and morphology was characterized by using SEM and DLS techniques. From FTIR it was realized that in the synthesis of nanoparticles there is some authentic association of nanoparticles with the carbonyl and hydroxyl groups. It was realized that infusions from Lycopersicon esculentum were the source of elegant and frequent fabrication of Ag and Au nanoparticles.

Fabrication by eco-friendly method is most wanted in nanotechnology because of the violence of toxicity produced from the chemical route. Moreover it is also needed in medical applications to avoid hazardous and toxic effects. Silver nanoparticles were fabricated by using fruit (Solanum lycopersicums) infusion. After the fabrication silver nanoparticles were characterized by different techniques to know their morphology and other phenomenon. Characterizations include FTIR, UV visible spectrophotometry, Raman spectroscopy, SEM and TEM. Spherical shaped Ag nanoparticles having size of about 10 nm were noticed and identified. Resonance peak of about 445nm was observed. Infusions contained reducing and stabilizing agents that were main keys in the fabrication of silver nanoparticles.

Fabrication of nanoparticles of metals from green route is casting so much importance for its eco-friendly and cost effective nature. Most of them are single step reactions also. By using fruit extract silver nanoparticles were fabricated by using the infusion of Morinda tinctoria (the medicinal plant), appraising the microbial assets in it. The fabrication of silver nanoparticles was confirmed by using characterization techniques such as SEM, TEM, FTIR and UV- visible spectrophotometry. DDA technique was used to identify the antibacterial activity of Ag nanoparticles. By UV-visible spectroscopy maximum absorption peak was noted at 420-25 nm. From FTIR technique it confirmed that silver nanoparticles converted from monovalent to zerovalent metal atom. Morphology of Ag nanoparticles was noted from SEM and TEM techniques. It was realized that fruit infusion of M.Tinctoria promotes the synthesis and anti microbial nature of Ag nanoparticles. Moreover it showed antibacterial activity against many plant species.Environmental friendly and cheap method for the fabrication of silver nanoparticles has been reported by using infusion of pomegranate seeds. As the infusions play role in the reduction process of metals as they are rich source of reducing and capping agents. Fabrication of silver nanoparticles was realized to be easy to handle. This method was studied by UV technique. Shape and size of silver metal nanoparticles was studied under TEM and XRD techniques.

These nanoparticles were suggested for the cure of infections and therapeutic fields.

Fabrication of silver nanoparticles has been done by different routes i.e chemical, physical and natural. Infusion from Daturaalba nees is use for the fabrication of Ag nanoparticles. Plant extract treated with the solution of AgNO3, the colour change from colourless to reddish brown was noted that indicated the reaction proceeded. Absorbance peek was noted at about 444nm. XRD analysis showed that the morphology of the Silver nanoparticles was crystalline and the size about 28.42nm. Spherical shaped nanoparticles were observed under SEM analysis. Anti-bacterial activity was noted against Chlostridium diptheriae by counting the inhibitory region.Ag nanoparticles were synthesized by using many plants sources.it has been thought that there may be a detailed procedure involved in the conversion of Ag valent to Ag zero-valent using plant infusions. Characterization techniques including TEM, SEM UV-visible spectroscopy, XRD and FTIR showed their morphology, size, absorbance, bond stabilization etc. Accumulation of the concentration, desired amount of AgNO3 solution was added to the biotic infusions. Colour change from colourless to brown indicated the occurrence of the reaction and fabrication of Ag nanoparticles. Absorbance between 425-435nm was noted. Antibacterial activity from silver nanoparticles has been considered by the zone cleared by gram positive and gram negative bacteria along with some pathogenic fungi.By using infusions of clove, copper nanoparticles were synthesized. Solution of CuSO4 was allowed to anti oxidized in the presence of clove aqueous extract. Morphology of Cu nanoparticles was proved by TEM. TEM showed the particle size from 5 to 40 nm. For the proval of crystalline nature of copper nanoparticles XRD was used. SEM showed throw imagination that the particles are of spherical shape and have grainy nature. UV-visible spectrophotometry showed the peak for absorbance.In the field of nanotechnology solid impression proved its importance. It has been estimated that the metal nanoparticles have 1-100 nm area of dimensions. Copper nanoparticles are important of their importance in the industry, agriculture and many other fields. The necessity of biological components in plants revolutionized its role in the nanotechnology. Nanoparticles have been synthesized by combining physical, chemical and biological technique. Copper nanoparticles were found to be effective for anti-bacterial activities in the field of agriculture as it has facilitated the researchers to fabricate eco-friendly, inexpensive and clean Cu nanoparticles.

In medical sciences copper nanoparticles are being used for the cure of ulcers and to inhibit infections. Nanoscience have revolutionized important role in the medical field due to its major utilities. Cu nanoparticles were synthesized by using leaf infusion of Nerium oleander that acted as anti-oxidant. Cu nanoparticles after fabrication were subjected to characterization to know their morphology, size and motif structures. Characterization techniques included FTIR and UV-visible spectroscopy. Cu nanoparticles were used for the study of five different bacterial diseases. Iron nanoparticles were synthesized by using fruit infusions. Mango infusion was treated with Iron chloride solution in moderate atmospheric conditions by green route for fabrication. Sample of iron nanoparticles was characterized by different techniques to know their morphology, absorbance and structural nature. Characterization techniques involved SEM, UV-V spectroscopy and XRD. Diameter of Fe zerovalent particles was noted about 50-100 nm.Prathna and fellows used lemon infusion as anti-oxidant source for the fabrication of Ag nanoparticles. Different parameters involved in the process were studies including antioxidant concentration, ratios and molarity of AgNO3 used. By taking ratio of range 1:4 lemon infusion was treated with 2 to 10 M solution of AgNO3 for about 4 hours. Fabrication of Ag nanoparticles has been confirmed by characterization techniques including UV visible spectroscopy, XRD and TEM. Absorption spectrum was in the range of 400 to 500 nano-meters. In lemon infusion citric acid played the main role for the reduction for the fabrication of nanoparticles. TEM analysis confirmed the spherical and spheroidal morphology with 50 nm size. Another example of ecofriendly method of fabricating nanoparticles is fabrication by orange peels infusions. Orange peels were the source of anti-oxidants because of large number of phenolic compounds present in the peels. At atmospheric conditions and basic pH nanomaterials were prepared which was starch supported. Affluence of sugars, carotenoids, pectins etc. also investigated as anti-oxidants that helped in the fabrication of Ag nanoparticles. SPR (surface Plasmon resonance) crest was noted of about 3 to 12 nanometre. Researcher have selected plants components from the peels of citrus sinensis that exposed to sunlight for drying. These phyto components were used in the fabrication of Ag and Au nanoparticles. The extract was rich in poly phenolic compounds that play role in maintenance of nanomaterials. Fabricated nanomaterials were stabilized and spherical shaped particles having size of 14 to 20 nm. SPR bands of silver nanoparticles were studied that appeared at 445 nm and 424 nm. The reaction was carried out at moderate conditions providing 60 C. From FTIR phenolic components were confirmed which play their role in anti-oxidation of metals. TEM analysed the nanosize of the synthesized nanoparticles was estimated 2 to 35 nm along average size 1 to 10 nm. XRD characterization showed 3 to 33 nm and average size 2 to 8 nm of silver nanoparticles respectively. XRD technique results for silver nanoparticles were supported by EDAX technique results. Elemental detection concluded the presence of silver. By this technique conversion of Ag ions in to non-ionic form was confirmed. Silver nanomaterials were considered to have efficient activity against gram negative as well as Gram positive bacteria. Although further research proved that the antibacterial activity is more adjacent to Gram negative bacteria as compared to the Gram positive bacteria. Ag nanoparticles that were fabricated at 60C showed high antibiotic activity in contrast to the nanoparticles synthesized at 25C due to their small size. Anti-bacterial activity is mainly because of Ag cationic species that came from silver nanoparticles. Banana peels and pulp is discarded mostly because the pulp got oxidized and peels were wasted as useless material. It was realized that the waste material from agriculture department that many be rich source of polymers, can be utilized for the fabrication of metal nanoparticles such as palladium. Banana peels were dried in the presence of air and were used as anti-oxidant source in the fabrication of Ag nanoparticles. Reaction conditions were change i.e. pH, concentration and temperature of incubation. Colour of the reaction mixture was changed from colourless to brown. The sample was characterized by UV-visible spectroscopy that indicated the presence of silver content. SEM results confirmed the crystalline structure and size of particles in nanoscale, after short intervals of incubation. In 2010 palladium nanoparticles were fabricated by using waste peels of banana. UV-visible spectrophotometry confirmed the presence of palladium by showing definite peak at 400nm. Banana peels infusions have also been used for the fabrication of Au nanoparticles as they are the rich source of reducing agents. Array of bright colours and UV-Visible spectra was noted by changing the conditions of reaction. Peaks observed at (111) and (200) indicate the existence of frame structure of microwires along with microcubes. Banana peels infusions helped to moderate these structures. Anti-microbial activity is also reported by making tests of fungi and bacteria cultures by using Au nanoparticles.In green method of fabrication of nanoparticles NEEM KERNAL infusion was used as a source of reducing agents. Characterization techniques XRD and TEM confirmed the fabrication and morphology of the silver nanoparticles. Size of silver nanoparticles was noted about 1.37 nm to 8.25 nm. Silver nanoparticles were found to be good sensors for the confirmation of H2O2 in aqueous media. Pd nanoparticles were obtained by using the infusions of Annona squamos and spherical shaped of size 5 to 80 nm were reported. It was reported that there were some hydroxyl group containing agents present that were responsible for the reduction of Pd (II) metal into zerovalent form. Size of the spherical shaped nanomaterials was noted from 20-60 nm. reaction between the infusion of Annonasquamosa and the aqueous solution of Pd (II) was also provided. Further research was made by synthesizing TiO2 nanomaterials using the same source for anti-oxidants. XRD results showed that the TiO2 nanocrystals were spread to the area of 2 to 23 nm range. Mechanism of the reaction of fabrication was also determined. A squamosa has been suggested to have the efficiency of the fabrication of alloy and its oxides. Infusion of another seed named as B.hispida was also proved to be great fountain for anti-oxidants. In spectrophotometry spectrum gave information about the morphology and the study of interaction of nanoparticles with the infusion. Is has been noted that at moderate conditions the particles size is broad because the anti-oxidation rate is low at room temperature. With the passage of time the SPR tape moved from 548 nm to 544 nm which indicated that the particle size got smaller.

X-ray diffraction bands turned broad that showed the fabrication of Ag nanoparticles. Penta bands in XRD indicated the cubic structured nanogold. TEM high resolution technique showed that the resolution at (111) is more intensive and dominating. Crystal structure of nanoparticles was indicated by bright round dots in SAED form. Proteins in infusions were realized the rich source and responsible for the reduction and stabilization of metal nanoparticles . Fabrication of Ag nanoparticles by green method was reported in 1 hour, confirmed by the colour change from colourless to yellowish brown. Synthesis of nanosilver was insured by cubic morphology and adsorption band at the range of 457 nm. Characterization techniques showed that silver nanoparticles were shaped like leaf and the size of nanoparticles was estimated about 38 nm. Fabrications of Ag and Au nanoparticles have been reported for getting particles of different morphology by using leaf infusions. Morphology of silver nanoparticles was based on the changing concentration of Ag salt and the infusion. In the green route for fabrication of Au nanoparticles by using warm aqueous infusion of the leaves of olive, phenolic components in infusion were realized to be the rich source of reducing agents. These reducing species were responsible for the conversion of cationic gold into non-ionic form. From the characterization techniques it was reported that the shape and size of the nanomaterials was based on different parameters such as pH and the conc. of the metal salt and infusion that was used. Cellulosic nanoparticles were fabricated by using the aqueous infusion of corn cob that was reported as a rich source of reducing agents. Raw material was treated with 0 to 24 % of NaOH solution. The fabricated nanomaterials were washed by using dist. water, shattered and filtered. Resultant product was treated with NaCl and allowed to dry in oven. Dried sample was meshed and separated with the help of 270 mesh screens. TEM technique reported the size of nanoparticles 22nm.

Fabrication of Ag nanoparticles was done by using different varieties of cereal and pulse crops. Extracellular fabrication of Ag nanomaterials was studied by using natural sources. Stabilized Ag nanoparticles were fabricated at 50C to 95C by the treatment of plant leaf infusions with AgNO3 solution. 50 to 200 nm sized Ag nanoparticles were confirmed by SEM and EDX techniques. In the fabrication of Ag NPs guava leaf infusion was used as a main source of anti-oxidizing agents. Guava leaf infusion was reported to have many phenolic compounds that were responsible for the reduction of metal cations into non-ionic form. Microwave method was used for this application. Fabricated silver nanoparticles were reported of about 5-26 nm. Fabrication of nanosilver was occurred in very short time about 90 seconds so called as rapid reaction. It indicated the presence of the efficient reducing agents in guava infusion. Fabricated Ag nanoparticles have been reported for their larvicidal activity by using malaria and filariasis vectors. SEM results showed that silver nanoparticles have size from 35 to 60 nm and were easily noted. Fabricated Ag nanoparticles were treated with larvae by changing the concentrations for about whole day. Major capability was noted against C.quinquefasciatus. More evident species of plants sustain in dry climate the Prosopis juliflora was used in the fabrication of metal nanoparticles. Although its pharmaceutical applications were not reported. Researchers have used the infusion f leaves of this plant in the fabrication of Ag nanoparticles. The anti-fungal, anti-viral and anti-bacterial activeness has also been studied.

In green method of fabrication, the use of agricultural waste for the synthesis of metal nanoparticles has caste an impression. Eco-friendly synthesis of Ag nanomaterials was reported by using A.hypogaea leaves infusion. By this method the ordinary size of nanoparticles was reported from 7-8 nanometre. The fabricated nanomaterials were analysed by SEM for their morphology and were coated on glass. Prevention of Ag nanoparticles against antibiotics has also been studied .

Copper oxide nanoparticles were synthesized by utilizing the waste from agriculture. It was realized to be a low cost, eco-friendly and non-toxic method of fabrication. Barfoed reagent was reduced by using infusion of A. hypogaea leaf. Leaf infusions were rich source of aldehyde group present in sugars that was used superb anti-oxidant agent for the reduction of metal cations. Anti-bacterial activity of Cu2O nanomaterials was tested in contrast to Gram negative bacteria. After fabrication, copper nanoparticles were characterized by different techniques such as FTIR, SEM, XRD and UV-visible spectrophotometry for their morphology and other characteristics .

Ag nanomaterials have been prepared by using fruit infusions. The fabrication of nanoparticles of silver by using pomegranate peel infusion has been reported. Infusions of pomegranate peel are treated with aqueous solution of silver nitrate for the fabrication of nanoparticles. Pomegranate peels were considered as the rich source for poly phenolic compounds that played their further role in the reduction by acting as reducing agent in the fabrication of silver nanoparticles. Simple procedure for the fabrication of Ag nanomaterials was reported. Characterization techniques were applied for the sake of morphology and crystalline nature of nanoparticles. TEM technique reported the size of nanoparticles of about 5-15 nm.

Researchers made efforts to fabricate Au nanoparticles by cost effective and non-toxic method. Palm oil effluence from mill was used for the fabrication of gold nanoparticles beyond any other stabilizing agent. Characterization techniques were used for knowing the morphology, size and other characteristics of the fabricated nanoparticles. Samples were characterized by TEM, FTIR, XRD and spectrophotometry. Hexagonal and triangular nanomaterials of gold have been reported.

Coconut palm reported as very useful plant as its fibrous nature. Its leaves from agriculture waste were used for the fabrication of Ag nanoparticles. Leaves infusion was realised as the excellent source for the fabrication purpose of Silver nanoparticles. The synthesized nanoparticles were further characterized for confirmation and identification of morphology. TEM characterization showed that the located nanoparticles having size about 2 to 23 nm. Ag nanoparticles were also used further against pathogen activity of microbes.3.1. Chemicals All the chemicals were of analytical grade and were used without any further pre-treatment. Salts used for the synthesis of nanoparticles include ferric chloride hexahydrate (FeCl3.6H2O), copper sulphate pentahydrate (CuSO4.5H2O) and cobalt nitrate hexahydrate () were purchased from Sigma Aldrich.

Other materials Fresh Punica granatum were procured from local market and their peels having polyphenol were used as reducing and stabilizing agent for the fabrication of nanomaterials. 3.2.Preparation of Salt Solutions1Molar solution of ferric chloride was prepared by adding a weighed amount (16.2 gm) of FeCl3 in 100 ml of volumetric flask containing 30 ml of de-ionized water. After shaking, volume was made up to the mark with deionized water.

Similarly, 1M solution of CuSO4 was prepared by adding 16 gm of CuSO4 in 100 ml of deionized water. For the preparation of 1M solution of Co(NO3)2.6H2O, weighed amount of salt was dissolved in 100 ml flask and made up to the mark with deionized water.3.3.Preparation of Pomegranate Aqueous Extract Extract of fresh peels of pomegranate was prepared by reported method with slight modification. Peels of pomegranate were washed and weighed. 194 gm of fresh peels of pomegranate were mixed with 1300 ml of deionized water and grinded with the help of electric grinder. Then mixture was heated along with continuous stirring with the help of magnetic stirrers on the hot plates at about 80-85 C for -----minutes .The resultant mixture was filtered with the help of suction assembly. The filtrate was collected and named as extract.3.4.Synthesis of Nanoparticles3.4.1.Synthesis of Zero-valent Iron Nanoparticles100 ml of freshly prepared pomegranate peels extract was added in 1M solution of FeCl3. FeCl3 solution color change was observed from yellow to black that indicated the reaction. Then this reaction mixture was allowed to heat for about 10 minutes at 60-70 C along with stirring. Magnetic stirrer was used for this purpose. After this treatment stirrer was removed and the solution was filtered with the help of suction assembly. Sample was collected, washed by ethanol and dried in oven at about 40-45 C. Then this sample was grinded with the help of pastel and mortar and preserved in glass voiles.

3.4.2.Synthesis of Copper Nanoparticles100 ml of freshly prepared pomegranate peels extract was added to 1M solution of CuSO4.CuSO4 solution colour change from blue to green indicated the reaction occurrence. Then this reaction mixture was allowed to heat for about 10 minutes at 60-70 C along with continuous stirring with the help of magnetic stirrer. After heating along with stirring, stirrer was removed with the help of magnetic bar and filtered with the help of suction assembly and sample was collected, then allowed to dry in oven at about 40-45 C after washing with ethanol. Then this sample was grinded with the help of pastel and mortar and saved in glass voiles.

3.4.3.Synthesis of Cobalt Nanoparticles100 ml of freshly prepared extract of pomegranate peels was mixed with 1M solution of Co(NO3)2.6H2O, color change was observed from light to dark red that indicated the reaction occurred. Then this mixture was allowed to heat at about 60-70 C for about 10 minutes along with continuous stirring with the help of magnetic stirrer. After heating, stirrer was removed with the help of magnetic bar. Then this reaction mixture was allowed to filter by using suction assembly. Sample was collected and washed with ethanol and allowed to dry in oven at about 40-45 C.After drying the sample was grinded with the help of pastel and mortar and finally saved in cleaned glass voiles.3.5.Characterization of NanoparticlesNanoparticles of Iron, Copper and Cobalt were prepared from pomegranate peels extract and were characterized by different techniques in order to confirm the successful formation of nanoparticles and to further elucidate the morphology and particle size of synthesized particles. For this purpose, samples were subjected to Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Energy Dispersive X-Ray Spectroscopy (EDX).RESULTS

4.1.Selection of MethodMany routes have been applied for the fabrication of metal nanoparticles such as physical, chemical and biological methods. Due to toxic, expensive and eco-hazardous nature of chemical method researchers molded their research route to green method of fabrication for nanoparticles of metal, metal oxides and their alloys. Green route is considered as eco-friendly, cost effective and non-hazardous. As the nanoparticles have applications in almost all fields of life and among them in medical field in cure and early detection of cancer nanoparticles are gaining much importance. Green route prevents the waste of time, waste of money, toxicity, hard handling etc. Metal nanoparticles are synthesized in just 90 seconds that is an amazing rising fact. In green method of fabrication metal nanoparticles are fabricated by using plants, microbes and agriculture waste. Fresh fruits extracts as well as wasted and rotten fruits extract is used after screening, in the fabrication. In plants, roots, stems, leaves and flowers are being used but most of them an interesting and fruitful source is the use of fruits. Edible fruits waste such as seeds and peels are wasted for none. Hence researchers made their research on utilizing seeds and peels in the fabrication of nanomaterials. On further research it is reported that the extracts of peels and seeds of fruits contain an excellent amount of poly-phenolic compounds that play their role in reduction. These poly-phenolic components are called as reducing or anti-oxidants for metals. These reducing agents change the cationic form of metals into non-ionic form due to anti-oxidant activity. Moreover we can say that by using fruits we are gaining fruitful results in favour of nanotechnology.

Fabrication of nano materials was done by different routes including chemical and natural compounds. Admitting that fabrication of nano materials by using chemical route do not take more time for production at large scale. Metal nano materials need reducing and stabilizing agents that are provided by chemical and biological methods. But chemical route is considered as toxic, hazardous, expensive. So the desire of the formation of nanoparticles by non-toxic, cheap and eco-friendly method was produced. Green route of fabrication of nano particles gained a lot of importance because of eco-friendly, inexpensive and non-toxic nature. Extracellularly and intra-cellularly occurring nano material fabrication has been accounted in biological organisms e.g plants, fungi and microorganisms, up till now. Plants are considered to be more suitable for the fabrication of nano materials because they are non-toxic and are source of reducing agents. Furthermore, plant infusions are regarded as inexpensive source of capping agents as compared to microorganisms in nano fabrication. Employing different plants and their infusions in nano fabrication is more beneficial as compared to other biological methods of fabrication.Various experimentations are being done for the fabrication of varied metal nano corpuscles utilizing fungi e.g. Fusarium oxysporum, Penicillium and a few bacteria like Bacillus subtilis. Fabrication of nano materials by plant infusions is more adoptive in producing environmentally-friendly synthesis of nano specks. This is also profitable as plants are cosmopolitan, safe in handling, easily accessible and reservoirs of different metabolites.

4.2.Choice of Materials

Pomegranate (Punica granatum ) is one of those widely grown crop because it has been used in different industries like food, cosmetics and medicines from ancient times. This plant is a rich source of poly phenolic compounds. Ellagic acid, punicalagins A and B, ellagitannins, gallic acid and gallotannins are the main compounds which are found in pomegranate. Structure of these poly phenolic compounds are given in figure 3.Among all these compounds ellegic acid (EA) has been of prime importance due to its role in different pharmaceutical products. EA has the unique characteristics and it has been used as anti-tyrosinase, anti-inflammatory, antioxidant anti-mutagenic. Furthermore, EA has been effectively applied to suppress skin pigmentation induced by UV radiations, which suggests that EA could be used to prevent skin pigmentation after sunburn. Therefore, EA is considered to be a promising skin whitening agent in cosmetics industry. In recent studies it has been reported that extract produced from the pomegranate peels contains upto 13% ellagic acid and this extract has quite comparable features like anti-inflammatory, anti-allergic and antibacterial agent to the pure EA. Due to the presence of all these poly phenolic compounds and flavonoids in the pomegranate peels, which act as reducing agents as well as stabilizing the nanoparticles, this plant was selected for extract preparation. Before selecting, it was thoroughly reviewed that this plant was not commonly used for nanoparticle preparation.Iron is one of the most important metals in the periodic table, due to its versatile properties, natural abundance in nature, variable oxidation state and application in every field of research and technology. Iron exists in three different oxidation states, zero valent, divalent and trivalent. Due to this variable oxidation state it has huge diversity in compounds. Metal nanoparticles prepared from conventional methods has been very reactive and could not be stored for longer period of time, but green synthesis approach have the ability to overcome this difficulty because of the presence of biological compounds in extract which act as stabilizing agents. Hence, nanoparticles prepared by this method could be stored for a longer period of time. Apart from this, iron nanoparticles prepared by chemical methods cannot be applied in biomedical field due to their toxic effects. Similarly, magnetic iron nanoparticles lose some of their magnetic property in air. Green synthesis method has removed all these problems.

Metal nanoparticles synthesized by green approach have many applications in different areas of research. Iron nanoparticles have been used in bio-medical (drug carrier, anti-cancer drugs, MRI), removal of pollutants from water and many other fields. Copper is an essential microelement which required by the plants for their growth and development. Optimum concentration is required for plants. If concentration is below certain limit then it causes deficiency while on the other hand if concentration exceeds certain limit then it is considered to be toxic. Copper plays its role in protein regulation, hormone signaling, mitochondrial respiration, participates in photosynthetic electron transport, oxidative stress response, cell wall metabolism and cofactor for many enzymatic reactions. Deficiency of Copper in plants is results inlight chlorosis, petioles bent downwards, curled leaves and permanent loss of turgor pressure in the young leaves. Similarly Cobalt was selected due to its widely growing applications in the field of catalysis and to make inexpensive multilayer ceramic capacitors. At nanoscale the electrical conductivity increases due to greater surface area.4.3.Reaction Mechanism The mechanism of synthesis of metal nanoparticle from plant extracts can be divided into 3 major steps. (A) 1st step is the activation phase in which metal ions are reduced followed by the nucleation of the reduced metal atoms takes place. (B) In 2nd step is named as growth phase in which the small neighboring particles simultaneously merge into particles to form larger size which increase the thermodynamic stability of nanoparticles. (C) 3rd and last phase is known as termination phase which determines the shape of nanoparticles. With the increase duration of growth phase, nanoparticles shape changes which are transformed in to nanotubes, nanoprisms, nanorods. Apart from these regular shapes, a variety of other irregular shaped nanoparticles can also be formed. During the termination phase, synthesized nanoparticles attain the most energetically favorable conformation.This process also being strongly affected by the stabilizing ability of a plant extract. The mechanism of formation of nanoparticles from pomegranate extract is shown in figure 4.

Gallic Acid, Ellagic Acid

Stabilizing Agents

H+Figure 4. General mechanism of nanoparticle fabrication from pomegranate peel extractPrimary components of pomegranate extract are alkaloids, flavonoids and polyphenols. These phenolic compounds present in pomegranate are also known as anthocynadins which are well-known scavengers of free-radicals reactions. Antioxidant properties of these phenolic compounds is primarily due to their high inclination towards chelate metals. Phenolic compounds present in the extract contain hydroxyl and carboxylic groups which have very high tendency to bind heavy metals. When metal ions present in the salt solution come in contact with the any of the poly phenolic compound (ellagic acid), p track conjugation effect is formed from ester oxygen atom and ortho-phenolic hydroxyl group present on ellagic acid. This esterification process of carboxylic and hydroxyl groups of ellagic acid results in the loss of hydrogen from ortho-phenolic hydroxyl group. A semi-quinone structure is formed after the loss of hydrogen. H+ radical is formed due to the electron losing property of ellagic acid.Size of metal ions is reduced to nano during this whole process. Phenolic compounds in the pomegranate have excellent free radical scavenging capability. As they are water soluble and very high bioavailability, they possess remarkable pharmacological activity due to the presence of hydroxyl groups. The scavenging capability of punicalagin against different oxidizing radicals (OH, N3 and NO2) is different. Different techniques like time-resolved kinetic spectrophotometric techniques have been used to study rate constants of scavenging reactions and formation of transient. Antioxidant mechanism of punicalagin in nanoparticle formation is expressed through its scavenging reactions along with the formation of H+ species which results in reduction of the size metal particles to nano level.4.4.Characterization of Fabricated Nanoparticles4.4.1.Fourier Transform Infrared Spectroscopy (FTIR)FTIR is one of the most commonly used tool for the characterization of nanomaterials. It is a non-invasive and non-destructive technique. Instrument consists of a light source and a beam splitter which is composed of up of cesium iodide (CsI), potassium bromide (KBr), mirrors and a detector. Radiations emitted from light source are passed through the sample and reach the detector. Sample absorbs some of the radiation while transmitted radiations reach the detector where intensity is measured. In detector, excitations are generated from the vibrations produced by the radiations, where a signal is produced and peaks appear.FTIR spectrum usually gives information about the functional groups present in the compound; stability and quality of samples can also be analyzed by this technique. Newly fabricated nanoparticles of iron, copper and cobalt was analyzed by FTIR spectroscopy to get the information about the compounds which are responsible for the reduction of metal cations to zero valent metal nanoparticles. Figure 5 shows the FTIR spectra of iron nanoparticles which a broad peak appeared in the range of 3229 cm-1. This peak corresponds to OH group which are present in poly phenolic compounds. Peak appears to be broad due to hydrogen bonding present in the OH groups of phenolic compounds. In plane bending of OH groups appeared in the region of 1322 cm-1. Very small curves present in the region of 2900 cm-1 were representative peaks of CH stretching. Peaks appearing in the region of 1573 cm-1and 1600 cm-1are due to the presence of aromatic groups of phenolic compounds. And in range of 1073cm-1 is due to C-O stretching of alcohols and carboxylic groups. There were some other minor peaks were observed due to the presence of some other biomolecules. All these peaks confirm the presence of different types of biological compounds which were present in the pomegranate extract which act as reducing agents during the nanoparticle formation and after that stabilizing and capping agents to protect the fabricated nanoparticles from degradation in air.

Figure 5.FTIR spectra of Iron nanoparticles synthesized from pomegranate peelsSimilarly, copper nanoparticles were also characterized by FTIR (Figure 6). Broad peak present at 3134 cm-1 is due representative peak of OH groups. Broadness in the peak is due to intermolecular hydrogen bonding of polyphenols. A band appearing in the region of 1669 cm-1 is due to aromatic rings. 1068 cm-1 is the peak due to C-O of alcohols and carboxylic acids. All these peaks confirm the presence of different biological compounds around the copper nanoparticle which act as capping agents.

Figure 6.FTIR spectra of copper nanoparticles.

To analyze the formation of cobalt nanoparticles and presence of stabilizing agents around the cobalt nanoparticles, FTIR analysis was carried. Quite similar peak pattern was observed when which was expected before the analysis. FTIR analysis of Co nanoparticles synthesized from pomegranate peel extract is shown in figure 7. A wide broad peak around 3225 cm-1 is characteristic peak of OH groups of poly phenolic compounds of pomegranate extract. Bands present in the regions of 1569 cm-1, 1313 cm-1 and 1049 cm-1 are the representative peaks of aromatic rings, in plane bending of OH and C-O stretching of alcohols and carboxylic acids. All these results confirm the successful synthesis of metal nanoparticles from green synthesis approach by using pomegranate peels extract which is rich source of different phenolic compounds.

Figure 7.FTIR spectra of cobalt nanoparticles synthesized from Punica granatums peels4.4.2.Scanning Electron Microscopy

Progress in the field of optical microscopy and revolution in the field of nanoscience and technology are going parallel to each other and these two fields are considered to be complementary to each other and indeed are fuelling each other. Size and shapes are two major characteristics which play a vital role in electrical and mechanical properties of nanoparticles. Scanning Electron Microscopy (SEM) has been one of the most of the powerful and reliable source for the characterization of nanomaterials due to its high resolution and easy access. Materials in the range of 10th of millimeter to sub nanometer level can be characterized by this versatile technology. Apart from surface morphology this technology has the ability to determine particle size of material particles. Sample is subjected to a strong beam of high energy electrons accelerated through high voltage which passes through the sample and image of those particles is developed on the detector. These electron microscopes have the ability to scan the image by detecting the reflected electrons from the sample. This technology has revolutionalized the field of material science and nanotechnology.Iron nanoparticles were subjected to SEM analysis to access the particle size and shape of the particles. Figure 8 shows SEM image of iron nanoparticles. Scale to measure the particle size of nanoparticles is also given in the image. It is quite clear from the image that the size of fabricated nanoparticles is in the nanometer range. Estimated particle size distribution is from 40 nm to 80 nm. Particles are spherical in shape with variable size. Particles are well dispersed and are quite homogeneous. These particles have been engineered from metal cation reduction into zero-valent metal nanoparticles and have coatings of the different biological compounds which have surface hydroxyl groups. Due to intermolecular hydrogen bonding of these protecting agents, these particles appear to be agglomerated. This image is evidence of the successful formation of nanoparticles .

Figure 8.Scanning Electron Microscopy image of iron nanoparticles synthesized from pomegranate peel extract.Similarly, copper nanoparticles were also characterized by scanning electron microscopy for morphology analysis. Figure 9 shows the SEM micrograph of newly fabricated copper nanoparticles from pomegranate peels extract. Different phenolic compounds are present in the extract, which contributes in the reduction of metal cations into the metal nanoparticles, as well as protecting agents. Due to surface hydroxyl groups, these particles are closely attached to each other, hence agglomerates formation seems to be occurring in these nanoparticles, which is quite evident from the image as well. Size of these copper nanoparticles is also in the nanometer range. Shape of these particles is semi-spherical. Particle size is estimated to be less than 80 nm.

Figure 9.SEM micrograph of copper nanoparticles synthesized from pomegranate peel extract.

To analyze the surface morphology and particle size of the cobalt nanoparticles, sample was analyzed by scanning electron microscopy. Quite similar peak pattern was observed as it was estimated. Figure 10 is the representative spectra of cobalt nanoparticles. It can be seen that particles are round shape, some are irregular structures with particle size distribution in nanometer range. From scale, the calculate particle size is varying from 60nm to 80 nm. Agglomeration in the particles depends upon the nature of the extract and the compounds present in the extract. Reactivity and attraction of the functional group results in the formation of larger size particles.From all these results it can be concluded that method adopted for synthesis of metal nanoparticles is quite successful and efficient enough to use it for this purpose. This method has yielded nanoparticles with desired size and shape. All the SEM results confirm the successful formation of nanosized composites.

Figure 10.Scanning Electron Microscopy image of cobalt nanoparticles synthesized from pomegranate peel extract.4.4.3.Energy Dispersive X-Ray Spectroscopy

Energy Dispersive X-Ray Spectroscopy (EDX), also known as EDS, is another versatile and reliable technique which is frequently being used for the characterization of nanoparticles. This technique is complementary to electron microscopy. In EDX, elemental composition of materials could be determined during the SEM analysis. During SEM analysis, when electron beam interacts with the sample, X-Rays are produced. Every element have different elemental structure, hence X-rays emitted from different types of elements are characteristic for each element. From X-ray emission spectrum, elemental composition of any sample can be accessed. EDX results are in the form of spectra with energy at X-axis and counts at Y-axis.Nanoparticles fabricated from pomegranate peel extract were also subjected for EDX analysis to determine the chemical composition of the nanoparticles.

Figure 11 is the EDX spectra of iron nanoparticles. This spectrum was recorded by using internal standard at energy range from 0-20 KeV. High intensity peaks of iron, carbon and oxygen can be seen in the spectra t different energy levels. These high intensity peaks are the major constituents of the samples under analysis. Composition of each of these elements is also recorded. Highest composition of iron in mass percent is 41.90%, carbon is 31.70% and oxygen is 14.12%. Very small percentage of some other elements like calcium, aluminium and zinc are also present in the sample in trace quantity which could be impurity present in the salt used for the nanoparticle formation. From these results it is quite clear that major constituent of this sample is iron. Presence of carbon and oxygen in relatively high percentage is due to the presence of polyphenolic compounds present in pomegranate peels extract. These results confirm the successful formation of nanoparticles and protecting coating of phenolic compounds around the metal nanoparticles.

Figure 11.Energy dispersive X-ray micrograph of iron nanoparticles synthesized from Punica granatums peelsEDX spectrum of copper nanoparticles is given in figure 12. In this figure it is quite obvious that oxygen and carbon are also present in higher percentage along with copper. According to mass percent of the sample 46% of samples consist of copper which came from the salt solution and reduced in the presence of pomegranate peels extract. Carbon is present upto 27% and oxygen 11% along with some impurities of sulphur. These results indicate copper nanoparticles have been successfully fabricated by green synthesis route.

Figure 12.Energy dispersive X-ray spectra of copper nanoparticles

Similarly, EDX analysis cobalt nanoparticles were carried out by using internal standard at energy from 0-20keV. Percentage of cobalt was found to be 39%, carbon 22% and oxygen 37%. Presence of all these constituents in the sample confirms the successful synthesis of nanoparticles and also indicates the presence of outer capping agent. Figure 13 shows the EDX spectra of cobalt nanoparticles.

Figure 13.EDX spectra of cobalt nanoparticles synthesized from pomegranate peel extract4.4.4.Atomic Force MicroscopyAtomic Force Microscopy is another characterization tools which is currently being used in the field of science and technology. This technique has the property that it can give three dimensional image of the nanoparticles, which is very helpful to judge the morphology of the nanoparticles. In AFM, a laser beam is used instead of electron beam. Laser beam is focused on the back of a cantilever. Tip of that cantilever interacts with a surface which is reflected and reach the detector. As it provides three dimensional image of the nanoparticles, height measurements provides the height of nanoparticles with great precision and accuracy. If the particles are spherical in shape then height measurement relates to the size/diameter or of the particle. AFM has some special features of high spatial resolution so it is being used in material science as well as for the imaging of biological specimen. Particles with sub nanometer size can also be analyzed with AFM.So, further confirmation of the particle size and morphology of fabricated nanoparticles was carried out by atomic force microscopy. Figure 14 shows the AFM image of iron nanoparticles along with their particle size distribution. Figure 14B is three dimensional view of the nanoparticles. From the grooves present in the image morphology of the particles can be assessed. Height of the mountains (grooves) is different which means the particles size is not quite homogenous which is evident in Figure 14A which represents is particle size distribution of nanoparticles. Particles are separated over a broad range, but majority of the particles are in between 40-80 nanometer. Variation in size is attributed to the presence of poly phenolic compounds which have strong attractive forces between them, holds the particles together which results in the formation of larger size particles. Agglomerates formation is particularly due to the presence of intermolecular hydrogen bonding between hydroxyl groups of different phenolic compounds.

Figure 14. Atomic force microscopy of iron nanoparticles fabricated from pomegranate peels extract (A) particle size distribution graph (B) three dimensional view of the nanoparticles.When copper nanoparticles were analyzed by AFM, a quite similar pattern was observed because of similar nature of the extract. Figure 15B shows three dimensional AFM image of copper nanoparticles indicating the semi-spherical morphology of the nanoparticles. Different shapes of the grooves is also clearly visible confirming the varying size of the fabricate particles. Region where agglomerate formation is large, grooves are larger, hence resulting in larger particles and vice versa. This pattern is confirmed by particles size distribution graph (figure 15B). Copper nanoparticles prepared from the extract of pomegranate peels are in the range of 50-80nm.

Figure 15. Atomic force microscopy of copper nanoparticles (A) particle size distribution curve (B) three dimensional image.Similarly, AFM analysis of cobalt nanoparticles was carried and results are shown in figure 16. Particle size distribution of copper nanoparticles is from 40-80nm. Grooves are not homogenous here as well in three dimensional image of nanoparticles which is mainly due to agglomeration caused by phenolic compounds present around the nanoparticles. Presence of the phenolic compound has stabilized the nanoparticles and has protected them from being degraded by air but on the other hand, they are the major reason for larger particle size and agglomeration amongst the particles.Results obtained from AFM analysis has been complementary to previously discussed SEM, EDX and FTIR. These results have confirmed the successful fabrication of metal nanoparticles in the presence of pomegranate peels extract. This extract has contributed to be a rich source of biological compounds which acted as reducing agents as well as stabilizing agents for the fabricated metal nanoparticles.

Figure 16. Atomic force microscopy of Cobalt nanoparticles (A) particle size distribution (B) 3D image of the nanoparticles4.5.Conclusion

In present work we have successfully fabricated three different metals (Fe, Cu, Co) nanoparticles by using green synthesis method. Punica granatums peels extract was used as reducing medium for reduction of metal ion from their respective salt solution. Punica granatums peels has proved to be a rich source of flavonoids, alkaloids and poly phenolic compounds. Poly phenolic compounds were the major participants in the reduction of metal ions to metal nanoparticles. All the synthesis procedure was carried out at room temperature. Resulting metal nanoparticles were characterized by different techniques in order to confirm the formation of nanoparticles and to access the morphology and particle size. FTIR, SEM, AFM and EDX analysis were carried out for this purpose. Peak pattern in FTIR, SEM and AFM showed the successful formation of these nanomaterials, while EDX analysis showed the elemental composition of the samples. Representative peaks in FTIR analysis justified the synthesis process. SEM, AFM results showed that the particle size was in nanometer range. All the results from different characterization tools were found complementary to each other. All these materials could be used is different fields of research such as biomedical fields, removal of pollutants from environment particularly for the biotransformation of synthetic dyes. Punica granatums peels have an excellent potential to be a fruitful reducing medium for the fabrication of different type of nanomaterials. By optimizing the conditions, particle of different size and shape could be obtained by using Punica granatums peels extract as reducing medium.Nucleation

Reduction

M

M

M+

Nanoparticles with organic coating

Metal ions

Metal nanoparticles

Chemical Nature

Metals, Metal oxides, Biomolecules, Alloys, Ceramics, Carbon, Semiconductors

Shape

Nano rods, Nano cones, Nanowires,Nanotubes, Nano disks, Nano flowers

Synthesis Methods

Green synthesis, Co-precipitation, Sol-gel, Hydrothermal, Sonochemical, Microemulsion

Applications

Environmental, Biomedicine, Food, Electronics, Energy, Catalysis, Sensing, Drug delivery

Classification

Nanoparticles, Nano composites, Core-shell, Magnetic Nanofluids

Nanomaterials

Characterization

FTIR, SEM, AFM, XRD, EDX, TEM, XPS, TGA

Top Down Approach

Bulk Material

Powder

Nano

Particles

Cluster

Atoms or Molecules

Bottom up Approach