experimental and characterization techniques for lsmo...

Chapter 3

Experimental And

Characterization Techniques For

LSMO Nanoparticles

Chapter 3

Centre For Interdisciplinary Research, DYPU, Kolhapur 70

3.1 Introduction

This chapter focuses on the synthesis and experimental techniques

employed for characterizations. There are many routes in the synthesis of

MNPs. However, synthesis of the sample in single phase is the most

imperative part of the research work. According to the literature survey,

several methods have been embraced for the synthesis of LSMO which are

reviewed in last chapter. In current research work, SPM LSMO NPs are

synthesized by combustion method due to enormous advantages which has

been discussed in below. The different analytical techniques used for phase

analysis, composition study, elemental analysis, structural and morphological

analysis of the prepared samples are described with principle and working.

3.2 Experimental

3.2.1 Mechanism of combustion method

Combustion synthesis (CS) is a versatile, simple and rapid process,

which allows efficient synthesis of a variety of nanomaterials. In this process

mainly a self-sustained reaction in homogeneous solution of different

oxidizers (e.g., metal nitrates) and fuels (e.g., urea, glycine, hydrazides) takes

place. It occurs at low temperature which offers a unique mechanism via a

highly exothermic redox reaction to produce oxides. Mainly, depending on

the type of the precursors, and on conditions used for the process

organization, the CS may occurs as either volume or layer-by-layer

propagating combustion modes. Several processing factors such as C/H ratio

(type of fuel), the fuel to oxidizer ratio (F/O), and the water content of the

precursor mixture and the ignition temperature mainly influences the

combustion reaction.

CS is also recognized as self-propagating high-temperature synthesis

(SHS). In CS there is the exothermic oxidation of a fuel. The word

„combustion‟ itself suggests flaming (gas-phase), smouldering heterogeneous

Chapter 3


as well as explosive reaction. In this method, the powder characteristics such

as crystallite size, surface area, size distribution and nature of agglomeration

are basically controlled by enthalpy or flame temperature generated during

combustion which itself dependent on the nature of the fuel and fuel-to-

oxidizer ratio [1]. Main advantages of CS are (i) Use of relatively simple

equipment (ii) Formation of high-purity products (iii) Stabilization of

metastable phases (iv) Formation of different sizes such as micro, nano and

shape like spherical, hexagonal and rod like products. Uniform distribution of

dopants takes place through the host material due to the atomic mixing of the

reactants in the initial solution. This method has been proven to be a best

technique to achieve various types of oxides at the nanometre scale and is

used for a variety of technological applications and this broad range of oxides

is prepared with an eye on their magnetic, mechanical, dielectric, catalytic,

optical and luminescent characteristics. Out of various combustion sub types

the solution combustion (SC) method of preparing oxide materials is a fairly

recent development compared to SSC or SHS techniques. In present research

SC is employed for synthesis of LSMO NPs.

The important parameters of combustion that have been broadly

studied are: type of flame, combustion temperature, evolved gases, air-fuel-

oxidant ratio and chemical composition of the precursor reagents. The

characteristic of the SC method is, after initiation locally, the self- sustained

propagation of a reaction wave through the heterogeneous mixture of

reactants. Under controlled conditions, SC reaction, generates a peculiar kind

of burning or smoldering type flame, depending on the used fuel and oxidizer-

fuel ratio. The burning flame may be capable of endure for seconds or even

minutes, while the smoldering flame does not rise or is extinguished in a few

seconds. The type of flame in the combustion plays a vital role in controlling

the particle size of as-synthesized materials. There are four important

temperatures which can affect the reaction mechanism and final product

Chapter 3


properties during the CS reaction. These are initial temperature, ignition

temperature, adiabatic flame temperature and maximum flame temperature.

Initial temperature is defined as the average temperature of the reagent

solution before the reaction is ignited. Ignition temperature is the point at

which the combustion reaction is dynamically activated without an additional

supply of external heat. Adiabatic flame temperature represents the maximum

combustion temperature achieved under adiabatic conditions. And the

maximum flame temperature is the highest temperature reached in the actual

configuration, i.e., under conditions that are not adiabatic [2].

Evolution of gases is another important parameter which affects on

combustion reaction. Mainly, in CS, the powder morphology, particle size and

surface area are directly related to the amount of gases that escape during

combustion. The difference in particle size, using different fuels, depends on

the number of moles of gaseous products released during combustion. Pores

between the particles are produced when gases breaks large clusters. In this

process one of the most important parameters in determining the properties of

synthesized material is fuel-oxidant ratio. A fuel is a substance capable of

breaking and burning the CH bonds (electrons acceptor). An oxidant is a

substance which helps in burning, supplying oxygen (electrons donor) [3].The

effect of gases on the morphology of the particles is studied by the oxidant

fuel ratio. Excellent quality product homogeneity is accomplished by the use

of chemical precursors intimately mixed. The type and amount of chemicals

used in the reactions influences on the characteristic features of the resultant

powders. The solubility of the fuel, the presence of water and type of fuel

used, are essential. Mainly, in solution, mixtures of metal nitrates (oxidizers)

and urea or glycine (fuel) are broken down quickly via deflagration burning or

combustion. To realize the highly exothermic nature of this reaction, concepts

used in propellant chemistry were used. A solid propellant contains an

oxidizer like ammonium perchlorate and a fuel like carboxyl terminated

Chapter 3


polybutadiene together with aluminium powder and some additives. The

specific impulse (Is) of a propellant, which is a measure of energy released

during combustion, is given by the ratio of thrust produced per pound of the

propellant. It is expressed as,

(3.1)

The highest heat T (chamber temperature in the rocket motor) is

produced when the equivalence ratio (ɸe= oxidizer/fuel ratio) is unity. The

equivalence ratio of an oxidizer and fuel mixture is expressed in terms of the

elemental stoichiometric coefficient,

(3.2)

A mixture is said to be stoichiometric when ɸe = 1, fuel lean when ɸe >

1, and fuel rich when ɸe < 1. Stoichiometric mixtures produce maximum

energy. The oxidizer/fuel molar ratio (O/F) required for a stoichiometric

mixture (ɸe = 1) is estimated by summation of the total oxidizing and

reducing valences in the oxidizer compounds and dividing it by the

sum of the total oxidizing and reducing valences in the fuel compounds. In

this kind of calculation, oxygen is the only oxidizing element; carbon,

hydrogen, and metal cations are reducing elements and nitrogen is neutral.

The oxidizing elements have positive valences and reducing elements have

negative valences.

3.2.2 Synthesis of LSMO nanoparticles

The strontium doped perovskite LSMO NPs were synthesized by

solution combustion method by using PVA as a fuel. Our previous research

(Coefficient of oxidizing element in specific formula)×(valency)

( 1) (Coefficient of reducing element in specific formula)×(valency)e

sp

TI k

Molecularwtofgaseousproduts

Chapter 3


work reported that PVA fuel as a potential candidate for preparation of LSMO

in biomedical field [4-5].

La (NO3)3·6H2O, Sr (NO3)2.4H2O and Mn

(NO3)2·4H2O were used as the starting reactant precursors and were obtained

from Sigma Aldrich (99.9%). The stoichiometric amounts of all these

precursors which act as oxidants were dissolved separately in double distilled

water (DDW) under constant stirring for about 10 minute to form the solution

of uniform mixture of 0.1 M. The solution of PVA was prepared by same way

by dissolving in DDW under constant stirring about an hour. The mixture was

then kept in beaker and stirred for 30 min at 100 ºC to achieve the

homogenous solution. The solution was then converted in to yellowish gel

which was further subjected to hot plate preheated to 300 ºC. During this

process, foams of LSMO produced along with sparks. Foam does not initiate

ignition only helps to sustain combustion. At the time of ignition CO2, H2O

and N2 gases evolved which were responsible for combustion to escape the

foam, yielding a voluminous fluffy black coloured product. The maximum

exothermic temperature of the redox reaction was reached between 1000-

1500 ºC.

Assigning the +4, +1, +3, +2 and +2 valencies to the C, H, La3+

, Sr2+

,

and Mn2+

, reducing elements, respectively, the -2 valency to O2-

oxidizer and

assuming nitrogen with the valence 0, 4 then the ɸe is determined according to

the equation (3.2). For this stoichiometric combustion (ɸe =1), 2.29% of the

PVA solution is required to balance the oxidation and reduction valences in

the solution. The resulting black coloured powder crushed with mortar and

pestle, annealed at 800 ºC for 5 hour, and then used for further

characterization. The possible combustion reaction is as follows:

(3.3)

3 3 3 2 3 2 2 4 2

0.7 0.3 3 2 2 2

0.7La (NO ) + 0.3Sr (NO ) + Mn (NO ) + 2.29(-C H O) + 1.5O

La Sr MnO + 4.58H O + 2.35N + 4.58CO

Chapter 3


3.3 Characterization techniques

In order to investigate different properties of LSMO NPs various

characterization techniques are required. Different techniques provide

different information, some about chemical and physical properties, and

others about structure, morphology and geometry. The different techniques

are used in the present work which includes X-ray Diffraction (XRD), Fourier

Transform Infrared Spectroscopy (FT-IR), Inductively Coupled Plasma

Optical Emission Spectroscopy (ICP OES), Field Emission Scanning Electron

Microscopy (FE-SEM), Vibrating sample Magnetometer (VSM),

Transmission electron microscopy (TEM), Zeta measurement, Dynamic light

scattering (DLS) and Induction Heating system etc.

3.3.1 Structural and phase analysis

3.3.1.1 XRD

XRD is a versatile non-destructive technique which is one of the

important fundamental tools employed in solid state chemistry and materials

science. This technique is used to identify crystalline phase, chemical

composition, strain state, grain size preferred orientation and defect structure.

X-rays are the electromagnetic radiations having wavelength 1 A˚ The basic

principles of X-ray diffraction are found in classic textbooks, e.g. by

Cullity [8] and Guinebretiere [9].

The diffraction of X-rays mainly occurs only when the wavelength

of the wave motion is of the same order of magnitude as the repeat distance

between scattering centres. The scientist Bragg studied the diffraction from

crystalline material and formulated the mathematical expression known as

Bragg‟s law and is expressed as [10],

(3.4)

2 sind n

Chapter 3


Where, d is interplanar spacing, θ is diffraction angle, λ is wavelength of X-

ray and n is order of diffraction.

Fig. 3.1 (a) Schematic of X-ray diffractometer [6] (b) Benchtop X-ray

diffraction instrument [7].

There are three methods used for the diffraction of X-rays as Laue

method, rotating-crystal method and Powder method. Out of these three

powder method is employed for whole proposed work, where, λ is fixed and θ

is variable. Particularly, in present method, the samples to be studied is

crushed to a very fine powder and then put in a rectangular plate of glass or

aluminium. X-rays beam of single wavelength (monochromatic) is incident on

the sample. Each particle of the sample is a small crystal oriented randomly

with respect to the incident X-ray beam. The information about the atomic

arrangements can be analysed by detecting the directions of diffracted

X-rays beam. Typically, the crystal and phase structure can be confirmed

by X-ray diffraction analysis. The schematic representation of XRD system

is shown in the Fig. 3.1. (a) and (b).

Identification of phases in a sample is studied from the d spacing using

the standard JCPDS powder diffraction file and the reflections can be

indexed with Miller indices. However, if the size of the diffracting tiny

Chapter 3


crystal is small, there is no more complete destructive interference at θ±dθ,

which broadens the peak corresponding to diffracted beam in proportion to

the size of the tiny crystal and that can be used to calculate the particle size.

The relation for the same is given by Debye Scherrer formula,

(3.5)

Where, D is particle size, θ is diffraction angle, λ the wavelength of X-rays

and is Full Width at Half Maxima (FWHM).

Fig. 3.2 A typical XRD pattern LSMO NPs.

The phase composition, lattice parameter and the mean size of the

crystallites of LSMO NPs were determined by XRD (RIGAKU Miniflex 600)

equipped with a crystal monochromator employing Cu-Kα radiation of

wavelength 1.54 Å and applied scanning rate of 3ºmin-1

, ranged from 20 to

80°. The patterns were analysed by X'Pert High score software and compared

with standard JCPDS (reference code: 00-051-0409). The average crystallite

size was calculated from the broadening of the XRD peaks using the

Scherrer‟s equation. Fig. 3.2 show a typical XRD pattern of LSMO NPs.

0.9

cosD

Chapter 3


3.3.1.2 FT-IR

The main aim of this technique is to find the changes in the intensity of

beam of infrared radiation as a function of wavelength or frequency after it

interacts with the specimen. Quantitative and qualitative measurements of the

organic and inorganic samples can be determined with this technique. The

spectra provides information about the identification of the chemical bonds in

molecule by recording an infrared absorption spectrum that is like a molecular

"fingerprint" [11-13]. In biomedicine the MNPs must be coated with organic

or inorganic material in order to enhance stability, dispersibility and

biocompatibility. The FT-IR analysis helps to understand the successful

attachment of coating agent on the surface off MNPs. The schematic of the

FT-IR spectrometer is shown in Fig. 3.3 (a).

Fig. 3.3 (a) Block diagram of optical FT-IR Spectrometer [14], (b) FT-IR

spectrum of LSMO NPs.

Michelson interferometer is generally used in FT-IR spectrometry. The

interferometer consists of two perpendicularly plane mirrors, one of which

can travel in a direction perpendicular to the plane. A semi-reflecting film,

known as beam splitter, bisects the planes of these two mirrors. The beam

splitter material is decided according to the region to be examined. For the

Chapter 3


mid- or near-infrared regions materials like germanium or iron oxide are

covered onto an „infrared-transparent‟ substrate as like potassium bromide or

caesium iodide to produce beam splitters. In this technique mainly, a chemical

substance shows selective absorption in the infrared region. The wavelength

of absorption depends on relative masses of the atoms, force constants of the

bonds and geometry of atoms. Then the molecules of the chemical substance

vibrate at many rates of vibrations, giving rise to close packed absorption

bands. The IR absorption spectrum may extend over a wide range of

wavelength. In spectrum each band corresponds to the characteristic

functional groups and bonds present in a compound.

FT-IR spectra of the synthesized LSMO NPs were collected on a

Perkin Elmer spectrometer model no. 783 USA. FT-IR spectrum of a LSMO

NPs is shown in Fig. 3.3 (b).

3.3.2 Elemental analysis

3.3.2.1 ICP OES

ICP/OES is one of the most powerful and popular analytical technique

for the determination of traces elements. In this method when plasma energy

is given to an analysis sample from outside, the component elements (atoms)

are excited. When the excited atoms return to low energy position, emission

rays (spectrum rays) are released and the emission rays that correspond to the

photon wavelength are measured. The element type is determined based on

the position of the photon rays, and the content of each element is determined

based on the rays intensity.

To generate plasmas first, argon gas is supplied to torch coil, and high

frequency electric current is applied to the work coil at the tip of the torch

tube. Using the electromagnetic field created in the torch tube by the high

frequency current, argon gas is ionized and plasma is generated. This plasma

has high electron density and temperature (10000K) and this energy is used in

Chapter 3


the excitation-emission of the sample. Solution samples are introduced into

the plasma in an atomized state through the narrow tube in the center of the

torch tube [15].

Fig. 3.4 Schematic of ICP-OES system [16].

The block diagram of ICP/OES system is relatively simple as shown in

Fig. 3.4. A portion of the photons emitted by the ICP is accumulated with a

lens or a concave mirror. This focusing optic forms an image of the ICP on

the entrance aperture of a wavelength selection device such as a

monochromator. The particular wavelength exiting the monochromator is

translated to an electrical signal by a photo detector. The signal is amplified

and processed by the detector electronics, then displayed and stored by a

personal computer.

3.3.2.2 X-ray photoelectron spectroscopy (XPS)

XPS has broadly used technique for studying the properties of

atoms, molecules, solids and surfaces. XPS spectra are gained by irradiating a

material with a beam of X-rays while simultaneously measuring the kinetic

http://en.wikipedia.org/wiki/Photoelectron

http://en.wikipedia.org/wiki/Spectroscopy

http://en.wikipedia.org/wiki/Spectrum

http://en.wikipedia.org/wiki/X-ray

http://en.wikipedia.org/wiki/Kinetic_energy

http://en.wikipedia.org/wiki/Kinetic_energy

Chapter 3


energy and number of electrons that escape from the top 0 to 10 nm of the

material being analyzed. It is based on the principle of photoelectric effect. In

the working of XPS, electrons are released from the sample as a result of a

photoemission process. An electron is emitted from an atomic energy level by

an X-ray photon, mostly from an Al-Kα or Mg-Kα primary source, and its

energy is analysed by the spectrometer. The schematic representation XPS

process is as shown in Fig. 3.5 (a).

Fig. 3.5 (a) Block Diagram of XPS system [17], (b) Typical XPS survey of

LSMO NPs.

The experimental quantity that is measured is the kinetic energy of the

electron, which depends on the energy hν of the primary X-ray source. The

typical factor for the electron is its binding energy.

The relation between these parameters is expressed by the equation,

(3.6)

Where, EB and EK are respectively the binding and the kinetic energy of the

released photoelectron, hν is the photon energy, and W is the spectrometer

B E kh E W

http://en.wikipedia.org/wiki/Electron

http://en.wikipedia.org/wiki/Nanometre

Chapter 3


work function. In a first approximation, the work function is the major

difference between the energy of the Fermi level EF and the energy of the

vacuum level EV, which is the zero point of the electron energy scale,

(3.7)

This quantity has been calculated by calibration for the spectrometer used.

From Equation (3.7) it is obvious that only binding energies lower than the

exciting radiation (1486.6 eV for Al-Kα and 1253.6 eV for Mg-Kα) are

probed. Each element has a distinguishing electronic structure and thus a

characteristic XPS spectrum. Fig. 3.5 (b) shows the XPS spectrum of LSMO.

3.3.3 Morphological Study

3.3.3.1 FE-SEM

Field emission microscopy (FEM) is a powerful tool used in materials

science to investigate molecular surface structures and their electronic

properties. In FE-SEM a field-emission cathode in the electron gun

provides narrower probing beams at low as well as high electron energy.

It consists of a metallic sample in the form of a sharp tip and a conducting

fluorescent screen enclosed in ultrahigh vacuum. This typical arrangement

over SEM results in to improved spatial resolution and minimized

sample charging and damage. In actual working process electrons are

liberated from a field emission source and accelerated in a high electrical field

gradient. In the high vacuum region these so-called primary electrons are

focussed and deflected by electronic lenses to produce a narrow scan beam

that bombards the object. As a result secondary electrons are emitted from

each spot on the object. The angle and velocity of these secondary electrons

communicates to the surface structure of the object. A detector collects the

secondary electrons and produces an electronic signal. This signal is

F VW E E

http://en.wikipedia.org/wiki/Materials_science



Chapter 3


amplified and transformed to a video scan-image. Then images can be

displayed on monitor and processed further. Fig. 3.6 (a) represents schematic

of the FESEM system. Fig. 3.6 (b) shows FE SEM image of LSMO NPs.

Fig. 3.6 (a) Schematic of the FESEM system [18], (b) FE SEM images of

LSMO NPs.

3.3.3.2 TEM

TEM is one of the important technique based on the use of electrons

rather than light to examine the structure and behaviour. The TEM image

gives more depth knowledge of morphology and direct estimation of its

size distribution of the nanomaterials. This method has been used for high

resolution imaging as well as chemical and structure study on atomic level.

Chapter 3


Fig. 3.7 (a) Ray diagram of Transmission electron microscope [20], (b) TEM

images of LSMO NPs.

Schematic 3.7 (a) shows ray diagram for transmission electron

microscope. In actual working of TEM, an electron gun at the top produces

the stream of monochromatic electrons. This stream of electrons is focused to

a small, thin, coherent beam by the use of two coherent lenses. When a beam

of electrons bombards on the sample, part of it gets transmitted. This

transmitted portion is focused by the objective lens into an image. There is a

mandatory requisite of sample for TEM analysis is, it must be thin enough to

allow the electrons to be transmitted. The recommended thickness of the

material for TEM is about 0.5 μm. For Preparation of sample for TEM

analysis, the sample to be analyzed is dispersed in some dispersive media

(inert to powder) to form colloidal solution, then a drop of solution is kept on

a conducting grid of copper or silver (sq. size is ~1 μm) and dried. This dried

grid is then act as specimen for analysis using TEM. In present work, the

shape, size and uniformity of the NPs were measured by TEM and high

resolution TEM (HR-TEM) with TECNAI F20 Philips operated at 200 KV.

The typical TEM image of LSMO NPs is shown Fig. 3.7 (b) [19].

http://www.rsic.iitb.ac.in/Tem.html

Chapter 3


3.3.4 Colloidal stability Study

3.3.4.1 Zeta potential

Fig 3.10 (a) Optical configurations of the Zetasizer Nano series for

zeta potential measurement [21], (b) Zeta potential measured for

LSMO NPs.

For biomedical applications MNPs should be stable in an aqueous

solution. Therefore, colloidal stability of MNPs is an important issues which

can be measured in terms of zeta potential. Zeta potential is defined as the

potential difference between the dispersion medium and the stationary

layer of fluid attached to the dispersed particle. When all the particles in

the suspension have a large negative or positive zeta potential, then they will

tend to repel each other and there is no tendency to flocculate and vice

versa. In present work, zeta potential of the nanofluid was measured using a

PSS/NICOMP 380 ZLS particle sizing system (Santa Barbara, CA, USA)

with a red He–Ne laser diode at 632.8 Å in a fixed angle 90° plastic cell.

Measurements were performed at 25 °C after a temperature

homogenization time of 5 min with varying pH from 2 to 12. For precise

data at least three measurements were conducted for each pH value. The

instrument calibration was examined before each experiment using a

latex suspension of known zeta potential (i.e. −55 ± 5 mV). Zeta

Chapter 3


potential is not measurable directly but it can be estimated using

theoretical models and an experimentally- determined electrophoretic

mobility or dynamic electrophoretic mobility. Fig. 3.10 (a) shows block

diagram for zeta potential measurement.

Major application of zeta potential is to determine surface charge of

NPs. Derivations show that the zeta potential is the double-layer potential

close to the particle surface. The liquid layer of a particle in suspension

migrating in an electric field moves at the same velocity as the surface

(shear surface). This shear surface occurs well within the double layer, likely

at a location roughly equivalent to the Stern surface. Although, the precise

location of the surface of shear is not known, it is assumed to be within a

couple of molecular diameters of the actual particle surface for smooth

particles. This thickness is associated with the zeta potential and

determines the ion atmosphere near a surface [22].

Electroacoustic phenomena and electrokinetic phenomena are the

typical sources of data for estimation of zeta potential. As the magnitude

of zeta potential gives an indication of the potential stability of colloidal

system. If all the particles in suspension have a larger negative or

positive zeta potential then they will tend to repel each other and there

is no tendency to flocculate. However, if the particles have lower zeta

potential values then there is no force to prevent the particles aggregation.

The general dividing line between stable and unstable suspensions is usually

taken at either +30mV or -30mV. Particles with zeta potentials more

positive than +30mV or more negative than -30mV are generally

considered stable. Fig. 3.10 (b) represents zeta potential for measured for

LSMO NPs.

Chapter 3


3.3.4.2 DLS

This technique is one of the most popular method in nanotechnology

used to determine the size of particles in suspension [23]. Particles suspended

in liquids show Brownian motion due to random collisions with solvent

molecules. It causes the particles to diffuse through the medium. The

diffusion coefficient D is inversely proportional to the particle size.

According to the Stokes-Einstein equation,

D=kBT/6πηa (3.8)

Where, a is the radius of the beads, kB is the Boltzmann constant, T is the

temperature in Kelvin degrees (in this experiment it will be considered as if it

is taking place at room temperature) and η is the viscosity of the

solvent.

Fig. 3.11 (a) Block diagram of typical DLS instrument [23], (b) DLS

histogram of LSMO NPs.

In working principle, radiating a monochromatic light beam, such as a

laser, onto a suspension with particles in Brownian motion causes a Doppler

Shift when the light falls the moving particle, changing the wavelength of the

Chapter 3


incoming light. This change is associated to the size of the particle. It is

probable to calculate the size distribution of particles and give a description of

the particle‟s motion in the suspension medium, measuring the diffusion

coefficient of the particle and using the autocorrelation function.

In present case, DLS of the LSMO NPs were measured using a

PSS/NICOMP 380 ZLS particle sizing system (Santa Barbara, CA, USA)

with a red He–Ne laser diode at 632.8 Å in a fixed angle 90° plastic

cell. All measurements were carried out at 25.0 + 0.1ºC using a circulating

water bath. Cylindrical cells of 10 mm diameter were used in all of the

light scattering experiments. Fig. 3.11 (a) shows block diagram of typical

DLS instrument and Fig. 3.11 (b) shows DLS histogram of LSMO NPs.

3.3.5 Magnetic Characterizations

3.3.5.1 VSM

Fig. 3.12 (a) Schematic of VSM showing the overall assembly [24], (b)

Magnetic measurement of as prepared LSMO NPs at temperature 27 ºC.

VSM is used to measure materials entire magnetic behaviour. Using

this method the magnetic characteristics of sample as a function of applied

field at different temperatures and as a function of temperature at different

applied field strengths can be measured. Operating principle of VSM is based

Chapter 3


on Faraday's Law of Induction [25]. It states that a changing magnetic flux

through the circuit will produce an electric field in any closed circuits.

This can be expressed as,

(3.9)

Where, N is the number of turns of wire in the coil, A is area coil, and θ is

the angle between the B field and the direction normal to the coil

surface. In VSM measurements the sample to be analysed is placed in a

constant magnetic field. If the sample is magnetic, this constant magnetic

field will magnetize the sample by aligning the magnetic domains or

the individual magnetic spins, with the applied field. Due to applied

magnetic field the magnetic dipole moment of the sample will create the

magnetic field around the sample. The oscillatory motion of the magnetized

sample will induce a voltage in the detection coils [26]. This voltage can be

used to identify a high resolution and accuracy by means of suitable

associated electronics. The induced voltage is proportional to the sample‟s

magnetization, which can be varied by changing the dc magnetic field

produced by the electromagnet. For application in biomedicine super-

paramagnetic behaviour of MNPs at room temperature is one of the essential

criteria. Field cooled zero field cooled measurements with the help of

Superconducting Quantum Interference Device (SQUID) i. e. SQUID -VSM

can gives the accurate data of about the blocking temperature of the sample.

The blocking temperature of the material can gives the information about the

superparamagnetism. Fig. 3.12 show vibrating sample magnetometer (a) and

magnetic measurements of LSMO NPs (b).

- cosd

N BA Jdt

Chapter 3


3.3.6 Biocompatibility study: Cytotoxicity assays

In the biomedical field especially, NPs are being utilized in diagnostic

and therapeutic tools to better understand, detect, and treat human diseases.

Human exposure to NPs is predictable as NPs become more extensively used

and, as a result, nanotoxicology research is recently focusing attention.

However, while the number of NPs, types and applications continues to

increase, studies to distinguish their effects after exposure and to address their

potential toxicity are few in comparison. For medical use exposure to NPs

involves intentional contact or administration; therefore, understanding the

properties of NPs and their effect on the body is vital before clinical use can

occur. For NPs to be used in clinical area, it is necessary that nanotoxicology

research uncovers and understands how these multiple factors influence the

toxicity of NPs so that their undesirable properties can be avoided [28].

Fig. 3.13 Schematic of cytotoxicity test [27].

Chapter 3


Selection of the appropriate cytotoxicity assay is crucial to the

accurate assessment of NPs toxicity. Several assays can be used to study the

toxic effects of NPs on cell cultures, including lactate dehydrogenase (LDH)

leak-age,3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-yltetrazolium bromide

(MTT) assay, Trypan blue Dye exclusion (TBDE) assay (and identification

of cytokine/ chemokine production etc. In deciding the correct assay, all

potential interferences must be considered to avoid obtaining false-positive

and false negative results. Interactions between the NPs and the chosen dye

have been cited as a major potential interference leading to inaccurate results.

In present investigation MTT and TBDE assays have been used to study

cytotoxicity. In TBDE assays cell viability analysis is done with trypan blue

dye exclusion staining. Cells were routinely counted manually with a

hemocytometer. Now a days advanced programmed instrumentation has been

introduced to supplement this traditional technique with the efficiency and

reproducibility of automated sample handling, computer control and advanced

imaging. The conventional method of performing TBDE analysis involves

manual staining and use of a hemocytometer for counting. Recent advances

in instrumentation have led to a number of fully automated systems that can

enhance the output and accuracy of this technique.

The MTT assay is a colorimetric assay for measuring cell viability.

The reduction of MTT (tetrazolium salts) is now extensively believed as a

reliable way to observe cell proliferation. The yellow tetrazolium MTT (3-

(4, 5-dimethylt hiazolyl-2)-2,5-di phenyltetrazoli um bromide) is reduced by

metabolically active cells through dehydrogenase enzymes, and make

reducing equivalents such as NADH and NADPH. The consequential

intracellular purple formazan can be solubilized and measured by

spectrophotometrically. The cell proliferation rate can be calculated by using

MTT cell proliferation assay. When metabolic events initiate apoptosis or

necrosis it results into the reduction in cell viability. The number of stages

Chapter 3


involved in assay has been minimized as much as possible to expedite sample

dispensation. The MTT reagent give ups low background absorbance values

in the absence of cells. Fig 3.13 shows schematic of cytotoxicity tests [27].

Cancer cells are more resilient towards NPs toxicity than normal cells

due to an increased rate of proliferation and metabolic activity. The difference

in toxic effects is even observed for NPs of the same material. Therefore,

selection of the appropriate cell type based on target introduction methods of

nanomaterials is an essential issue in cytotoxicity assays [29]. In the case of

biomedical applications, NPs are often introduced into the human body

through the intravenous, subcutaneous, intra-muscular or intraocular pathway.

Based on the variety of affected organs, numerous cell types ranging from

endothelium, blood, spleen, liver, nervous system, heart and kidney are all

of interest in NPs cytotoxicity studies [30].

3.3.7 Induction heating system for caner Hyperthermia study

Fig. 3.14 induction heating system for magnetic fluid hyperthermia.

Chapter 3


Heating ability of MNPs for cancer hyperthermia therapy has been

studied with induction heating system. Induction heating characteristics of

samples for hyperthermia application were carried out in Eppendorf tube

using instrument (Easy Heat 8310, Ambrell, UK).The coil having 6 cm

diameter (4turns) consisted of loops of copper (Cu) pipe was cooled by water

circulation in coil to keep the temperature constant. Suspensions of NPs were

prepared in DDW as well as different physiological media and placed at the

centre of the coil.

Particularly, magnetic induction heating system has generated a

growing interest in the medical field and oncology. Induction Heating offers a

controllable and localized method of heat without contact to the parts

(components) being heated [31-32]. It has been studied that the malignant

cells are more sensitive and responsive to induced heat than normal

cells. Using particular range of frequency and magnetic field strength, a

heating coil of a given shape and size in conjunction with other methods can

apply a hyperthermia effect on tumour cells. The basic principle of working

of induction heating is based on the faradays law, “The amount of voltage

created is equal to the change in magnetic flux divided by the change in

time”. The greater the change in the magnetic field, the larger will be amount

of voltage.

In this system, a source of high frequency electricity is used to drive a

large alternating current through a coil. This coil is known as the work coil

(Fig. 3.14). The passage of current through this coil generates a very

intense and rapidly changing magnetic field in the space within the work

coil. The work piece to be heated is placed within this intense alternating

magnetic field [33]. The intense alternating magnetic field inside the work

coil repeatedly magnetises and de-magnetises the crystals to be analysed. This

rapid flipping of the magnetic domains causes considerable friction and

heating inside the material.

Chapter 3


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Chapter 3


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Chapter 3


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