Q1.

Principle: Light microscopy, also known as optical microscopy, is a technique used to

visualize small structures by using visible light and a system of lenses to magnify images of
small samples. The basic principle involves the passage of light through or reflected from the
sample, which is then magnified by the lenses, enabling the detailed observation of structures
that are not visible to the naked eye.

Working: Light from a source (typically a tungsten or LED lamp) is directed onto the
specimen. The light passes through a condenser lens, which focuses the light onto the sample.
The light interacts with the sample, either passing through it or reflecting off its surface.
Depending on the sample's properties, it may absorb, refract, or scatter the light. After
interacting with the sample, the light enters the objective lens. This lens collects light and
magnifies the image of the sample. The objective lens is one of the most critical components,
with various magnification powers (e.g., 4x, 10x, 40x, 100x). The magnified image produced
by the objective lens is further magnified by the eyepiece lens (usually 10x). This final image
is what the observer sees when looking through the microscope. Coarse and fine focusing
knobs adjust the distance between the objective lens and the sample to bring the image into
sharp focus.

Utility in Exploring Microstructures:

The light microscope is a fundamental tool for exploring microstructures in various scientific
and industrial fields. Its utility spans across biology, medicine, materials science,
environmental studies, and forensic science etc.
Biological Research: Light microscopy is essential in biology for examining cells, tissues,
and microorganisms. Techniques like staining (e.g., Gram staining, hematoxylin and eosin
staining) help differentiate cellular components.
Medical Diagnostics: Used extensively in pathology labs to examine tissue samples and
diagnose diseases, such as cancer, infections, and other conditions.
Material Science: Helps in analyzing the microstructure of materials, including metals,
polymers, and ceramics, to study grain structure, phase distribution, and defects.
Environmental Studies: Assists in examining soil samples, water quality, and microorganisms
present in various ecosystems.
Forensics: Utilized in forensic science to analyze trace evidence, such as hair, fibers, and
biological samples.

Magnification: Magnification is the process of enlarging the appearance of an object. In

microscopy, it refers to the factor by which the image of the specimen is enlarged compared
to its actual size. The total magnification is the product of the magnifications of the objective
lens and the eyepiece lens.
 For example, if the objective lens magnifies 40x and the eyepiece lens magnifies 10x, the
total magnification is 400x.

Resolution: Resolution is the ability of a microscope to distinguish between two points that
are close together. It determines the level of detail visible in the image. Higher resolution
means finer detail can be observed. Factors such as numerical aperture of the lenses and the
wavelength of light used influence the resolution.

Aberrations: Aberrations are optical imperfections in the microscope's lenses that cause
distortion or blurring of the image. There are several types of aberrations:
a) Chromatic Aberration: Occurs when different wavelengths of light are focused at different
points, resulting in coloured fringes around the image.
b) Spherical Aberration: Happens when light rays passing through the edges of a lens are
focused on a different point than those passing through the centre, causing a blurred image.
c) Astigmatism: Caused by lenses that are not perfectly symmetrical, leading to images that
are sharp in one direction but blurred in another.

Q2.
Transmission Optical
Feature Microscope Transmission Electron Microscope (TEM)
(Light Microscope)

Uses a beam of electrons to illuminate the

Principle Uses visible light to illuminate
sample and electromagnetic lenses to
the sample and lenses to
magnify the image
magnify the image

Limited by the wavelength

Much higher than optical microscopes,
Resolution of visible light (around 200
typically in the range of 0.1 nm, allowing
nm)
atomic-scale imaging

Typically, up to 1000x
Magnification Can exceed 1,000,000x
to 2000x

Requires thin sections or Requires very thin sections (usually less

Sample transparent samples; than 100 nm thick); samples often need to
Preparation staining is often used to be specially prepared and can be stained
enhance contrast with heavy metals to enhance contrast
 Operates in a vacuum; samples must be
Imaging Air or glass; samples can be
compatible with vacuum conditions
Medium observed in a natural state

Contrast Based on the absorption, Based on electron scattering by the

Mechanism reflection, or transmission sample, which depends on the density and
of light by different parts of the atomic number of the elements present
sample

Diffraction Contrast in TEM

Diffraction contrast in Transmission Electron Microscopy (TEM) arises from the interaction
of the electron beam with the periodic crystal lattice of a sample. When the electron beam
passes through a crystalline material, it is diffracted according to Bragg's law. This interaction
produces a pattern of diffracted beams that can be used to form images, revealing details
about the crystal structure and defects within the sample.
Mechanism:
Crystal Orientation: When electrons are transmitted through a crystalline sample, they are
scattered by the atomic planes in the crystal. Depending on the orientation of these planes
relative to the incident electron beam, constructive interference (diffraction) occurs, creating
bright spots known as diffraction spots.
Defects and Dislocations: Regions with crystallographic defects, such as dislocations,
stacking faults, or grain boundaries, disrupt the regular atomic arrangement, causing
variations in diffraction intensity. These variations create contrast in the TEM image,
highlighting the defects.
Imaging Mode: By selecting specific diffraction spots using an aperture, certain
crystallographic planes or defects can be emphasized in the image. This is known as dark-
field imaging, where only the diffracted electrons contribute to the image, enhancing contrast
in regions with specific orientations or defects.

Mass Density Contrast in TEM

Mass density contrast in TEM arises due to variations in the mass-thickness or atomic
number of different regions within the sample. These variations affect the scattering of
electrons, with denser or higher atomic number regions scattering more electrons, leading to
differences in image intensity.
Mechanism:
Mass-Thickness: Areas with greater thickness or higher atomic mass will scatter electrons
more strongly, appearing darker in the TEM image. Conversely, thinner or less dense areas
will appear lighter.
Atomic Number (Z) Contrast: Elements with higher atomic numbers scatter electrons more
effectively than those with lower atomic numbers. This results in higher contrast between
regions composed of different elements.
Staining and Coating: Heavy metal stains or coatings (e.g., uranium, lead) can be applied to
biological samples to enhance contrast by increasing electron scattering in specific areas.

Q3.
AFM is a kind of scanning probe microscope in which a 3D (three dimensional)
topographical image of the sample surface on a nanoscale can be achieved based on the
interactions between a tip and a sample surface.
Principle:The AFM measures the forces acting between a fine tip and a sample. The tip is
attached to the free end of a spring cantilever and is brought very close to a surface. When the
tip is brought within the interatomic separation between the tip and sample, interatomic
potentials are developed between the atoms of the tip and the atoms of the surface, resulting
into attractive or repulsive forces. As the tip scans the surface of the sample, the force
between the tip and the sample varies which is sensed by the tip. The amount of force
between the probe and the sample is dependent on the spring constant of the cantilever and
the distance between the probe and the sample surface. This force can be characterized with
Hooke’s Law. F= - k·x ,
F = Force
k = spring constant
x = cantilever deflection
As the tip travels across the sample, it moves up and down according to the surface properties
of the sample (eg. topography). These fluctuations are sourced by the interactions
(electrostatic, magnetic, capillary, Van der Waals forces) between the tip and the sample.
Accordingly, the bending of tip is then detected by means of a laser beam, which is reflected
from the backside of the cantilever. The laser beam gets constantly reflected towards a
position-sensitive photodetector consisting of four side by-side photodiodes. This laser beam
detects the bend occurring in the cantilever and calculates the actual position of the
cantilever. The vertical deflection measures the interaction forces while the horizontal
deflection measures the lateral forces. Thus, AFM records a three dimensional image of the
surface topography of the sample under a constant applied force (as low as nano Newton
range).
i.Probe and its Motion Sensor
Probe:
Function: The probe in AFM consists of a sharp tip mounted on a flexible cantilever. It is
used to scan the surface of the sample.
Motion Sensor:
Function: The motion sensor detects the deflections of the cantilever as it scans the surface.
ii. Scanner and Vibration Isolated System
Scanner:
Function: The scanner, usually a piezoelectric tube, precisely moves the sample or the probe
in three dimensions (X, Y, and Z axes).
Vibration Isolated System:
Function: The vibration isolation system minimizes external vibrations and noise that can
affect the accuracy and resolution of the AFM measurements. vibration isolation ensures that
the data collected reflects the true surface topography and properties of the sample, rather
than artifacts introduced by external disturbances.
Q 4. The importance of FTIR is that it used for identification of unknown materials as well as
analysis of material composition. The specific absorption peaks in the spectrum provide
information about the functional groups present in the molecule.
The instrumentation of an FTIR spectrometer consists of following components:
Infrared Source:Typically a Globar (silicon carbide) or Nernst glower (zirconium oxide and
yttrium oxide) that emits broad-spectrum infrared radiation.
Interferometer:
Michelson Interferometer: The core component of an FTIR spectrometer. It splits the infrared
beam into two paths using a beam splitter, reflects them off a fixed mirror and a movable
mirror, and then recombines them to create an interference pattern (interferogram).
Moving Mirror: The mirror moves back and forth, changing the path length difference
between the two beams, which modulates the intensity of the combined beam over time.
Sample Compartment:
The combined beam passes through the sample, where certain wavelengths are absorbed
depending on the molecular composition of the sample.
The sample can be in various forms, including solid, liquid, or gas. Accessories like
attenuated total reflectance (ATR) can be used for direct analysis of solids and liquids.
Detector: Common detectors include Deuterated Triglycine Sulphate (DTGS) and Mercury
Cadmium Telluride (MCT). These detect the intensity of the transmitted or reflected infrared
radiation after interaction with the sample.
Computer and Fourier Transform Algorithm: The detected signal (interferogram) is digitized
and processed using a Fourier transform algorithm to convert the time-domain data into a
frequency-domain spectrum.
The resulting spectrum displays absorption intensity versus wavenumber, providing the
molecular fingerprint of the sample.
Q5.
i .Dynamic Light Scattering:

DLS, also known as Photon Correlation Spectroscopy (PCS) or Quasi-Elastic Light

Scattering (QELS), is a technique used to measure the size and size distribution of particles
suspended in a liquid solution.

Principle:

DLS relies on the phenomenon of Brownian motion. Particles in suspension undergo random,
microscopic movements due to collisions with solvent molecules.When a laser beam hits
these particles, the light scatters in all directions.The intensity of the scattered light fluctuates
due to the Brownian motion of the particles.By analysing these fluctuations, DLS can
determine the rate of diffusion of the particles, which is related to their size. Smaller particles
diffuse faster, causing faster fluctuations.

Construction:

A DLS instrument consists of:

Laser source: Provides a monochromatic light source to illuminate the sample.

Sample cell: Holds the liquid containing the particles or molecules to be analysed.

Detector: Measures the intensity of the scattered light at a specific angle.

Correlator: Analyses the fluctuations in the scattered light intensity.

Working:

The laser beam shines on the sample cell.The particles in the sample scatter the light.The
detector measures the intensity of the scattered light at a specific angle (usually 90
degrees).The intensity of the scattered light fluctuates due to the Brownian motion of the
particles.The correlator analyzes these fluctuations and calculates the autocorrelation
function (ACF). The ACF describes how the intensity of the scattered light is correlated at
different time delays.The decay rate of the ACF is related to the diffusion coefficient of the
particles.Using the Stokes-Einstein equation, the diffusion coefficient is converted to the
hydrodynamic radius of the particles. This provides information about the size of the
particles.

KT
D H=
3 π ηD

Where, D-translational diffusion coefficient, k - Boltzmann's constant, T - absolute

temperature, n - viscosity of solvent.

ii. Polydispersity Index (PDI)

The Polydispersity Index (PDI) is a measure of the distribution of molecular mass in a given
polymer sample. It provides an indication of the degree of non-uniformity in the size of the
polymer chains.
Calculation:
PDI is defined as the ratio of the weight-average molecular weight (Mw) to the number-
average molecular weight (Mn):
Mw
PDI= Mn
Where, M w considers the mass of each molecule, giving more weight to larger molecules and
M n takes the simple average of the molecular weights of the polymer chains.
If
PDI = 1: Indicates a monodisperse sample, where all polymer molecules have the same
molecular weight. This is ideal but rarely achieved in practice.
PDI > 1: Indicates a polydisperse sample, where there is a distribution of molecular weights.
The higher the PDI, the broader the distribution of molecular weights within the sample.
Q 6.
Quantitative analysis in X-ray Fluorescence (XRF) spectroscopy involves determining the
concentrations of elements in a sample by measuring the intensities of characteristic X-rays
emitted when the sample is irradiated with high-energy X-rays. This non-destructive
technique requires proper sample preparation to ensure homogeneity, followed by calibration
using standards with known element concentrations to create calibration curves. During
measurement, the sample's emitted X-rays are detected, and the resulting intensities are
analyzed and corrected for matrix effects and inter-element interferences. The corrected
intensities are compared to the calibration curves to quantify the elements present. XRF is
widely used in various fields, including environmental analysis, mining, materials science,
and quality control, due to its rapid, efficient, and multi-element detection capabilities.
Q7.
Generation of X-rays
X-rays are generated when high-energy electrons interact with a target material. There are
two primary mechanisms through which X-rays are produced: Bremsstrahlung radiation and
characteristic X-ray emission.
1.Bremsstrahlung Radiation:
When high-energy electrons are decelerated or deflected by the electric field of atomic nuclei
in the target material, they emit X-rays.This process generates a continuous spectrum of X-
rays because the energy loss of the electrons can vary continuously. The intensity of
Bremsstrahlung radiation increases with the atomic number of the target material and the
energy of the incident electrons.
2.Characteristic X-ray Emission:
When an incident electron has enough energy to eject an inner-shell electron (usually from
the K or L shell) of an atom in the target material, an electron from a higher energy level fills
the vacancy. The energy difference between these levels is released as an X-ray photon. This
process produces X-rays with discrete energies, known as characteristic X-rays, specific to
the elements in the target material. These X-rays appear as sharp peaks superimposed on the
continuous spectrum of Bremsstrahlung radiation.
Existence of X-ray Characteristic Spectrum
The existence of the X-ray characteristic spectrum is due to the discrete energy levels within
atoms. When an electron from an inner shell is ejected, an electron from a higher shell falls
into the lower energy state to fill the vacancy. The energy of the emitted X-ray photon
corresponds to the difference in energy levels of the shells involved.
Characteristic X-rays:
K α and K β Lines: When an electron transitions from the L shell to the K shell, a K α X-ray is
emitted. When an electron transitions from the M shell to the K shell, a K β X-ray is emitted.
Lα , L β, etc.: Similar transitions can occur between other shells, leading to the emission of L,
M, and N characteristic X-rays with specific energy lines like Lα , L β, and so on.
In an X-ray spectrum, the continuous background from Bremsstrahlung radiation
and sharp peaks from characteristic X-rays can be observed. The position (energy) of the
peaks identifies the elements present, while the peak intensity indicates the concentration of
these elements.
Q8.
i. Thermo Gravimetric Analyzer:
Thermogravimetric analysis (TGA) is a method of thermal analysis in which the
mass of a sample is measured over time as the temperature changes. This
measurement phenomena, provides information such as phase transitions,
absorption and desorption as well as chemical phenomena including
chemisorption…, thermal decomposition, and solid-gas reactions (e.g., oxidation
or reduction)

Principle:
In thermo-gravimetric analysis, the sample is heated in given environment (air, N2,
CO2, He, Ar, etc.) at controlled rate. • The temperature is increased at a constant rate
for a known initial weight of the substance and the changes in weights are recorded as
a function of temperature at different time interval. • This plot of weight change
 against temperature is called thermo-gravimetric curve or thermo-gram, this is the
basic principle of TGA.

Construction:
A TGA typically consists of following components:
Recording balance: A microbalance is used to record a change in mass of sample/ substance.
Sample holder: The sample to be studied is placed in sample holder or crucible. It is attached
to the weighing arm of microbalance.
Furnace: The furnace should be designed in such way that it produces a linear heating range.
Recorder: The balance is calibrated in a manner that a change in weight of the sample
produces a proportional electrical signal. A data processing system then collects this electrical
signal and converts it into weight or weight loss, which is then plotted on the y axis of the
thermal curve.
Working:
A small, weighed sample is placed in the sample pan.The TGA chamber is purged with an
inert gas to remove any air.The desired temperature program is selected, specifying the
starting, ending, and ramp-up/cool-down rates.The experiment commences, and the TGA
continuously records the sample's mass and temperature data.After the experiment, the data is
analysed to determine the weight changes and their corresponding temperatures. This data
can be plotted as a thermogravimetric curve (TGA curve) to visualize the material's
behaviour.
By analyzing the TGA curve, valuable insights can be gained in various material properties,
such as:Thermal stability,Moisture content,Volatile component content,Decomposition
products etc.

ii. Differential Thermal Analyzer (DTA):

A Differential Thermal Analyzer (DTA) is a thermo analytical technique used to study the
thermal behavior of materials. It measures the difference in temperature between a sample
and an inert reference material as they are heated or cooled at a controlled rate.
Principle
The basic principle of DTA is that when a material undergoes a physical or chemical change
(e.g., melting, decomposition), it will absorb or release heat. This heat flow will cause a
difference in temperature between the sample and the reference material. The DTA
instrument measures this temperature difference and plots it as a function of temperature or
time.

Construction
A DTA instrument typically consists of the following components:
Furnace: A furnace that can be heated or cooled at a controlled rate.
Crucibles: Two crucibles, one for the sample and one for the reference material. The
crucibles are usually made of a material that is inert to the sample and has good thermal
conductivity.
Thermocouples: Two thermocouples, one placed in each crucible. A thermocouple is a device
that converts a temperature difference into a voltage. The differential thermocouple measures
the temperature difference between the sample and the reference material.
Computer: A computer that controls the furnace temperature and records the output from the
thermocouples. The computer also plots the DTA curve.
Working
The sample and reference material are placed in their respective crucibles.The crucibles are
placed in the furnace.The furnace temperature is programmed to increase or decrease at a
constant rate.As the temperature changes, the sample may undergo physical or chemical
changes.These changes will cause the sample to absorb or release heat.The temperature
difference between the sample and the reference material is measured by the differential
thermocouple.The voltage output from the thermocouple is sent to the computer.The
computer converts the voltage signal into a temperature difference. The computer plots the
temperature difference as a function of temperature or time to create a DTA curve.
The DTA curve can be used to identify the thermal properties of the sample, such as melting
point, boiling point, and decomposition temperature. It can also be used to study the kinetics
of reactions.