Nanotechnology Subject Code:

IA Marks: 50

Hours/ Week: 04 Exam Hours: 03

Total Hours: 40 Exam Marks: 50

Module – 2

Syllabus
Characterization of Nanomaterials
Basic principles and instrumentations of Electron Microscopy –Transmission Electron
Microscope, Scanning, Electron Microscope, comparison of SEM and TEM, AFM and STM,
AFM and SEM. Basic principles of working of X-ray diffraction, derivation of Debye-
Scherrer equation, numerical on Debye Scherrer equation, Optical Spectroscopy-
Instrumentation and application of IR, UV/VIS (Band gap measurement)
Introduction
Microorganisms are too small to be seen by our unaided eyes and the microscopes are
of crucial importance as they help to view the microbes. A microscope is an optical
instrument consisting of one or more lenses in order to magnify images of minute objects.
Thus it is important to gain a preliminary knowledge about the principles of microscope and
its types. This chapter gives a brief introduction to microscopy

Properties of Light

To understand how a light microscope operates, one must know something about the way
in which lenses bend and focus light to form images.

When a ray of light passes from one medium to another, refraction occurs, i.e., the
ray is bent at the interface. The refractive index is a measure of how greatly a substance
slows the velocity of light, and the direction and magnitude of bending is determined by
the refractive indexes of the two media forming the interface.

When light passes from air into glass, a medium with a greater refractive index, it is
slowed and bent toward the normal, a line perpendicular to the surface. As light leaves
glass and returns to air, a medium with a lower refractive index, it accelerates and is bent
away from the normal. Thus a prism bends light because glass has a different refractive
index from air, and the light strikes its surface at an angle. Lenses act like a collection of
prisms operating as a unit. When the light source is distant so that parallel rays of light
strike the lens, a convex lens will focus these rays at a specific point, the focal point. The
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Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 1
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distance between the center of the lens and the focal point is called the focal length. Our
eyes cannot focus on objects nearer than about 25 cm or 10 inches. This limitation may be
overcome by using a convex lens as a simple magnifier (or microscope) and holding it
close to an object. A magnifying glass provides a clear image at much closer range, and the
object appears larger. Lens strength is related to focal length; a lens with a short focal
length will magnify an object more than a weaker lens having a longer focal length.

Principles of Light Microscopy

The light is the primary source on which magnification is based in light microscopes. The
magnification is obtained by a system of optical lenses using light waves. Magnification
refers the number of times a specimen is appeared to be larger than its original size.

Basic Units For Microscope

1 meter = 1000 millimeter

1 millimeter = 1000 micrometer ( m) = 10-6 meter

1 micrometer = 1000 nanometer (nm) = 10-9 meter

1 Angstrom (1 A) = 10-10 meter

1 nanometer = 10 Angstrom

Relative size of the microorganisms and their visibility. Man can see about 0.5 mm
sized object whereas the light microscopes can be used to visualize upto 1 m and EM
(electron microscopes) can be used to view 1 nm objects.
Basic Quality Parameters of Microscopic Images
The microscopic images should have four basic quality parameters, through which the
microscopes can be graded.

1. Focus: It refers whether the image is well defined or blurry (out of focus). The
focus can be adjusted through course and fine adjustment knobs of the microscope
which will adjust the focal length to get clear image. The thickness of specimen,
slide and coverslip also decide the focus of the image. (Thin specimens will have
good focus).

2. Brightness: It refers how light or the dark the image is. Brightness of the image is
depends on the illumination system and can be adjusted by changing the voltage of
the lamp and by condenser diaphragm.

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3. Contrast: It refers how best the specimen is differentiated from the background or

the adjacent area of microscopic field. More the contrast will give good images. It

depends on the brightness of illumination and colour of the specimen. The contrast

can be achieved by adjusting illumination and diaphragm and by adding colour to

the specimen. The phase contrast microscopes are designed in such a way that the

contrast can be achieved with out colouring the specimen.

4. Resolution: It refers the ability to distinguish two objects close to each other. The

resolution depends on the resolving power, which refers minimum distance

between the two objects which can be distinguishable.

Magnification And Resolution

The total magnification of compound microscope is the product of the

magnifications of objective lens and eyepiece. Magnification of about 1500x is the upper

limit of compound microscopes. This limit is set because of the resolution.

Resolution refers the ability of microscopes to distinguish two objects close to each

other, it depends on resolving power, which refers the minimum distance. Ex : Man has the

resolving power of 0.2 mm (meaning that he can distinguish two objects with a distance of

0.2 mm close to each other) If he want to see beyond the limit of his resolving power,

further magnification is necessary.

Resolving power = -------------------

n (sin ᶿ )

where, µ is the wave length of light source and n (sin ᶿ ) is the numerical aperture (NA).

For compound microscopes, resolving power is µ/2NA. The resolving power of an

microscope can be improved either by reducing the wave length of light or by increasing

the n(sin ᶿ) value.

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Numerical aperture (n sinᶿ) measures how much light cone spreads out between
condenser & specimen. More spread of light gives less resolving power means better
resolution. The numerical aperture depends on the objective lens of the microscope. There
are two types of objective lenses are available in any compound microscope.

The Limit of Resolution

The limit of resolution refers the smallest distance by which two objects can be separated
and still be distinguishable or visible as two separate objects.

Optical Instrument Resolving Power RP in Angstroms

Human eye 0.2 millimeters (mm) 2,000,000 Ao

Light microscope 0.20 micrometers (µm) 2000 Ao

Scanning electron microscope (SEM) 5-10 nanometers (nm) 50-100 Ao

Transmission electron microscope (TEM) 0.5 nanometers (nm) 5 Ao

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Assistant Professor, Dept of Physics,
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Types of Microscopes

Microbiologists use a variety of microscopes, each with specific advantages and

limitations. Microscopes are of two categories.

a. Light Microscope: Magnification is obtained by a system of optical lenses using

light waves. It includes (i) Bright field (ii) Dark field (iii) Fluorescence (iv) Phase

contrast and (v) UV Microscope.

b. Electron Microscope: A system of electromagnetic lenses and a short beam of

electrons are used to obtain magnification. It is of two types: (I) Transmission

electron microscope (TEM) (ii) Scanning electron microscope (SEM).

ELECTRON MICROSCOPE

In electron microscope, short beam of electrons and magnetic condenser lenses are

employed to produce the image. The electrons have short wavelength, which helps in better

resolution. It is possible to resolve objects as small as 10°A, which is 100 times more than

that of light microscope. It can magnify object up to 200,000X.

In electron microscope, a hot tungsten filament forms the source of electrons. The

object is placed in the path of moving electrons. Since electrons move only in the vacuum,

the entire path of electrons should be kept under vacuum. The magnetic condenser lens

causes the primary magnification. A second magnetic lens amplifies the primary image and

this image is viewed on a fluorescent screen or captured on photographic plates. There are

two types of electron microscopes.

a. Transmission electron microscope (TEM)

b. Scanning electron microscope (SEM)

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Assistant Professor, Dept of Physics,
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Scanning Electron microscope (SEM):

Principle, working, and applications

In optical microscope, light is made to illuminate objects. Wavelength of visible light

is of the order of 10-7m. In electron microscopes, accelerated electrons are made to illuminate
objects. According to de-Broglie, wavelength of accelerated electron beam is of the order of
10-12m. This is 10-5 times smaller than visible light. Resolving power of a microscope is
2Sin
(R.P= 1.22
) inversely proportional to wavelength of incident light and hence when electron

beam is used to illuminate the object, R.P increases 105 times to that of ordinary microscope.

Scanning Electron Microscope (SEM)

SEM gives high resolution images of surfaces as it uses electron to produce images.
Principle:
The surface of a sample is scanned using a high energy beam of electrons. This gives
rise to secondary electrons, back-scattered electrons and X-rays. The secondary electrons
give topographical information of the surface. Back-scattered electrons give topographical
information and chemical composition. X-rays give the elemental composition. Typically, the
entire process will take place in ~10-6 Torr vacuum condition.
Construction:
An electron gun is used to produce high energy electrons. Two magnetic lenses are
used as condenser lenses to convert the diverging electron beam into a fine beam of spot
diameter of the order of a few nanometers. A scanning coil is used to deflect the electron
beam to scan the sample. The objective lens is used to focus the scanning beam on a desired
spot on the sample. The intensities of secondary electron, back-scattered electrons and the X-
rays are recorded using detectors. The images are then displayed on TV monitor.
Working:
When the high energy electron beam strikes the sample, some electrons are scattered
due to elastic scattering (the back-scattered electrons), some electrons are knocked off from
the surface (the secondary electrons) and some electrons penetrate deep into the inner shells
of the sample atoms to knock off inner shell electrons due to which characteristic X-rays are
produced. These are detected using detectors and the signals are amplified and displayed on a
TV monitor.

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Samples are required to be conducting. Non-conducting samples are coated with thin
conducting material such as gold.

OR

Magnification
➢ Magnification in a SEM can be controlled over a range of up to 6 orders of
magnitude from about 10 to 500,000 times.
➢ Unlike optical and transmission electron microscopes, image magnification in the SEM is
not a function of the power of the objective lens. In a SEM, display screen has a fixed
size, higher magnification results from reducing the size of the raster on the specimen,
and vice versa. Magnification is therefore controlled by the current supplied to the
scanning coils, or the voltage supplied to the deflector plates, and not by objective lens
power.

Applications:
➢ Used for the morphological analysis of the surface
➢ To identify cracks, imperfections, or contaminants on the surfaces
➢ To determine the size and shape of tiny particles
➢ Used to study chemical compositional information
Advantages:
➢ SEM can provide digital image resolution as low as 50 nm
➢ It is easy to determine the thin-film thickness, grain size and particle size because of the
calibrated imaging technique.
➢ SEM provides great depth of focus

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Disadvantages
➢ SEM samples must be solid, and vacuum-compatible
➢ Samples that are strong insulators must be coated—usually with gold before testing.
➢ SEMs are expensive and bulky

Transmission Electron Microscopy (TEM)

The transmission electron microscope is a very powerful tool for material science. A
high-energy beam of electrons is passed through a very thin sample, and the interactions
between the electrons and the atoms can be used to observe features such as the crystal
structure and features in the structure such as dislocations and grain boundaries.
Principle
A beam of electrons passes through the sample, the transmitted beam reproduces the
pattern of the material, forming an enlarged image when it falls on the detector. The electron
beam transmission depends to a large extent on the properties of the material being tested.
These properties are density, composition, etc.
Construction and working
TEM has five major components such as an electron gun to produce electrons, an
electron optical system to probe electrons, a specimen stage to place the specimen, imaging
screen to get the image of the sample.
Electron beam source:
The primary part of TEM is the electrons beam source which is a filament made up of
LaB6 or tungsten (W). When the Negative potential is applied to the electrode, a small area
of the filament emits a beam of electrons.
Electron optical system:
A combination of several magnetic lenses and physical apertures provides the
controlled electrons deflection along the column. Finetuning of the magnetic lens system can
generate collimated (parallel) or convergent illuminations with different spot sizes at the
sample.
Condenser Lens: The electrons beam from the electron beam source is made to focus on a
thin and small sample by using the condenser lenses. The first condenser lens helps in
determining the “spot size” and the second condenser lens helps in changing the spot size on
the sample surface.

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Condenser Aperture: A condenser aperture is a thin disk made up of metal with a small
circular through-hole. This aperture is to limit the electrons beam and filter out unwanted
scattered electrons.
Objective Lens: Objective lens is used to focus the electrons transmitted from the sample
onto the screen where it forms the image of the sample.
Objective Aperture: Objective aperture is used to increase the contrast of the image by
blocking the electrons that are diffracted at high angles.
Specimen stage (TEM grid):
The TEM grids are placed into the microscope using the sample holder. This system,
together with the goniometer, can provide sample movement along 6 axes horizontal
movement (x,y), vertical movement (z), tilting movement and rotation.
Imaging screen:
The transmitted electrons are made to strike on the phosphor screen where they form
an image by generating light. The darker area of the image represents that part of the sample
that allowed fewer electrons to pass through and the lighter area corresponds to the area
having more transmittance towards electrons.
OR

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Applications:
➢ TEM is used to study topographical, morphological, compositional, and crystalline
information.
➢ To analyse the structure and texture of the specimen.
➢ TEM is used in semiconductor industries
➢ Used in technology-based companies to identify flaws, fractures, and damages to micro-
sized objects.
Advantages
➢ TEM has high resolving power and can produce high-quality and detailed images with 2
nm resolution
➢ It can yield information on surface features, shape, size and structure
➢ No stain coating is needed for imaging, thus, it is convenient for the structural imaging
of organic materials
➢ TEM can also offer phase identification of the crystal, symmetry determination, lattice
parameter measurement, disorder, and defect identification.
Disadvantages
➢ TEM setup is bulky, require special housing and expensive
➢ It involves laborious sample preparation
➢ Operating the TEM setup and analysing the data require special training
➢ Samples are limited to those that are electron transparent
Difference between SEM and TEM:

Scanning Electron Transmission Electron

Parameters
Microscopes (SEM) Microscopes (TEM)
Electron stream Fine, focused beam Broad beam
Image taken Topographical/surface Internal structure
Resolution Lower resolution Higher resolution
Magnification Up to 2,000,000 times Up to 50,000,000 times
Image dimension 3-D 2-D
Sample thickness Thin and thick samples okay Ultrathin samples only
Penetrates sample No Yes
Sample restriction Less restrictive More restrictive
Sample preparation Less preparation required More preparation required
Cost Less expensive More expensive
Speed Faster Slower
More complicated; requires
Operation Easy to use
training

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X-Ray diffractometer (XRD)

German physicist Max von Laue first observed the phenomenon of X-ray diffraction.
The detailed analysis of crystal structure was given by Sir William Henry Bragg and William
Lawrence Bragg, who were regarded as founders of X-ray diffraction science. X-ray
scattering and Bragg diffraction can access the structural information of the investigated
nanomaterials.
The “crystal structure” of material commonly refers to its composition at different levels of
complexity, ranging from the basic molecular formula (revealing which elements are present
and in what ratio) to the exact positions of all atoms in the molecule. XRD is a widely used
technique to derive materials information like lattice constant, preferred orientation, lattice
strain, chemical composition, dislocation density, crystallite size, etc.
Principle
X-ray diffraction is based on constructive interference of diffracted monochromatic X-rays
from a crystalline sample. The incident X-ray on the sample produces constructive
interference if it satisfies Bragg’s law. That is 𝑛𝜆 = 2𝑑𝑠𝑖𝑛𝜃 where ‘d’ is lattice spacing, ‘λ’ is
the X-ray wavelength, ‘n’ is the order of diffraction and ‘θ’ is the diffraction angle.
Construction and working
X-ray diffractometers consist of three basic elements: A source X-ray, a sample holder, and
an x-ray detector.
a. A source of X-ray
X-rays are generated in a cathode ray tube by heating a filament to produce electrons,
accelerating the electrons toward a target by applying a voltage, and bombarding the target
material with electrons. These spectra consist of several components, the most common being
Kα and Kβ. Filtering, by crystal monochromators, is required to produce monochromatic X-
rays needed for diffraction.

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b. Sample holder
X-rays produced from the x-ray tube collimated to a narrow beam by two fine lead slits S 1
and S2. This collimated beam is made to fall on the nanomaterial C mounted on the centre of
the turntable. The position of the table can be read using a Vernier scale. X-ray beam and X-
ray detector rotates at an angle of θ with the sample. The instrument used to maintain the
angle and rotate the source and detector is termed a goniometer. The diffracted rays are made
into a narrow beam using the slits S3 and S4.
c. X-ray detector
In the detector, the x-ray produces ionization of the gas in chamber D and the ionization
current is measured by an electrometer. This X-ray signal is further converted into a count
rate which is then output to a device such as a computer monitor or a printer. Ionization
current is measured for different values of glancing angle. A sudden rise in the ionization
current indicates an X-ray beam at an angle of incidence satisfying the Braggs law.
Applications:
1. It is a popular technique to identify the crystalline phase
2. It is used to determine the structural properties such as lattice parameter, crystal size,
crystal strain etc.
3. It is used to determine atomic arrangement
Advantages
1. It is a rapid technique for identifying unknown materials
2. It only requires preparation of a minimal sample for analysis
3. It is a non-destructive technique
4. Easy to interpret the resulting data
Limitations (Disadvantages)
1. The sample should be homogeneous.
2. Interpretation requires access to standard reference data
3. Preparation of samples often requires grinding them down to a powder or as a film

Crystal size determination by Scherer equation

The pattern obtained from the X-ray diffractometer is as shown in the figure below where
peak broadening β (Full width half Maximum – FWHM) can be calculated for all the peaks.
Scherrer derived a formula relating the average crystallite size, D, of a powder to the
Kλ
broadening, β, of its powder diffraction peaks. That is D = βcosθ where K is the constant
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Assistant Professor, Dept of Physics,
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depends on the geometrical shape of the crystallite, θ is the Bragg angle, λ is the X-ray
wavelength. Thus, β and D are reciprocally related: the greater the broadening the smaller the
crystallite size and vice-versa.

Numerical problems
BRAGG’S LAW:
1. First order spectrum is formed when X-rays of wavelength 1.5Å is incident on
crystal at 12°. Calculate the interplanar spacing of the crystal.
Data: Order of diffraction, n= 1

To find

Solution:

2. Using a Bragg’s spectrometer, the glancing angle for the first order spectrum
was observed to be equal to 6°. Find the wavelength of the X-rays if d=2.82 .
Data:

Order of diffraction, n= 1
To find:
Solution: From Bragg’s law, we have

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3. Calculate the glancing angle for incidence 0f X-rays of wavelength 0.58Å on the
plane (132) of NaCl which results in second order diffraction maxima taking the lattice
constant as 3.81Å.
Data:

(h,k,l) = (1, 3, 2)
Order of diffraction, n= 2

To find: Glancing angle, θ=?

Solution: ………………………………………… (1)

4. First order Bragg reflection occurs when a monochromatic beam of X-rays of

wavelength 0.675Å is incident on a crystal at a glancing angle of 4°51’. What is the
glancing angle for third order Bragg reflection to occur?
Data:

Order of diffraction, n= 1

To find:

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5. GaAs has its principles planes separated at 5.6534Å. The first order Bragg’s reflection is
located at 13°40’. Calculate the wavelength of the X-ray and angle for second order
Bragg reflection.
Data:

Order n =1

Angle of reflection θ = 13°40’

To find: and

Solution: For First order n =1

For second order, n=2

SCHERER’S EQUATION:
6. Determine the crystal size when the peak width is 0.5° and peak position 30° for a
cubic crystal. The wavelength of X-rays used is 100Å and Scherer’s constant
K=0.92.
Data:

To find:

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Solution:

7. Determine the crystal size given the wavelength of X-rays 12nm, the peak width 0.5° and
peak position 23° for a cubic crystal. Given K=0.94

Data:

To find:

Solution:

8. Determine the wavelength of X-rays for the crystal size of , peak

width is and the peak position of 30° for a cubic crystal. Given that Scherer’s

constant K=0.92.

Data:

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Optical spectroscopy
Introduction
The most challenging task of a material scientists is to determine the chemical structure of an
unknown compound. There are many ways by which we can identify the unknown substance.
A person can use physical methods such as boiling point, melting point, spectroscopy as well
as chemical methods such as functional group testing and others to determine the structure of
compounds. Spectroscopy is one of the best methods to identify a substance, which may
include UV, IR, NMR, Raman and others. Here we will discuss about the various aspects of
different spectroscopic techniques and more specifically about UV-spectroscopy and its uses

Electromagnetic Radiation and Spectroscopy

Electromagnetic spectrum covers a wide range of electromagnetic radiations, ranging from γ-

rays having wavelength which are the order of a fraction of an angstrom, to radio waves
having wavelength in meters or kilometers. The arrangement of all types of radiations in the
order of their increasing wavelength or decreasing frequencies is known as electromagnetic
(EM) spectrum. The electromagnetic spectrum includes radio and TV waves, microwaves,
infrared, visible light, ultraviolet, X-rays, γ-rays, and cosmic rays, as shown in the Figure 1.

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Spectroscopy:
Spectroscopy is the study of interaction of electromagnetic radiations with matter.
Electromagnetic radiations can interact with matter in various ways. Each interaction gives us
insights about certain properties of the matter and use of electromagnetic radiations of
different energies can give different information about the matter under study.

It is the motion of electrically charged particles that give rise to electromagnetic radiations.
There are various forms of electromagnetic radiation e.g. radio waves, X-rays, gamma rays,
infrared, visible, ultraviolet etc. All the types of radiations travel with the same velocity but
differ from each other in terms of frequency and wavelength. They do not require any
medium for their propagation and can travel through empty space as well as through air and
other substances. Each type of electromagnetic radiation has a dual nature- wave like nature
and particle like nature. The particle nature has been established by the fact that the energy of
particular radiation occurs in discrete packets or quanta known as photons. Each photon
contains a certain amount of energy. The different types of radiation are defined by the
amount of energy found in the photons. The energy associated with particular
electromagnetic radiation is directly proportional to its frequency. The photons with the
highest energy correspond to the shortest wavelengths
Absorption of Electromagnetic Radiations by matters

When electromagnetic radiations are passed through an matter, some of the part gets
absorbed, while the remaining is transmitted. The absorption of energy can bring about
translational, rotational or vibrational motion, electronic transition or ionization of the
molecules depending upon the frequency of the electromagnetic radiation they receive (Table
1).

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Table 1: Energy changes depending on the type of radiation

The energy required for these transitions is quantized. Excited molecules are unstable and
quickly come back to the ground state by releasing the energy they had received as
electromagnetic radiation.

Infra-red Spectroscopy
Introduction
`The most frequent spectroscopic technique used by organic and inorganic chemists is
Infrared (IR) spectroscopy. It deals with the absorption of radiation in the infrared region of
the electromagnetic spectrum. IR spectrum gives sufficient information about the structure
(identification of functional groups) of a compound and can also be used as analytical tool to
assess the purity of a compound. The absorption of infrared radiation by a molecule causes
changes in their vibrational and rotational energy levels and hence IR-spectroscopy is also
known as vibrational-rotational spectroscopy.
The infrared spectrum can be divided into three main regions: the far infrared (<400
cm−1), the mid-infrared (4000–400 cm−1) and the near-infrared (13000–4000 cm−1). The
mid IR region is of greatest practical use to the organic chemist, but the near- and far-infrared
regions also provide important information about certain materials.

IR-spectroscopy gives the information about molecular vibrations or more

precisely on transitions between vibrational and rotational energy levels. Since the
absorption of infrared radiation leads to transition between vibrational and rotational
energy levels, it is also vibrational-rotational spectroscopy.

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When a molecule absorb IR-radiation below 100 cm-1, transitions between rotational
energy levels takes place. Since these energy levels are quantized, a rotational spectrum
consists of discrete lines. If a molecule absorbs radiation in the range 100-10,000 cm-1, it
causes transitions between vibrational energy levels. These energy levels are also quantised
but vibrational spectra appear as bands rather than discrete lines. Each vibrational energy
level is associated with a large number of closely spaced rotational energy levels or we can
say that the energy difference between various rotational energy levels is very short than the
energy difference between various vibrational energy levels. Therefore the vibrational spectra
appear s vibrational-rotational bands instead of discrete lines. Organic chemists are mainly
concerned with these transitions especially with those occur in the range 4000-667 cm-1.

Molecular vibrations

The atoms in a molecule do not remain fixed at certain positions. The two nuclei can
vibrate backwards and forwards or towards and away from each other around an average
position. There are two types of fundamental molecular vibrations viz stretching (change in
bond length) and bending (change in bond angle).

Stretching vibrations: It involves a rhythmic movement along a bond axis with a subsequent
increase and decrease in bond length. Stretching vibrations are of two types viz Symmetrical
Stretching and asymmetrical stretching

Bending vibrations: It involves a change in bond angle or movement of a group of atoms

with respect to the rest of the molecule. Bending vibrations are of four types.
i) Rocking
ii) Scissoring
iii) Wagging
iv) Twisting

Instrumentation
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The basic components of a dispersive IR spectrometer include a radiation source,

Sample and reference cells, monochromator, detector, amplifier and recorder. A schematic
diagram of a typical dispersive spectrometer is shown in Fig.

Radiation source: The common IR radiation source are inert solids that are heated
electrically to 1000 to 1800 °C to promote thermal emission of radiation in the infrared
region of the EM spectrum. The most common sources are Nernst filament (composed of
rare-earth oxides such as zirconium, cerium and thorium), Globar (composed of silicon
carbide), and Nichrome coil. They all produce continuous radiations, but with different
radiation energy profiles. The beam from the source is divided into two equivalent beams,
one passing through the sample and the other as reference beam.

Sample and reference cells: Like UV sample tubes (cuvettes) glass or quartz cannot be used
to make the sample cells for IR-spectroscopy, because they absorb strongly in most of the IR
region. Alkali metal halides such as KCl, NaCl are commonly used as they are transparent to
the IR- region.

Monochromator: The monochromator is a device used to disperse or separate a broad

spectrum of IR radiation into a continuous spectrum of frequencies of Infrared light. The
monochromator consists of rapidly rotating chopper that passes the two beams alternately to a
diffraction grating. The slowly rotating diffraction grating varies the frequency or wavelength
of radiation and sends it the individual frequency to the thermocouple detector which
generates an electrical signal as a response.

Detectors and Amplifier: Detectors are devices that convert the radiant energy into an
electrical signal. The detector determines the ratio between the intensities of the reference and
sample beams. Due to the difference in the intensities of the two beams falling on the
detector, an alternating current starts flowing from the detector to the amplifier, where it is
amplified and relayed to the recorder.
Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 21
Nanotechnology Subject Code:

The detectors used in IR spectrometers can be categorized into two classes: thermal
detectors and photon detectors. Thermal detectors consists of several thermocouples
connected together to produce greater sensitivity. They measure the heating effect produced
by infrared radiation that causes the flow of current. The current produced is proportional to
the intensity of radiation falling on the thermal detector. Photon detectors rely on the
interaction of IR radiation and a semiconductor material. Non-conducting electrons are
excited to a conducting state and therefore producing a small current or voltage.

Recorder: It records IR-spectrum as a plot of frequency of absorbed radiation and intensity

of absorption in terms of transmittance.

Transmittance (T) = I/I0

Percent transmittance (%T) = I/I0 X 100

Where I0 is the intensity of the incident radiation and I is the intensity of the radiation
emerging from the sample.

Applications

• Identification of functional group and structure elucidation.

• Identification of substances. ...
• Studying the progress of the reaction.
• Detection of impurities.
• Quantitative analysis.

Advantages

• Qualitative and quantitative analysis: One of the key advantages of Infrared

spectroscopy is without destroying the sample it can provide qualitative and
quantitative chemical information.
• Sample Preparation: The major advantage of infrared spectroscopy is that the sample
does not need any particular preparation.
• Sensitive and Time-saving technique: IR spectroscopy is very sensitive, hence it
required minimum sample quantity to scan the sample spectrum and it takes a few
seconds to scan a whole range of IR.
• It's versatility: Solid, liquid, gases and semisolid samples can be analyzed by the IR
spectroscopy.
Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 22
Nanotechnology Subject Code:

• Easy for interpretation: The Peak intensities, peak positions, peak widths, shapes, and
functional groups provide all helpful information.

Disadvantages

• Disadvantages include sometimes difficult handling procedures and maintenance of

the sample cells.
• There are no infrared spectra in atoms or monatomic ions, hence it cannot analyses.
• To use infrared spectroscopy is that it requires very sensitive and properly tuned
devices.
• The sample having aqueous solutions and complex mixtures are complicated to
analyze by infrared spectroscopy.

Ultraviolet-Visible Spectroscopy
UV-Visible spectroscopy deals with the study of the electronic transitions of
molecules as they absorb light in the UV (190-400 nm) and visible regions (400-800 nm) of
the electromagnetic spectrum. The absorption of ultraviolet or visible radiation lead to a
transition among electronic energy levels, hence it is also often called electronic spectros
Principle:
A light beam is passed through an object and wavelength of the light reaching the
detector is measured. The measured wavelength provides important information about
chemical structure and number of molecules (present in intensity of the measured signal).
Thus, both quantitative and qualitative information can be gathered. Information may be
obtained as transmittance, absorbance or reflectance of radiation in 160 to 3500 nm
wavelength range. The absorption of incident energy promotes electrons to excited states or
the anti-bonding orbitals. For this transfer to occur, photon energy must match the energy
needed by electron to be promoted to next higher energy state.

Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 23
Nanotechnology Subject Code:

Fig 2: Electronic Transitions in UV/VIS spectroscopy

Figure 2. The exact energy differences between the orbitals depend on the nature of
atoms present and the nature of the bonding system You can see, in each possible case, an
electron is excited from a low energy, ground state orbital into a higher energy, excited state
anti-bonding orbital (Figure 2). Each transition requires a defined amount of energy. The
larger the gap between the energy levels, the greater the energy required to promote the
electron to the higher energy level, resulting in electromagnetic radiation of higher frequency,
and therefore shorter wavelength, being absorbed. The important modes of electronic
transitions are described here.
a) σ to σ∗

A transition involving excitation of an electron from s bonding orbital to σ anti-

bonding orbital is called σ to σ* transition.
b) π to π∗

Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 24
Nanotechnology Subject Code:

The transition of an electron from a π bonding orbital to a π∗ antibonding orbital is

designated as π to π∗ transition. These types of transitions take place in compounds
containing one or more unsaturated groups like simple alkenes, carbonyl, aromatics, nitriles,
nitro etc
c) n to σ∗

The transition of an electron from a non-bonding orbital to the antibonding sigma

orbital is designated as n to σ∗ transition

d) n to π∗
The transition of an electron from a non-bonding orbital to a π∗ antibonding orbital is
designated as n to π∗ transition. This transition involves the least amount of energy in
comparison to all other transitions and therefore gives rise to an absorption band at longer
wavelength.

Types of Spectrophotometer
There are two types of spectrophotometers, namely, single beam spectrophotometer or
double beam spectrophotometer

Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 25
Nanotechnology Subject Code:

A single beam spectrophotometer has only one beam of light, which passes through
the sample, therefore it requires taking reading for the reference and sampling separately.
On the other hand, in a double-beam instrument, the light is split into two beams
before it reaches the sample. The two beams move simultaneously, one passing through a
reference solution and the other through the sample. The reference beam intensity is taken as
100% Transmission (or 0% Absorbance), and the measurement displayed is the ratio of the
two beam intensities.
Of the two types of spectrophotometer, double beam spectrophotometers are faster to
operate and in their performance. They provide more reproducible results because they
perform an automatic correction for the loss of light intensity as the beam passes through the
sample and reference solution.

Instrumentation and working

a) Light Source
The most suitable sources of light are
• Deuterium lamp which emits the light in the UV-region (160-375 nm)
• Tungsten lamp or tungsten-halogen lamp which emits radiation in the Visible and
near infrared regions (350-2500 nm)
• Xenon arc lamp which emits radiation in the range 190-800 nm
• Light emitting diodes (LED) which emits radiation in the visible range 400-800 nm
b) Monochromator
The main function of the monochromator is to disperse the beam of light obtained from
the primary source, into its component (Figure 3). The principle components of
monochromator are
• An entrance slit
• A collimating lens
• A dispersing device
• A focusing lens
• An exit slit

Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 26
Nanotechnology Subject Code:

The radiation emitted from the primary source (polychromatic radiation) enters the
monochromator through the entrance slit. The beam is collimated and then strikes the
dispersing element (Prism or grit) at a particular angle. Two types of dispersion devices
viz. prisms and holographic gratings (Figure 4) are commonly used in UV-visible
spectrophotometers
Light falling on the prism is reflected at different angles, depending on the
wavelength. The beam is split into its component colors. By moving the dispersing
element or the exit slit, radiation of only a particular wavelength can be obtained, which
leaves from the exit slit and can be used for the recording purpose (Figure 3). The beam
selected by the slit is monochromatic and further divided into two beams with the help of
another prism, which then passes through the sample and reference solutions.
c) Cuvettes

The cells are usually made of glass, plastic as well as silica or quartz. Of these, glass
cells cannot be used for the UV region as they absorb light in the UV region but can be
Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 27
Nanotechnology Subject Code:

used satisfactorily in the visible region. Quartz is transparent in all (200-700 nm) ranges
and is the best choice and hence can be easily used in UV as well as visible region.
Monochromator source is used; before reaching sample, light is divided in two parts
of similar intensity with a half-mirror splitter. One part (or sample beam), travels via the
cuvette having the solution of material to be examined in transparent solvent. Second
beam, or reference beam, travels via similar cuvette having only solvent. Reference and
sample solution containers have to be transparent towards passing beam.
d) Detectors
A detector converts a light signal into an electrical signal. After the beams are passed
through the sample under study as well as the reference cell, the intensities of the respective
transmitted beams are then compared over the whole wavelength range of the instrument.
Generally two photocells are used as detector in UV spectrometer to record the spectra. One
of the photocell receives the beam from sample cell and second detector receives the beam
from the reference. The intensity of the radiation from the reference cell is stronger than the
beam of sample cell. This results in the generation of pulsating or alternating currents in the
photocells.
Spectrophotometers consist of either a photomultiplier tube detector or a photodiode
detector.The commonly used detector in UV-Vis spectroscopy is photomultiplier tube (Figure
6). It is m consists of three components:
• a cathode which emits electrons when struck by photons of radiation known as
photo emissive cathode
• several dynodes which emit several electrons for each electron striking them
• an anode

Applications of UV-Vis spectroscopy

• DNA and RNA analysis
• Pharmaceutical analysis
• UV-Vis spectroscopy is utilised frequently in bacterial cultivation. To assess cell
concentration and monitor growth, 600 nm OD readings are routinely and rapidly
acquired at a wavelength of 600 nm.
• Another common application of UV-Vis spectroscopy is the determination of specific
chemicals in beverages. Caffeine content must be under specified legal limits which
can be measured with UV light.

Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 28
Nanotechnology Subject Code:

• UV-Vis spectroscopy is used to study the electronic and optical properties of

materials, such as semiconductors, dyes, and pigments.
• UV-Vis spectroscopy is used to study the properties of blood, to monitor the level of
glucose in blood, and to study the photochemistry of biological systems.
• UV-Vis spectroscopy is used to analyze trace evidence, such as fibers and paint, to
identify the source of a sample.
• UV-Vis spectroscopy is used to measure the concentration of food ingredients, to
monitor the quality of food products, and to detect contaminants.
Absorption Laws
When the radiation passes through a solution, some amount of light is absorbed.
Suppose I0 is the intensity of the incident radiation and I, the intensity of the transmitted
radiation.
The amount of radiation absorbed can be measured by Transmittance T = I/I0
% Transmittance = T×100 Absorbance A = Log10 I0/I= Log10 1/T
If the entire light passes through a solution without any absorption, then absorbance is zero.
In this case, the percent transmittance is 100%.
If all the light is absorbed, then absorption is infinite and the percent transmittance is zero %.

Beer-Lambert Law: It gives a linear relationship between absorbance and concentration of an

absorbing species. This law states that the absorption is directly proportional to the
concentration of absorbing substance and the length of the path of radiation through the
sample. It is independent of the intensity of the incident light and each successive unit layer
absorbs an equal fraction of light incident on it.
A = ɛcb Log10 I0/I= ɛcb
Where A is the sample’s Absorbance value at specific wavelength or frequency
ε is the molar absorptivity or the molar extinction coefficient of the substance b is the path
length of the sample in cm
c is the concentration of the solute in mol L-1.
The value of ε absorptivity coefficient is constant for a particular material at a particular
wavelength.
Limitations of the Beer-Lambert Law
Under certain conditions Beer-Lambert law fails to maintain a linear relationship between
absorbance and concentration of the solution.

Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 29
Nanotechnology Subject Code:

1. Deviations in absorptivity coefficients at high concentrations (>0.01M) due to

electrostatic interactions between molecules in close proximity.
2. Scattering of light due to particles present in the sample.
3. Chemical deviations due to the specific chemical species of the sample under
investigation.
4. Non-monochromatic radiation
5. Fluorescence or phosphorescence of the sample

Dr. Yashwanth H J
Assistant Professor, Dept of Physics,
Nitte Meenakshi Institute of Technology, Bangalore. 30