On National Teacher Day, meet the 2024-25 Kenan Fellows
Spectroscopy Technique
1. SPECTROSCOPY TECHNIQUE
Submitted to
Dr. Dhananjay Kumar Singh
Assistance Professor
Department of Pharmacy
School of Health Science
Presented by
Mr. Sree Prakash Pandey
(CUSB2006122010)
M.Pharm 1st year (1st Sem)
Pharmacuetics
School of Health Science
Central University of South Bihar, Gaya
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3. SPECTROSCOPY
INTRODUCTION
This term originated for the study of visible light dispersed by a
prism as per wavelength of different colors present in the visible
region.
The study of interaction between matter and electromagnetic
radiation, termed as spectroscopy.
The measurement of spectroscopy is a function of the wavelength of
radiation being observed.
It allows to determination of composition, physical and electronic
structure of various particles at molecular or atomic levels.
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4. What is Electromagnetic radiation?
Electromagnetic radiation (EMR) refers to the waves of
electromagnetic field composed by perpendicular oscillations
of electric and magnetic fields.
EMR is a form of energy having dual nature, i.e. to explain
some phenomenon such as interference it can be considered as
waves whereas, in some cases it can be considered as particle.
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5. WAVE NATURE OF EMR
An electromagnetic wave is described by several basic properties such as velocity, frequency, amplitude,
polarization, Phase angle and direction of propogation.
In the vacuum, electromagnetic waves travel at the speed of light commonly denoted as c.
The relationship between wavelength and frequency is-
𝛌 =
𝒄
𝛎
The amplitude can be defined as the maximum height attained by wave. The amplitude of an oscillating
electric field at any given point along the direction of propogation wave is given by relation
At = Ae Sin (2πνt + ψ)
Where, At = Magnitude of e.f. at time t.
Ae = Maximum amplitude of wave
ν = Frequency
ψ = A Phase angle
Similarly, for magnetic field-
At = Ae Sin (2πνt + ψ)
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7. PARTICLE NATURE OF EMR
According to the Newton’s corpuscular theory, the light radiation is made up of small
packets of energy travelling in the straight line. These packets are known as Photons.
When these Photons strikes the matter it imparts its energy to the matter which may give
rise to various effect such as Absorption, Transmission, Reflection, Refraction, and
Emission.
The relationship between energy and frequency is-
E = h ν
Or, 𝐄 =
𝐡𝒄
𝝀
Where, h = Planck’s constant = 6.63 x 10-34 J-s
EMR includes Radio waves, Microwaves, Infrared (IR), Visible light, Ultraviolet, X-Rays,
and Gamma rays.
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9. Basic Terminologies
Wavelength, λ
The wavelengths that are absorbed by electrons are related to the energy-level of
the molecule.
Wavelength can be defined as the distance between two successive crests or
troughs of a wave.
It is measured in the direction of the wave.
The energy content E of photons is inversely proportional to the wavelength λ of
the electromagnetic wave according to following equation:
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11. Wave number, ν
The reciprocal of the wavelength, λ, or the number of waves per unit length along
the direction of propagation.
𝝂 =
1
𝝀
The SI unit is m–1, but a commonly used unit is cm–1.
Frequency
Frequency, f is the number of occurrences of a repeating event per unit of time, T.
𝒇 =
𝟏
𝑻
Unit of frequency is Hz
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12. Spectrometer
The spectrometer that measures the interaction of EMR from the
sample or the emission of EMR by various sample.
Spectra
Singular spectrum, in optics, are the colors observed when the white
light is dispersed through a prism.
Spectrum refers to the range of various variables associated with
light and other waves.
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13. Classification of Method
Absorption spectroscopy
The absorption spectroscopy refers to absorption of electromagnetic radiation by
sampling atoms or molecules.
The atoms or molecules undergo transition from higher energy state to lower energy state.
The most common examples are Infrared and Ultraviolet-Visible spectroscopy.
Emission spectroscopy
It refers to radiation or emission of energy/photons by analytical molecules or atoms.
It means atoms or molecules that are excited to high energy level can decay to low energy
level by Emitting radiation.
The most common examples are Atomic emission spectroscopy, Flame emission
spectroscopy and Fluorescence spectroscopy.
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14. Elastic/scattering spectroscopy
The Scattering spectroscopy deals with the certain physical properties by measuring
the amount of light or radiation that a substance scatters at certain wavelength.
The most useful application of light elastic spectroscopy is Crystallography.
Inelastic spectroscopy
The phenomena involve an exchange of energy between the radiation and the matter
that shifts the wavelength of the scattered radiation.
One of the most useful application of light inelastic spectroscopy is RAMAN
SPECTRUM.
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15. VARIOUS SPECTROSCOPY TECHNIQUES
There are several types of spectroscopy techniques-
Type of Energy Transfer Region of Electromagnetic
Spectrum
Spectroscopic Technique
Absorption Gamma ray Mossbauser spectroscopy
X-Ray X-Ray absorption spectroscopy
UV/Vis UV-Visible spectroscopy
Atomic absorption spectroscopy
IR Infrared spectroscopy
Raman spectroscopy
Microwave Microwave spectroscopy
Radiowave Nuclear magnetic resonance spectroscopy
Electron spin resonance spectroscopy
Emission UV/Vis Atomic Emission spectroscopy
Photoluminescence X-Ray X-Ray fluorensence
UV/Vis Fluorensence spectroscopy
Phosphorensence spectroscopy
Atomic fluorensence spectroscopy
Chemiluminescence UV/Vis
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16. ULTRAVIOLET-VISIBLE SPECTROSCOPY
UV-Visible spectroscopy is routinely used in the quantitative determination of solution of
transition metal ions and highly conjugated organic compound.
THE ABSORPTION LAWS
There are two laws which govern the absorption of light by the molecules.
Lambert’s Law – when a beam of monochromatic radiation passes through a
homogeneous absorbing medium, the rate of decrease of intensity of radiation with
thickness of absorbing medium is proportional to the intensity of the incident radiation.
Mathematically,
-dI/dx = KI
Where, I = intensity of radiation after absorption.
dx = small thickness of absorbing medium
K = Absorption constant
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17. Beer’s Law – when a beam of monochromatic radiation is passed through a solution of an
absorbing medium, the rate of decrease of intensity of radiation with the thickness of the
absorbing solution is proportional to the intensity of incident radiation as well as the
concentration of the solution.
Mathematically,
-dI/dx = KI0C
Where, I0 = intensity of radiation before absorption.
C = Concentration of solution in Mol/litre
K = Molar absorption coefficient
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18. On the combining the Beer-Lambert Law-
log I0/I = ε.c.t. = A
Where, A = Absorbance
I0 = Intensity of incident light
I = Intensity of transmitted light
c = Concentration of the solution in Mol/litre
t = Path length or thickness of absorbing media (usually 1 cm)
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19. Spectrophotometer
A spectrophotometer is an instrument used to measure absorbance or transmittance of a
material in EM radiation. The major components of a spectrophotometer are
1. Source: The component used to generate radiation is known as source.
Sun is the best example of radiation source as it emits full spectrum of
electromagnetic radiations.
In the instrumentation these radiations are generated in a particular band of
wavelengths.
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20. 2. Slits
A slit is used to control the light beam and to collimate it. It plays a
vital role in controlling unwanted radiations and attaining high
resolutions in the spectrometer.
Slit is nothing but a fixed or variable opening and is generally used at the
entrance and exit of light path.
3. Monochromator
This is used to separate out monochromatic radiation from bundle of
polychromatic radiation. In the visible range a glass prism is it’s a best
example as it splits white light into a spectrum of VIBGOYR.
In spectroscopy the prism is very effectively used as a monochromator.
The other monochromator frequently used is dispersion grating.
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21. 21
4. Detector: A device which converts the radiation energy into the electrical
signal. It is the intensity of radiation which is subsequently digitally
processed and produced at output of instrument. These detectors may
work on different principles but ultimately.
They show us the amount of output radiations. A range of detectors for
ultraviolet, visible, infrared and X-rays radiation
Tansducer Wavelength Range
Photon Detectors
Phototube 200-1000 nm
Photomultiplier 110-1000 nm
Si photodiode 250-1100 nm
Photovoltaic cell 400-5000 nm
Photoconductor 750-6000 nm
Thermal Detectors
Thermocouple 0.8-40 μm
Thermistor 0.8-40 μm
Pneumatic 0.8-1000 μm
Pyroelectric 0.3-1000 μm
X-Ray Detectors
Geiger counter
Proportional counter
Semiconductor detector
24. APPLICATIONS OF UV/VIS SPEC
Detection of Impurities
Detection of Functional group
Distinction of conjugated and non-conjugated compounds
Identification of unknown compound
Structure elucidation of organic compounds
Determination of configuration of Geometrical isomers.
Determination of strength of Hydrogen bonding.
Quantitative analysis
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25. INFRARED SPECTROSCOPY
INTRODUCTION
Infrared radiation compasses of waves with wavelengths between the visible and
microwave regions of the EM spectrum.
The IR region is further subdivided into three regions namely Near-IR, Mid-IR
and Far-IR with following ranges
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26. FOURIER TRANSFORM INFRARED SPECTROSCOPY
(FTIR)
FTIR is a technique used to obtain an infrared spectrum of absorption or
emission of a solid, liquid or gas.
INSTRUMENTATION
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27. Principle of Infra-red Spec
The Absorption of IR radiation causes an extinction of molecules
from lower to the higher vibrational level, and each vibrational level
is associated with a number of closed spaced rotational levels, so,
the IR spectra is considered as vibrational-rotational spectra.
All the bonds in a molecule are not capable of absorbing IR energy
but only those bonds which are accompanied by a change in Dipole
Moment will absorb the IR region.
Such vibrational transitions which are accompanied by a change in
the Dipole-moment of the molecule are called IR active transition.
For example- vibrational transitions of C=O, N-H, O-H, etc.
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28. Theory of IR spectroscopy
A. Molecular Rotations
Rotational transitions are mainly observed in the gas samples as
line spectra. However, in liquids and solids it is observed as
band due to interaction of neighbor molecules and collisions.
B. Molecular Vibrations
There are total 3n degrees of freedom consisting of the
translational, rotational and vibrational motions of the molecule.
3n degree of freedom = Translational + Rotational + Vibrational
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29. There are two kinds of fundamental Vibrations -
1. Stretching: The distance between two atoms increases or decreases but the atoms
remains in the same bond axis.
1. Bending: The positions of atoms change with the respect to the original bond axis.
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30. Type of Stretching vibrations-
1. Symmetrical Stretching: The movement of the atoms with respect to a
particular atom in a same direction.
2. Asymmetrical Stretching: Here, one atom approaches the central atom
while the other departs from it.
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31. Type of Bending vibrations-
1. Scissoring: The two atoms are approaches to each other.
2. Rocking: The movement of two atoms take place in the same direction.
3. Wagging: The two atoms move ‘Up & Down’ the plane with respect to the
central atom.
4. Twisting: In this type, one of the atoms moves up the plane while the other
moves down the plane with respect to the central atom.
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32. VIBRATIONAL FREQUENCY
The value of vibrational stretching frequency of a bond can be calculated by
Hook’s law-
Where, μ = Reduced mass
k = Force constant
ῡ = Vibrational frequency
mA & mB are the masses of atoms in grams.
c = Velocity of radiation or light.
Note: The value of vibrational frequency or wave number depend upon-
(i) Bond strength (ii) Reduced mass
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33. Factor influencing vibrational frequencies
I. Fermi Resonance
II. Electronic Effect
III.Hydrogen Bonding
IV.Bond Angle
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35. MASS SPECTROSCOPY
INTRODUCTION
Mass Spectroscopy is the most appropriate method to determine the molecular
mass of the compound and its elemental composition.
In this technique, the molecular bombardment with a beam of energetic
electron.
The molecule gets ionized and broken up into many fragments, some are which
positive ions.
And each kind ion has a particular ratio of mass to charge, i.e. m/e ratio.
Why mass spec important?
It can give the exact molecular mass.
It can give a molecular formula.
It also provides the relevant & certain structural units in a molecule.
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36. THEORY OF MASS SPEC
⁕ A parent ion results when one electron is removed from the parent
molecule of the substance.
M (g) + e —> M+ (g) + 2e
⁕ The m/e ratio of the parent ion is equal to the molecular mass of the
compound.
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39. NUCLEAR MAGNETIC RESONANCE (NMR)
INTRODUCTION
Nuclear magnetic resonance NMR spectroscopy involve the interaction
between an oscillating magnetic field of electromagnetic radiation and
magnetic energy of the hydrogen nucleus.
The nucleus of hydrogen atom proton behaves as a spinning bar
magnet, it possesses both electric and magnetic spin.
The sample absorbs EMR in radiowave region at different frequencies
since absorption depends upon the type of protons or certain nuclei
contained in the sample.
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40. Theory of NMR
Consider a spinning on the top, it also performs a slower Waltz like motion, in
which the spinning axis of the top moves slowly around the vertical. This is
precessional motion and the top is said to precessional around the vertical
axis of the Earth’s Gravitational field.
The precessional arises from the interaction of spin with earth’s gravity acting
vertically downwards. This is called Gyroscopic motion.
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41. It has found that the proton (tiny magnet) precesses about the axis of the external
magnetic field in the same manner in which a spinning gyroscope precesses under the
influence of gravity.
It has found that the
Angular precessional velocity, ω = γH0
Here, H0 = Applied field in Guess
γ = Gyroscopic ratio =
𝟐𝛑𝛍
𝒉𝑰
Here, μ = Magnetic moment of the spinning bar magnet.
I = Spin quantum number
h = Planck’s constant
The precessional frequency is defined as the number of revolutions per second made
by magnetic moment vector of the nucleus around the external field H0 .
According to the fundamental NMR equation, correlation between electromagnetic
frequency and the magnetic field, we have-
ω = γH0 = 2πν here, ν = Frequency of EMR
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42. Spin quantum number (I)
The spin quantum number (I) is related to the atomic and mass number
of the nucleus.
I states that -
The element with either odd mass or odd atomic number have a
property of ‘nuclear spin’.
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43. Orientation of Proton
When a proton is placed in a magnetic field, it starts precessing at a certain
frequency under the EMR. Thus, it capable of taking up one of the two orientations
with respect to the external field.
1. Alignment with the field.
2. Alignment against the field.
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44. INSTRUMENTATION OF NMR
NMR Spectrophotometer consist of a –
1. Magnet
2. Coils
3. Sample tube holder
4. Radio frequency transmitter
5. Radio frequency receiver
6. Amplifier
7. Detector
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47. APPLICATIONS OF NMR SPEC
Identification of structural isomers
Detection of Hydrogen bonding
Detection of aromaticity
Detection of electromagnetic groups
Detection of Cis-Trans Isomers
Detection of double bonds
Quantitative analysis
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48. ATOMIC ABSORPTION SPECTROMETRY
INTRODUCTION
Atomic Absorption Spectrometry (AAS) is an analytical
technique that measures the concentrations of elements.
It is very common technique to detecting metals and metalloids in
the samples.
It is very reliable and simple to use.
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49. PRINCIPLE OF AAS
The principle of this technique is that free atoms (gas) generated
in antomizer can absorbed radiation at specific frequency.
Atomic absorption spectrometry quantifies the absorption of ground
state atoms in the gaseous state.
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51. Applications
Qualitative and quantitative analysis
Determination of metallic elements in biological system
Determination of metallic element in food industry
Determination of Ca, Mg, Na, K in serum
Determination of lead in petrol
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52. FLUORESCENCE SPECTROSCOPY
Fluorescence spectroscopy is a type of electromagnetic spectroscopy that
analyzes fluorescence from a sample.
It involves using a beam of light, usually ultraviolet light, that excites the
electrons in molecules of certain compounds and causes them to emit light;
typically, but not necessarily, visible light. A complementary technique
is absorption spectroscopy.
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53. Application of Fluorescence Spec
Determination of both organic and inorganic impurities.
Detector for HPLC
Laser induced fluorescence spectroscopy of human tissues for cancer
diagnosis.
Accurate determination of glucose.
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54. PHOSPHORESCENCE
Phosphorescence is a type of photoluminescence related to fluorescence.
The slower time scales of the re-emission are associated with
"forbidden" energy state transitions in quantum mechanics.
As these transitions occur very slowly in certain materials, absorbed radiation
is re-emitted at a lower intensity for up to several hours after the original
excitation.
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55. REFERENCES
Palvia Donald L.; Lampman Gary M.; Kriz George S.; Vyvan James R.;
“Introduction to Spectroscopy”; 5th Edition 2015; 200 First Stamford Place; 4th
Floor; Stamford; CT06902, USA.
Kemp William; “Organic Spectroscopy”; 3rd Edition 1991; Reprint 2002;
Published by- PALGRAVE; Houndmills, Basingstoke, Hamshite; Fifth Avenue;
New York; N.Y. 10010.
Hotlas Michael J.; “Mordern Spectroscopy”; 4th Edition 2004; John Wiley &
Sons Ltd; The Atrium; Southern Gate; Chichester; West Sussex; England.
Sharma K Ramesh; “Various Spectroscopy Technique”; January-2017;
ResearchGate.
Sharma Y.R.; “Elementary Organic Spectroscopy”; Multiedition; S. Chand &
Company Ltd; Ram Nagar; New Delhi-110055.
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