Although the inelastic scattering of light was predicted by Adolf Smekal in 1923, it was not observed in practice until 1928. The Raman effect was named after one of its discoverers, the Indian scientist C. V. Raman, who observed the effect in organic liquids in 1928 together with K. S. Krishnan, and independently by Grigory Landsberg and Leonid Mandelstam in inorganic crystals. Raman won the Nobel Prize in Physics in 1930 for this discovery. The first observation of Raman spectra in gases was in 1929 by Franco Rasetti.

1. Raman spectroscopy
Prepared by-
ROHAN JAGDALE
T. Y. B. Pharm SEM VI
2020-21
rohanjagdale235@gmail.com
Submitted to-
Prof. Amol kharche
Department of Pharmaceutical Analysis
Yadavrao Tasgaonkar Institute of pharmacy
University of Mumbai

2. Sir Chandrasekhara Venkata Raman

3. The first spectra taken by Prof. C. V. Raman
and Prof. K. S. Krishnan

4. ❖ Time lap
❖ Spectroscopy and light
❖ What is Raman Spectroscopy
❖ Raman spectrometers mechanism
❖ Raman scattering
❖ Raman shift
❖ Raman spectrum
❖ Vibrating atoms
❖ Raman Instrumentation
❖ Information provided by Raman spectroscopy
❖ Types of samples analysed with Raman spectroscopy
❖ Raman imaging
❖ Comparison between Raman & IR spectroscopy
❖ Applications
CONTENTS

5. Time lap
● 1923 - Inelastic light scattering is predicted by A. Smekel
● 1928 - Landsberg and Mendelstam see unexpected frequency shifts in
scattering from quartz.
● 1928 - C. V. Raman and K.S. Krishnan see “feeble fluorescence “ from neat
solvents.
● 1930 - C. V. Raman wins Nobel prize in Physics.
● 1961 - Invention of laser, makes Raman experiments reasonable.
● 1977 - Surface enhanced Raman scattering (SERS) is discovered
● 1997 - Single molecule SERS is possible.
● 2000 - in vivo applications, optical fiber probes

6. Spectroscopy and light
Light interacts with matter in different ways, transmitting through some
materials, while reflecting or scattering off others. Both the material and
the colour (wavelength) of the light affect this interaction. We call the
study of this light ‘spectroscopy'. Which parts of the visible spectrum
enter our eyes determines which colours we perceive.
A substance might appear blue, for example, if it absorbs the red parts of
the spectrum of light falling upon it, only reflecting (or scattering) the
blue parts into our eyes.

7. The different fundamental light processes during
material interaction.

8. What is Raman Spectroscopy?
● Raman spectroscopy is an analytical technique where scattered light is used to
measure the vibrational energy modes of a sample.
● Raman spectroscopy can provide both chemical and structural information, as
well as the identification of substances through their characteristic Raman
‘fingerprint’.
● Raman spectroscopy extracts this information through the detection of Raman
scattering from the sample.
● Raman spectroscopy is the measurement of wavelength and
intensity of inelastically scattered light from molecules. The
Raman scattered light occurs at wavelength that are shifted
from the incident. Raman spectroscopy is used to determine
the molecular motions, especially the vibrational one.

9. Raman spectrometers mechanism
▪When a substance (in any state) is irradiated with a monochromatic light of
definite frequency (v), the light scattered at right angle to the incident light
contains lines of - 1.Incident frequency and 2. Also of lower frequency
▪Sometimes lines of higher frequency are also obtained that of the incident
beam will be scattered. It is called Raman scattering.
▪The line with lower frequency are called Stokes lines
▪Also the line with higher frequency are called Anti-Stokes lines.
▪The line with the same frequency as that of the incident light is called
Rayleigh line

10. Rayleigh and Raman scattering energy diagram So, S1, S2 are electronic
energy levels, with higher energy vibrational levels.

12. The difference is called Raman frequency or Raman Shift

13. Raman Effect
The difference between the frequency of the incident light and that of
scattered light is constant and it depends only on the nature of
substance. It is completely independent of the frequency of incident light
The difference is called the Raman Effect. The Various observations
made by Raman are called Raman Effect and the spectrum obtained is
called Raman spectrum.

14. The Raman Effect/ Raman Scattering
Raman scattering or the Raman effect is the inelastic scattering of photons by
matter, meaning that there is an exchange of energy and a change in the light's
direction. Typically this involves vibrational energy being gained by a molecule as
incident photons from a visible laser are shifted to lower energy.
A ray of light is initially beamed onto a sample, which leads to absorption and
scattering of photons. Most of these dispersed photons have identical wavelengths
as the original photons and are termed “Rayleigh scatter”.
However, very small amount (~1/107) of the distributed radiation is moved to an
alternate wavelength, termed “Raman scatter”. The majority of these Raman
scattered photons are moved to greater wavelengths, which is called Stokes shift.
Alternately, a minute number are moved to inferior wavelengths, which is called
anti-Stokes shift.

16. When a substance interacts with laser beam, almost all of the light
produced is Rayleigh scattered light (elastic process). However, a small
percentage (about 0.000001%) of this light is Raman scattered (inelastic
process). Raman scattering is a process, where incident light interacts
with molecular vibrations in a sample.
All three different ways how light can be re-emitted: (a) Rayleigh scatter, (b) Stokes Raman
scatter, (c) Anti-Stokes Raman scatter.

17. The photons from the laser beam interact with the molecules and excites the
electrons in them. The excited electrons are in a “virtual state” which is not
stable, so they immediately fall down to the ground level. As electrons lose
energy and fall down to the ground state, they emit photons. There are three
different scenarios of how light can be re-emitted after energy had been
absorbed by an electron:
, ▪An electron falls down to the original ground state and there is no energy
change, therefore light of the same wavelength is re-emitted. This is called
Rayleigh scattering.
▪After being excited, an electron falls to a vibrational level, instead of the
ground level. This means the molecule absorbed a certain amount of energy,
which results in light being emitted in a longer wavelength than the incident
light. This Raman scatter is called “Stokes”.
▪If an electron is excited from a vibrational level, it reaches a virtual level with
higher energy. When the electron falls down to the ground level, the emitted
photon has more energy compared to the incident photon, which results in
shorter wavelength. This type of Raman scatter is called “Anti-Stokes”.

18. Energy level diagram explaining: (a) Rayleigh scatter, (b) Stokes Raman
scatter, (c) Anti-Stokes Raman scatter.

19. ▪In any case of Raman scatter, the energy change relative to
wavelength of the photons can be spectroscopically detected as a
colour change and is characteristic to the sample being investigated.
The output of Stokes and Anti-Stokes scatter measurements is called a
Raman Spectrum. Because Raman signal is quite weak, one of the ways
to enhance the intensity is SERS (Surface-Enhanced Raman
Spectroscopy).
▪In Raman spectroscopy, wavenumber (measured in cm-1) is used
instead of wavelength to characterise light. Wavenumbers are
convenient to use, because they are linearly related with energy and
independent of excitation wavelength. For example, the Raman peak of
diamond is always at about 1332 cm-1, no matter what the excitation
wavelength is. That is why every material has it’s own unique Raman
spectrum by which it can later be identified.

20. Jablonski Diagram showing the origin of Rayleigh, Stokes and Anti-Stokes
Raman Scatter

21. Raman frequency /Raman Shift
It is clear, that the wavelength of the Raman scattered light will depend on the
wavelength of the excitation light. This makes the Raman scatter wavelength an
impractical number for comparison between spectra measured using different
lasers. The Raman scatter position is therefore converted to a Raman shift away
from excitation wavelength:
(Δυ ̅) is the wavenumber Raman shift in cm-1, λ_(0 ) is the wavelength of the
excitation laser in nm, and λ_(1 )is the wavelength of the Raman scatter in nm.

22. SERS spectrum of drug XEFO. A SLM 785 nm, 1 mW laser was used as an excitation
source.

23. Raman spectrum
Plotting the magnitude of Raman scattered radiation as a function of the
frequency change from the original radiation (frequently in units of
wavenumbers, cm–1) produces a Raman spectrum.
This change is termed the Raman shift. As it is a difference value, this
change is not related to the frequency of the original radiation.
Usually, the only regions which are used are the Stokes region. Even
though the anti-Stokes spectrum has exactly the same pattern, its
intensity is much lower.

24. Raman shift in cm - 1= energy of photon - energy of photon out

25. Scattered light
The first step in producing a Raman spectrum is to illuminate your sample
with a monochromatic light source, such as a laser.
Most of the light that scatters off is unchanged in energy ('Rayleigh
scattered'). A minute fraction—perhaps 1 part in 10 million—has lost or
gained energy ('Raman scattered'). This Raman shift occurs because photons
(particles of light) exchange part of their energy with molecular vibrations in
the material.
Where energy is lost the Raman scattering is designated as 'Stokes'; where
energy is gained the Raman scattering is designated as 'anti-Stokes'. We
rarely use anti-Stokes Raman light as it is less intense than the Stokes,
however it does represent equivalent vibrational information of the
molecule.

26. Vibrating atoms
The change in energy depends on the frequency of vibration
of the molecule. If it is very fast (high frequency)—light
atoms held together with strong bonds—the energy change
is significant. If it is very slow (low frequency)—heavy
atoms held together with weak bonds—the energy change is
small.

27. Possible vibrational modes of atoms

28. Raman Instrumentation

29. Instrumentation
Instrumentation of modern Raman spectroscopy consists of three
components :-
▪A laser source
▪Sample illumination system
▪A suitable spectrometer

30. Laser or source of light
▪Lasers are generally the only source strong enough to scatter lots of light
and lead to detectable raman scattering.
▪Lasers operate using the principle of stimulated emission.
▪The sources used in modern Raman spectrometry are nearly always
lasers because their high intensity is necessary to produce Raman
scattering of sufficient intensity to be measured with a reasonable
signal-to-noise ratio.
▪Because the intensity of Raman scattering varies as the fourth power of
the frequency, argon and krypton ion sources that emit in the blue and
green region of the spectrum have and advantage over the other sources.

31. List of various laser source.
Sr. No. Laser Wavelength
1. Nd:YAG 1064 nm
2. He:Ne 633 nm
3. Argon ion 488 nm
4. GaAIAs diode 785 nm
5. CO2 10600 nm
6. Ti-Sapphire 800 nm

32. Sample illumination system
Sample handling for Raman spectroscopic measurements is simpler
than for infrared spectroscopy:-
▪because glass can be used for windows, lenses, and other optical
components instead of the more fragile and atmospherically less
stable crystalline halides.
▪In addition, the laser source is easily focused on a small sample area
and the emitted radiation efficiently focused on a slit.
▪Consequently, very small samples can be investigated.
A common sample holder for nonabsorbing liquid samples is an
ordinary glass melting-point capillary.

33. ● If the sample is colourless, it does not absorb a
visible laser.
● If the compound is colored, it can absorb the laser,
get hot and decompose. Some techniques are:
● Reduce the laser power (defocus) and/or change
wavelength;
● Dilute the sample into a KBr pellet;
● Cool the sample
● Rotate or oscillate the laser beam on the sample

35. Liquid samples
▪A major advantage of sample handling in Raman spectroscopy compared
with infrared arises because water is a weak Raman scattered but a
strong absorber of infrared radiation. Thus, aqueous solutions can be
studied by Raman spectroscopy but not by infrared.
▪This advantage is particularly important for biological and inorganic
systems and in studies dealing with water pollution problems.

36. Solid samples -
Raman spectra of solid samples are often acquired by filling a small
cavity with the sample after it has been ground to a fine powder.
Polymers can usually be examined directly with no sample
pretreatment.
Gas samples -
Gas are normally contain in glass tubes, 1-2 cm in diameter
and about 1 mm thick. Gases can also be sealed in small
capillary tube.

37. Fiber-optic sampling

38. ▪One of the significant advantages of Raman spectroscopy is that it is
based on visible or near- IR radiation that can be transmitted for a
considerable distance (as much 100 m or more) through optical fiber.

39. ▪Here, a microscope objective lens is used to focus the laser excitation beam on one end of an
excitation fiber of a fiber bundle.
▪These fibers bring the excitation radiation to the sample.
▪Fibers can be immersed in liquid samples or used to illuminate solids.
▪A second fiber or fiber bundle collects the Raman scattering and transports it to the entrance slit of
the spectrometer.
▪Several commercial instruments are now available with such probes.
▪Fiber-optic probes are proving very useful for obtaining Raman spectra in locations remote from
the sample site.
▪Examples include: hostile environments, such as hazardous reactors or molten salts; biological
samples, such as tissues and bacterial walls; and environmental samples, such as groundwater and
seawater.

40. Real time in-vivo Diagnosis of Nasopharyngeal Carcinoma using
Rapid-Fiber-optic Raman spectroscopy

41. Raman spectrometers
▪Raman spectrometers were similar in design and used the same type of
components as the classical ultraviolet/visible dispersing instruments.
▪Most employed double grating systems to minimize the spurious
radiation reaching the transducer.
▪Photomultipliers served as transducers.
▪Now Raman spectrometers being marketed are either Fourier transform
instruments equipped with cooled germanium transducers or
multichannel instruments based upon charge-coupled devices.

42. Block diagram of Raman spectrometer

43. Raman imaging and spectroscopy

44. Information provided by Raman spectroscopy
Raman spectra of ethanol and methanol, showing the significant spectral differences
which allow the two liquids to be distinguished.

45. ● Raman spectroscopy probes the chemical structure of a material and
provides information about:
● 1.Chemical structure and identity
● 2.Phase and polymorphism
● 3.Intrinsic stress/strain
● 4.Contamination and impurity
● Typically a Raman spectrum is a distinct chemical fingerprint for a
particular molecule or material, and can be used to very quickly
identify the material, or distinguish it from others. Raman spectral
libraries are often used for identification of a material based on its
Raman spectrum – libraries containing thousands of spectra are
rapidly searched to find a match with the spectrum of the analyte.

46. Mineral distribution
In combination with
mapping (or imaging)
Raman systems, it is
possible to generate images
based on the sample’s
Raman spectrum. These
images show distribution of
individual chemical
components, polymorphs
and phases, and variation in
crystallinity.

47. Type of samples analyzed with Raman
🔹Raman can be used to analyze many different samples. In general it is suitable for analysis of:
● Solids, powders, liquids, gels, slurries and gases
● Inorganic, organic and biological materials
● Pure chemicals, mixtures and solutions
● Metallic oxides and corrosion
● In general it is not suitable for analysis of:
● Metals and their alloys
● 🔹Typical examples of where Raman is used today include:
● Art and archaeology – characterization of pigments, ceramics and gemstones
● Carbon materials – structure and purity of nano-tubes, defect/disorder characterization
● Chemistry – structure, purity, and reaction monitoring
● Geology – mineral identification and distribution, fluid inclusions and phase transitions
● Life sciences – single cells and tissue, drug interactions, disease diagnosis
● Pharmaceutics – content uniformity and component distribution
● Semiconductors – purity, alloy composition, intrinsic stress/strain microscope.

48. Raman Imaging
Raman imaging is a powerful
technique that provides 3-D
spatial information and chemical
identification. Samples with
dimensions of micrometers to
millimeters can be analyzed in
just a few minutes. JASCO has
developed a technology called
QRI that increases the data
acquisition speed by up to 50
times compared with
conventional mapping, and also
offers a dramatic improvement in
sensitivity.
Raman imaging for spatial distribution and chemical identification

49. Comparison between Raman & FTIR
Spectroscopy
Although Raman and FTIR Spectroscopy give complimentary information
and are often interchangeable, there are some practical differences that
influence which one will be optimal for a given experiment. Most
molecular symmetry will allow for both Raman and IR activity. One
special case is if the molecule contains a center of inversion. In a molecule
that contains a center of inversion, Raman bands and IR bands are
mutually exclusive, i.e. the bond will either be Raman active or it will be IR
active but it will not be both. One general rule is that functional groups
that have large changes in dipoles are strong in the IR, whereas functional
groups that have weak dipole changes or have a high degree of symmetry
will be better seen in Raman spectra.

50. Difference between Raman and IR methods
Sr.
No.
Raman spectroscopy IR spectroscopy
1. It is due to scattering of light by
vibrating molecules
It is the result of absorption of light
by vibrating molecules
2. The vibration is Raman active if it
causes a change in polarisability
Vibration is IR active if there is
change in dipole moment.
3. The molecule need not possess a
permanent dipole moment
The vibration concerned should have a change
in dipole moment due to their vibration
4. Water can be used as a solvent Water cannot be used due to its
intense absorption of IR
5. Sample preparation is not very
elaborate, it can be in any state
Sample preparation is elaborate
Gaseous samples can rarely be used
6. Gives an indication of covalent
character in the molecule
Gives an indication of ionic character
in the molecule
7. Cost of instrumentation is high Comparatively inexpensive

51. Choose Raman Spectroscopy when:
● Investigating carbon bonds in aliphatic and aromatic rings are of
primary interest
● Bonds that are difficult to see in FTIR (i.e., 0-0, S-H, C=S, N=N, C=C
etc.)
● Examination of particles in solution is important, e.g. polymorphism
● Lower frequency modes are important (e.g. Inorganic-Oxides)
● Reactions in aqueous media are investigated
● Reactions in which observation through a reaction window is easier
and safer (e.g. high pressure catalytic reactions, polymerizations)
● Investigating lower frequency lattice modes is of interest
● Investigation of reaction initiation, endpoint, and product stability of
biphasic and colloidal reactions

52. Choose FTIR Spectroscopy when:
● Studying liquid-phase reactions
● Reactions in which reactants, reagents, solvents and reaction species
fluoresce
● Bonds with strong dipole changes are important (e.g. C=O, O-H, N=O)
● Reactions in which reagents and reactants are at low concentration
● Reactions in which solvent bands are strong in Raman and can swamp
key species signal
● Reactions in which intermediates that form are IR active

53. Applications in Pharmaceuticals
Light stability of ciprofloxacin tablets, xanthine derivative
tablets, quinolone delivative antibacterial tablets,
theophylline hydrates, anhydrates, crystalline polymorphs
of indomethacin, crystalline polymorphs of carbamazepine
(CBZ), crystalline polymorphs of ampicillin, crystal
structure and thermal stability of acetylsalicylic acid
(aspirin), active ingredients in drug substances and their
preparation (Jpn Pharmacopeia), qualitative/quantitative
evaluation of additives (Jpn Pharmacopeia), bronchodilator
(TBR, turobuterol) tape

54. Applications
Component distribution on white chocolate surface, butter/margarine
emulsion imaging , components of egg yolk, thermal change of trehalose
dihydrate, fatty acid in food oil, aaccharides solutions (saccharose,
glucose, xylitol, galactose, lactose), multilayer films for food packaging,
ethanol in glass bottles, caffeine, crystallinity of PET bottles
Food-
Carbon materials-
Carbon nanotubes, diamond-like carbon, fullerenes

55. Semiconductors
Power semiconductor (SiC) devices, crystallinity of polysilicon
Electronic devices-
Foreign matter in liquid crystal substrates, foreign matter in color
filters, diamond-like carbon on hard disk surfaces, solar cells
(crystalline silicon, amorphous silicon)
Polymer compounds-
3D imaging of cellophane tape, polypropylene-polyethylene multilayer
films,foreign matter on polyethylene films, polymer additives,
dispersion in blended polymers, crystallization of molten polymers,
curing of UV curable resin, dispersion of lubricant on films, orientation
of natural rubber, synthetic rubber

56. Biological materials
Visualization of sea-island structure in blended polymers, structural
changes in proteins (hemoglobin, lysozyme, cytochrome c), enzymes
(ribonuclease A), dental adhesive, collagen, chemical imaging of coral,
structure and orientation evaluation of spider silk
Cosmetics
Ingredients of lipstick marks, eye shadow
Gas -
Natural gas hydrates

57. Others
Imaging of bath powder (mixed powder samples), carbon nanotubes,
crystallinity of core of pencils, identification of fingerprints with
vermilion ink, iron rust, colored fibers, Nylon 6 fibers, wood (lignin),
quartz, calcite, nondestructive analysis of archaeological material
(mainly pigments)

58. C. V. Raman Research Institute Banglore

59. Reference
https://www.edinst.com/blog/what-is-raman-spectroscopy/
https://www.nanophoton.net/lecture-room/raman-spectroscopy/lesson
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