Raman Spectroscopy

Basic Principle and Applications

Himani Chandola
M.Sc. Final
2021-2023
Table of Contents
Discovery
Basic Principle
The Raman Spectrophotometer
Applications in Inorganic Chemistry
Applications in Organic Chemistry
Applications in Physical Chemistry
Biological Applications
In Food Analysis
Some Other Applications
Discovery
• An alternate and simple method than band spectra for obtaining vibrational and
rotational frequencies of molecules is through observation of the Raman Effect.
• A valuable adjunct to electronic and infrared spectra is the Raman Effect,
predicted theoretically by A. Smekal (1923) and discovered by Sir
Chandrashekhar Venkata Raman (1928).
• According to Raman, if any substance, gaseous, liquid or even solid is exposed to
radiation of definite frequency, then the light scattered at right angles contains
frequency different from the incident radiation, and is characteristic of the
substance under examination.
Basic Principle
The Raman effect is based on the scattering of light, which consists of elastic
scattering at the same wavelength as the incident light and inelastic light at
different wavelengths due to molecular vibrations. The elastic scattering is
Rayleigh scattering and inelastic scattering is Raman scattering.
Thus, the phenomena due to which the scattering light has a slightly different
frequency from that of incident light and there is a change in the atomic
oscillations within the molecules, it is called Raman Effect.
When scattering of light is observed with the help of a spectrophotometer, it is
found that spectrum (Raman Spectra) is made of lines of the incident light
together with a series of light on either side of them.
In the spectra of scattered
radiation, the Raman lines appear
in the spectrum on either side of
Rayleigh Scattering (spectral
lines having same frequency as
incident ray frequency), with
higher as well as lower
frequencies.

The Raman lines having

frequencies lower than the
incident frequency is called
Stokes lines and the lines having NOTE: Stokes lines are always more intense than Anti-Stokes lines and
frequencies higher than the Rayleigh scattering is much more powerful than either Stokes or Anti-Stokes
line.
incident frequency are called
Anti-Stokes lines.
The Raman
Spectrophotometer

A LASER source is needed to

excite the target species.
A filter collects the Raman scattered
light (Stokes) and filters out the
Raleigh and Anti Stokes light.
A diffraction grating bends the
Raman shifted light according to
wavelength.
A detector records the signal and
passes the signal to a computer for
decoding.
Applications of Raman spectroscopy

Raman Spectroscopy is an important tool for directly detecting the

frequencies of IR inactive vibrations and for solving the intricate
research problems concerning the constitution of compound.
The technique can be applied to investigate bond angles, structure,
ionic equilibria, nature of bonding, degree of dissociation of strong
electrolytes and the corresponding activity coefficients.
1. Applications in Inorganic Chemistry
Elucidation of Molecular Structure
It helps in finding the structure of molecule and complex ion from the activity
and depolarisation ratios of Raman band.
For example, XY₃ system can either be trigonal or pyramidal. Out of the six
modes of vibrations, only four are observable, since the two fundamental modes
are doubly degenerate.
From the activity of Raman bands listed in the table below, we can decide the
structure of XY₃ systems.

For example in SO₃, four observable modes of vibrations occur at 1332, 1068, 530
cm (Raman active) and 653 cm This confirms planar or trigonal structure of
-1 -1.

SO3. On the other hand, in the case of NH3 out of its four Raman observable
bands, viz., 3337, 950, 3414, 1628 cm , only two bands appear in the spectrum.
-1

The fact that the remaining degenerate vibrations have not been observed in
Raman Spectrum is
in agreement with the weakness of Raman lines corresponding to non-totally
symmetric vibrations. So NH3 has pyramidal structure.
To distinguish between linear and bent molecules
The vibrational Raman spectrum has been used to distinguish between linear
(CS2, HF2 , NO3 , ZnCl2) and bent (H20, H2S, NO2, OF2) structures.
- +

Identification of bonds
Consider homonuclear and heteronuclear molecules.
• The heavier the molecule, the lower is its vibrational frequency.
• Force constant may be divided into the ratio of 3:2:1, showing the presence
of triple, double and single bond between the atoms in diatomic molecules.
Nature of bonding in complexes
Totally symmetric vibrations of the tetrahedral complexes, ML4 and the
octahedral complexes, ML6 are Raman active only.
The Force constant for M-L bond can be estimated from the frequency of
totally symmetric vibration using the equation,
The high values of force constants for PO (5.46), SO4 (6.04), etc as
4
3- 2-

compared to those of ZnCl4 (1.02), SF6 (4.21), etc., indicate the presence of
2-

dπ-pπ bond between the central atom and oxygen atom in addition to σ bond in
the former systems.
Oxyacids of phosphorus
Raman spectra of anhydrous phosphorus and hypophosphorus acids show
frequencies corresponding to the P—H and O—H groups.
Analysis of the Raman spectra of these acids also reveal the presence of PO4 3-

which agrees well with the tetrahedral structure.

2. Applications in Organic Chemistry
Raman spectrum is a molecular spectrum and so it provides a wealth of
informations regarding the
• Constitution of organic compounds
• Presence or absence of specific linkages in a molecule
• Presence of impurities in dyes
• Study of isomers
• Classification od compounds
• Analysis of compounds
Raman lines generally lie in the region 500-3500 cm-1
Fixing the structures of simple molecules eg., Benzene
Bezene exhibits two strong Raman lines at 997 and 1050 cm-1 due to C-C and C=C
linkages respectively. This supports Kekule formula.
Determination of location of groups on a Benzene ring
Raman spectroscopy has been used to decide the position of substituent in benzene
derivatives. For examples, all meta-substituted benzene exhibit an intense, strongly
polarised line at 995 cm-1 which is not present in ortho- and para- compounds.
Ortho substituent can be distinguished from para-form by having a richer spectrum
while para isomer exhibits a line at 625cm-1.
Determination of geometrical isomerism
It is known that cis and trans isomers of 1,2-dichloroethylene exist in equilibrium
proportions. Out of them, trans configuration has centre of symmetry. Thus, the
coincident frequencies observed in IR and Raman spectra
of the sample can be assigned to cis configuration, thereby analysing the
mixture.

Mechanism of Tautomerism
All carbonyl compounds containing an α-H atom exist as keto and enol
tautomers. The existence of tautomeric forms is confirmed by Raman
spectroscopy.
Consider ethyl acetoacetic ether. In this compound, the appearance of two
Raman lines at 1632 and 1725 cm-1 are characteristic of ethylene bond. It
shows the existence of enolic form in it. These two lines are not present in
ethyl dimethyl acetoacetate because of the absence of α-H and ethylenic
bond in it.
Determination of conformational equilibria
Consider conformational equilibrium of mono-substituted cyclohexane.

The frequencies of the Raman band due to C—X depends on the fact whether X is
equatorial or axial. They differ in steric interactions with adjacent protons. It has been
observed that
vc—x (eq) > vc—x (axial)
It is also known that intensity of Raman lines is directly proportional to the amount of the
scattering species. That is, Ieq ∝ Ceq and also

Hence, by monitoring the intensities of vc—x bands corresponding to two conformations the
value of conformational equilibrium constant (k) can be determined.
Carbon Allotropes
Raman spectroscopy is an ideal tool for examining the geometric structure and determining
the bonding type within the carbon molecules. Because of its highly sensitive nature, it
gives an insight into carbon allotropes like fullerenes and graphene structures. Raman
spectra can also reveal the doping and stress in these allotropes that might be caused by
temperature or pressure changes.
 3. Applications in Physical Chemistry
Many problems of physical chemistry such as electrolytic dissociation, hydrolysis,
transition from crystalline to amorphous state etc. have been studied by making use of
Raman effect.
• The amorphous state of a substance gives rise to broad and diffused bands while
crystalline state gives fines sharp Raman lines.
• The degree of hydrolysis of a salt can be determined by measuring the relative
intensities of a set of lines characteristic of the base, the acid or the salt.
• In case of the phenomenon of electrolytic dissociation, the intensity of Raman lines
enables us to determine the number and nature of ions produced. Thus we can
decide whether the dissociation is complete or partial.
Study of Ionic Equilibria
Consider the following equilibria

Where k is dissociation constant of the acid. It is found that the scattering

intensity is directly proportional to the concentration of the scattering
species. Hence,

The value of k can be found by simply monitoring the scattering intensities

of the vibrational bands of X⁻ and HX respectively.
Semi-conductors
• Structure analysis: Raman spectroscopy gives detailed analysis of the geometry and
orientation of their structure.
• Any kind of stress and strain in the molecules of semiconductors can be detected by
observing the shift in the position of Raman band.
• To identify the contamination: here is a high possibility of contamination of
semiconductors due to various reasons. Raman spectroscopy provides the
information-rich Raman spectra that can be used to locate the contaminant’s
locations, and it also helps in identifying the source of contamination.
 4. Biological Applications
Raman spectroscopy is used in the identification of biochemical molecules, drugs,
proteins, metabolic intermediates and substrates.
Monitoring the contents of Proteins, Lipids, and Nucleic acid
Raman spectra provide more detailed information
about various biomolecules like proteins, lipids,
and nucleic acid than the infrared spectra. Raman
spectra bands are sharper while the infrared
spectra bands are broader, so we get better results
by using Raman spectroscopy. Hence, Raman spectroscopy is more beneficial for the
detailed analysis of complex biomolecules.
Identification and Classification of Cells
Raman Spectroscopy plays a significant role in the identification and classification of
cells as it uses laser beams that generate unique signals at various wavelengths that
can be further used to identify specific molecules like nucleic acid, protein, sugar, etc.
This process is accomplished by bombarding
a laser beam on a single cell and depending
on its nature, the cell may absorb, reflect, or
scatter the light waves that eventually
generate a pattern known as Raman spectra;
this is often termed as the signature or
fingerprints of the cells.
Bio-sensing
The changes in the behaviour of a cell that occurs upon its interaction with any toxic
agent can be observed with the help of Raman Spectroscopy. Different toxic agents
have different effects on the living cells, which results in the different Raman spectra.
The more advanced versions of Raman spectroscopy like Surface-Enhanced Raman
Spectroscopy (SERS) and Coherent Anti-Stokes Raman Spectroscopy (CARS) are
also used nowadays for analyzing living cells. These techniques enhance the Raman
signal of the chemical bonds present in cells and are widely used in the development
of sensors and biosensors.
Stem Cell Analysis
Stem Cell therapy is a widely used medical technology that can be used to cure
many chronic diseases like diabetes, Parkinson’s disease, and cardiovascular
diseases. This therapy process involves the replacement of the damaged cells and
tissues with healthy ones. This technology uses Raman spectroscopy to get the
fingerprints of the cells because different cells are specialized to perform specific
functions, which, with the help of Raman micro-spectroscopy, can be detected
easily.
To Diagnose Diseases
With the help of Raman spectroscopy, diseases can be easily diagnosed as the
molecular composition of healthy cells is different from that of diseased ones.
Raman spectroscopy is capable of diagnosing both the tissue samples, i.e., healthy
and diseased tissue samples. Each chemical bond of the sample molecule has
different vibrational frequencies, hence their fingerprints are also unique, and this
fingerprint help in diagnosing the ailment in living cells.
 5. Applications in Food Analysis
• Determination of fat in milk powder.
• Conjugated Linoleic acids (CLA) determination in cow’s milk by FT-Raman
spectroscopy.
• Determination of meat and fish quality parameters, including protein, fat and so
on. Also to check effect of external condition changes on them.
• Raman spectroscopy can be used in quality and security control of beverages.
• To detect and characterise pesticides or fungicides that are extracted from the
surfaces of fruits.
• SERS coupled with nanosubstrates to detect the presence of bacteria.
 6. Some Other Applications
Ceramics and Gemstones
The Raman spectra of gems help gemologists to determine their origin and chemical
properties by comparing them with the library spectra of gems obtained from various
sources.
Early Drug Development
It helps pharmacists design innovative drugs by doing preformulation studies that include
the physical, chemical, and analytical analysis of candidate drugs and develop required
drugs in the early stages.
Cosmetic Analysis
Nowadays, cosmetologists use the technology of Raman spectra to analyze their
cosmetics product’s permeability that how easily do the particular products absorb
through the skin. The better the permeability, the better is the product.
Drug Supply Surveillance
The technique of Raman spectroscopy is effectively used by police and various
surveillance agency to check the illegal moment of banned substances as Raman
spectra can easily distinguish between two similar-looking white powders, which
might look exactly the same to the human eye.
Process Analytical Technology
Raman Spectroscopy is a very important PAT tool in pharma-Industries. PAT is a
mechanism that includes designing, analyzing, and controlling pharmaceutical
manufacturing processes by measuring the critical parameters that affect the
quality of an active pharmaceutical ingredient (API).
Environmental Pollution Analysis
Raman spectrometer permits 3-Dimensional mapping of air pollutants as a function
of distance. The greatest advantage is the continuous monitoring of the quality of
air.
THANK YOU!
and have a great day ahead.