RAMAN SPECTROSCOPY

INTRODUCTION

• Raman spectroscopy has become more widely used since the advent of FT-Raman
systems and remote optical fibre sampling.
• Previous difficulties with laser safety, stability and precision have largely been
overcome.
 INTRODUCTION

• It is a spectroscopic technique used to observe vibration, rotational, and other

low frequency modes in a system.
• Commonly used in Chemistry to provide a finger print by which molecules can
be identified.
• It resolves most of limitations of other spectroscopic techniques and provides
both qualitative and quantitative information about the sample being
examined.
• Qualitative analysis involves measurement of the frequency of scattered
radiation, whereas measurement of the intensity of scattered radiation is the
part of quantitative analysis
 WHAT IS RAMAN SPECTROSCOPY
• When radiation pass through a transparent medium the species present scatter a fraction of the
beam in all directions.
• The visible wavelength of a small fraction of the radiation scattered by certain molecules differs
from that of the incident beam and furthermore that the shifts in wavelength depend upon the
chemical structure of the molecules responsible for scattering.
• Hence, scattered light is used to measure the vibrational energy modes of a sample.
• Raman scattering result from the same type of the quantities vibration changed associated with IR
• Can provide both chemical and structural information as well as the identification of substances
through their characteristic Raman fingerprint.
• It extracts this information through the detection of Raman scattering from the sample.
 PRINCIPLE

RAMAN EFFECT

• When a sample is exposed to monochromatic light in the visible region, the sample absorbs light.
• The major portion of the light gets transmitted to the sample
• However, a minute part of the light gets scattered in all directions.
• The scattered light can be observed at right angles to the incident beam.
 PRINCIPLE CONT...

•The incident light has a particular frequency

•When the scattered light has the same frequency as the incident light, then the scattering is called
elastic (energy of scattering particle is conserved) Rayleigh scattering.
•However, it has been noted that (approximately 1 in 10 million photons) of the total scattered intensity
occurs at frequencies different from the incident light, this is called Raman scattering, which is an
inelastic scattering process with a transfer of energy between the molecule and scattered photon.
PRINCIPLE…..

• When light is scattered by a molecule, the oscillating electromagnetic

field of a photon induces a polarisation of the molecular electron cloud
which leaves the molecule in a higher energy state with the energy of the
photon transferred to the molecule.
• This can be considered as the formation of a very short-lived complex
between the photon and molecule which is commonly called the virtual
state of the molecule.
• The virtual state is not stable and the photon is reemitted almost
immediately, as scattered light
 PRINCIPLE….

• If the molecule gains energy from the photon during the scattering
(excited to a higher vibrational level) then the scattered photon loses
energy and its wavelength increases which is called Stokes Raman
scattering (after G. G. Stokes).
• Inversely, if the molecule loses energy by relaxing to a lower vibrational
level the scattered photon gains the corresponding energy and its
wavelength decreases; which is called Anti-Stokes Raman scattering.
• Quantum mechanically Stokes and Anti-Stokes are equally likely
processes. However, with an ensemble of molecules, the majority of
molecules will be in the ground vibrational level (Boltzmann distribution)
and Stokes scatter is the statistically more probable process.
• As a result, the Stokes Raman scatter is always more intense than the
anti-Stokes and for this reason, it is nearly always the Stokes Raman
scatter that is measured in Raman spectroscopy.a
 INSTRUMENTATION

• Laser source
• Sample illumination system
• Suitable spectrometer
INSTRUMENTATION
 SOURCE

Lasers are used because of their high intensity to produce

Raman scattering of sufficient intensity to be measured with a
reasonable signal to noise ratio.
 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.
DIFFERENT TYPES OF RAMAN SPECTROSCOPY

• Resonance Raman Spectroscopy (RRS)

• Surface enhanced Raman Spectroscopy (SERS)
• Micro-Raman Spectroscopy
• Nonlinear Raman Spectroscopic techniques
• Fourier Transform Raman Spectroscopy
 RESONANCE RAMAN SPECTROSCOPY

• The excitation wavelength is matched to an Electronic transition of the

molecule or crystal , so that vibrational modes associated with the
excited electronic state are greatly enhanced.
• This is useful for the studying of large molecules such as polypeptides,
which might show hundreds of bands in conventional spectra.
• Useful in associating normal modes with their observed frequency
shifts.
 SURFACE-ENHANCED RAMAN SPECTROSCOPY
(SERS)

• Normally done in a silver or Gold colloid

• Surface plasmons of silver and gold are excited by the laser,
resulting in an increase in the electric fields surrounding the
metal.
• Given that Raman intensities are proportional to the electric
field, there is large increase in the measured signal (by up to
10^11).
 SURFACE-ENHANCED RESONANCE RAMAN
SPECTROSCOPY (SERRS)

• A combination of SERS and RRS that uses proximity to a surface to increase Raman
Intensity, and excitation wavelength matched to the maximum absorbance of the
molecule being analysed.
CHOHERENT ANTI-STOKES RAMAN SPECTROSCOPY
(CARS)
• Two laser beams are used to generate a coherent anti-stokes frequency beam, which can be
enhanced by resonance
 RAMAN OPTICAL ACTIVITY (ROA)

• Measures vibrational optical activity by mean of sall difference in the intensity of Raman
scattering from chiral molecules in right and left circularly polarised incident light or,
equivalently, a small circularly polarised component in the scattered light
SAMPLE ILLUMINATION SYSTEM
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.
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 pre-treatment.
Gas samples:
• Gas are normally contain in glass tubes, 1-2 cm in diameter and about 1mm thick. Gases can also be sealed in
small capillary tubes
 DATA INTERPRETATION

 Raman Spectra gives molecular fingerprint

 Different molecules have different Raman Spectra
 Rotational levels can be identified from the spectra- thus a particular molecule. This helps in performing qualitative
analysis.
 The concentration is determined from intensity of spectral lines, enabling quantitative analysis
 RAMAN SPECTRUM

• Typical Raman spectrum

• Plot of Signal Intensity vs Raman Shift
• Raman shift, in cm-1 = energy of photon in – energy of photon out
DATA INTERPRETATION
RAMAN SPECTRUM OF CARBON TETRACHLORIDE
MEASURED USING A 532NM LASER
 APPLICATION

• Basically, Raman spectroscopy is complementary to IR spectroscopy, but the sampling is more

convenient, since glass containers may be used and solids do not have to be mulled or pressed
into discs.
• Raman spectra tend to be less cluttered with peaks than infrared spectra. As a consequence,
peak overlap in mixtures is less likely, and quantitative measurements are simpler. In addition,
Raman sampling devices are not subject to attack by moisture, and small amounts of water in a
sample do not interfere.
BIOLOGICAL APPLICATIONS OF RAMAN SPECTROSCOPY

• Biocompatibility • Cell sorting

• DNA/RNA analysis • Characterisation of
• Drug/cell interactions Biomolecules

• Photodynamic therapy • Bone structure analysis

• Metabolic accretions
• Disease diagnosis
• Single cell analysis
PHARMACEUTICAL AND COSMETICS APPLICATION

• Blend uniformity • In vivo analysis and skin depth

profiling
• Compound distribution in tablets
• Crystallinity studies
• High throughput screening
• Polymorphic forms
• API concentration
• Powder content and purity
• Raw material verification
• Contaminant identification
MINEROLOGY AND GEOLOGY APPLICATIONS

• Gemstone and mineral identification

• Fluid inclusions
• Mineral and phase distribution in rock sections
• Phase Transitions
• Mineral behaviour under extreme conditions
 SEMICONDUCTORS

• Characterisation of intrinsic • Doping effects

stress/strain • Photoluminescence micro-analysis
• Purity • Defect analysis
• Alloy composition
• Hetero-structures
• Contamination identification
• Superlattice structure
DATA INTERPRETATION

• CCl4 is a tetrahedral molecule with 3 pronounced Raman active vibrational modes in the 100/cm to 500/cm
wavenumber region
• In the centre of the spectrum is the Rayleigh scatter peak at the laser wavelength
• This peak is million times more intense than the Raman scatter and is therefore, normally blocked by a notch or
edge filter in the Raman spectrometer but was included here for clarity
• Symmetrically placed on either side of the Rayleigh peak are the three stokes and three anti stokes peaks
corresponding to the three most intense Raman active vibrations of CCl4
• It can be seen that the Anti stokes lines are much weaker than the stokes due to the larger population of
molecules in the ground vibrational level of each mode
• CCl4 has one of the simplest Raman spectra but the same principle applies for all samples
• Raman spectroscopy is used to measure the unique vibrational fingerprint of the sample and from that
information, chemical, structural and physical properties can be determined
 ADVANTAGES OF RAMAN SPECTROSCOPY
• The technique is not sensitive to the presence of water or environment vapour
or gas
• Measurements can be performed on opaque or cloudy samples and directly
through glass or plastic containers making sample preparation much simpler
COMPARISON BETWEEN IR AND RAMAN SPECTROSCOPY
 COMPARISON BETWEEN IR AND RAMAN SPECTROSCOPY

RAMAN IR
• Due to scattering of light by the vibrating • Result of absorption of light by vibrating
molecules molecules
• Vibration causes a change in polarizability • Vibration causes change in dipole
• Molecule need not possess a permanent moment
dipole moment • Molecule has to change dipole moment
• Water can be used as a solvent due to the vibration
• Sample preparation in not very elaborate, • Water can not be used
it cab be in any state • Sample preparation elaborate. Gaseous
• Gives an indication of covalent character samples rarely used.
in the molecule • Gives an indication of ionic character
• Instrument cost very high
• Comparatively inexpensive
instrumentation
 COMPARISON CONT….

• Suitable for biological samples

in native state • Less instrument dependent than
• Standard detectors can be Raman because spectra is based on
used measurement of intensity ratio.
• Few intense overtones and • Lower detection limit than Raman
combination bands
• Totally symmetric vibrations
are observable
COMPARISON OF IR AND RS
• Both Raman and IR spectroscopy are based on molecular vibrations as shown in the previous slide

• IR is based on absorption of light energy corresponding to the vibrational energy of molecules

• Raman spectroscopy is based on scattering of incident light at an energy shifted by the vibrational energy (hv) of
the molecule
• Vibrational modes for the same functional groups are observed at the same wave number

• Therefore, both are forms of vibrational spectroscopy

• IR is based on the fact that molecular absorption at specific vibrational frequencies causes a change in the dipole
moment \
• Raman relies on the change in the polarizability of a molecule at the frequencies ( Raman shift) at which the
molecule scatters radiation
• IR is sensitive to hetero-nuclear functional group vibrations and polar bonds especially OH stretching in water

• Raman is sensitive to homo nuclear molecular bonds such as C-C, C=C, N-O, C-H