Transcript Raman Spectroscopy - School of Biotechnology

Komal Choudhary
Lecturer
School of Biotechnology
DAVV Indore
 When radiation passes through a transparent medium,
the species present scatter a fraction of the beam in all
directions.
 In 1928, the Indian physicist C. V. Raman discovered
that 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 the
scattering.
 Raman spectroscopy deals with the inelastic scattering
of light and not with its absorption.
 Electric polarizability(α)- it is defined as the ratio of the
induced dipole movement P of an atom to the electric field
E that produce this dipole movement.
 P= αE
 For asymmetric molecules, absorptions will give rise to
both types and virtually the same information could be
obtained from either. However for symmetrical
molecules(CO2,C2H2) having a centre of symmetry, the
fundamental frequencies that appear in the Raman do not
appear in the IR and vice-versa(“Mutual exclusion rule” ).
these two methods are truly complementary.
 For Exp.- the stretching vibration of homonuclear diatomic
molecule(such as H2, O2, N2) which are inactive in
IR(because the dipole movement is zero) are observed in
raman spectra.
 Rayleigh observed that if a substance is irradiated with
monochromatic light the scattered light is observed in
a direction at right angle to the incident light.
 Rayleigh scattering- The frequency of scattered light is
same as the frequency of incident radiation .
 Stokes scattering- The frequency of scattered light is
less than the frequency of incident radiation .
 Antistokes scattering- The frequency of scattered light
is greater than the frequency of incident radiation .
Energy Scheme for Photon Scattering
Energy
Virtual
State
hn0
hn0
hn0
hn0
hn0hnm
hn0+hn
m
E0+hnm
E0
IR
Absorption
Rayleigh
Scattering
(elastic)
Stokes
Scattering
Anti-Stokes
Scattering
Raman
(inelastic)
The Raman effect comprises a very small fraction,
about 1 in 107 of the incident photons.
 Stokes scattering: energy lost by photon:
 — 
(( —  )) 
Photon in
Photon out
No vibration
Vibration
 Anti-Stokes scattering: energy gained by photon:
(( —  ))
—  
Photon in
Photon out
Vibration
No vibration
 But dominant process is elastic scattering:
 Rayleigh scattering
 — 
Photon in
—  
Photon out
No vibration
No vibration
If incident photon energy E; vibration energy v,
then
in terms of energy, photon out has energy:
E-v Stokes scattering
E+v anti-Stokes scattering
E Rayleigh scattering (elastic scattering)
Representation in terms of energy
levels:
 Light source-Mercury arc, four low pressure mercury discharge
tube (increase intensity), laser.
 Sample holder- glass, Quartz
sample used for analysis in Raman spectroscopy may be
liquid(water used for solvent), solid, or gases(sample holder
generally bigger in size) .
 Collection optics- lens and Notch filter(NF)
lens directs the scattered radiation upon the slit of the
spectrograph and the Raman lines are obtained on the
photographic plate .
NF is used to suppress the Rayleigh scattering.
 Spectrograph- it should posses the following characteristics:
I.
It should have large gathering power.
II. Special prisms of high resolving power should be employed.
III. A short focus camera should be employed.
Differences between Raman spectra and IR spectra
Raman spectra
Infrared spectra
It is due to the scattering if light by the
vibrating molecule.
It is due to absorption of light by
vibrating molecule.
Polarizability of the molecule will
decided whether raman spectra will be
observed or not.
Presence of permanent dipole
movement
Water can be used as solvent.
Water can not be used as solvent
because it is opaque to infrared
radiation.
Accurate but not very sensitive.
Accurate and very sensitive.
Optical system are made of glass or
quartz
Optical system are made of special
crystals such as CaF2, NaBr, etc
In Raman effect vibrational frequencies
of large molecules can be measured.
In this, the vibrational frequencies of
large molecules cannot be measured.
As raman lines are weaker in intensity,
concentrated solution must be utilized
to increase the intensity of raman line.
Generally, dilute solution are preferred.
Application
 In inorganic chemistry
structure of CO2 and structure of N2O.
structure of mercurous salts.
 In physical chemistry
The amorphous state of a substance give rise to broad and
diffused bands while crystalline state gives fine sharp line.
The intensity of Raman lines enable us to determine the
number and nature of ion produced by electrolytic
dissociation, therefore, we can decide whether the
dissociation is complete or partial.
 In organic chemistry
it has been observed that each functional group will have
its own characteristic frequency
 In biochemical research Raman Spectroscopy mainly
used for intermediated sized molecule such as drugs,
metabolic intermediates and substrates for exp:
identification of substances such as penicillin and its
derivatives, small peptides and environmental
pollutants. It is an ideal rapid method for measuring
certain contaminants in foodstuffs.
 Use in study of photosynthesis and respiration in
plants, particularly for CO2 metabolism.