Raman spectroscopy in chemistry

3. hn
Excited states
Ground
(vibrational)
states
h(n1 - n0 ) h(n1 - n0)
h(n2 - n1) (overtone)
Infrared Absorption and Emission
n1
n2
n0
n3

4. Energy levels in Raman scattering
Rayleigh scattered light undergoes no energy change and corresponds to
the laser wavelength. Stokes scattering, the 'normal' Raman peaks, are
at lower energy relative to the laser (red shift) while anti-Stokes
scattering (usually much weaker) is at higher energy than the laser
wavelength.
excited
states
ground
(vibrational)
states
Rayleigh Stokes anti-Stokes
hn0 hn0 h(n0 - n1) h(n0 + n1 )
hn x
n
1
n
2
n
0
n
3
virtual states

5. • Typically, a sample is illuminated with a laser
beam. Light from the illuminated spot is
collected with a lens and sent through a
monochromator.
• Wavelengths close to the laser line due to
elastic Rayleigh scattering are filtered out
while the rest of the collected light is dispersed
onto a detector.

6. A simple Raman spectrograph
Laser
Sample Spectrograph
Detector
Laser / Rayleigh
Rejection notch filter (10-4 ~ 10-6)

7. • The Raman effect occurs when light impinges upon a
molecule and interacts with the electron cloud and the
bonds of that molecule.
• For the spontaneous Raman effect, which is a form of
light scattering, a photon excites the molecule from the
ground state to a virtual energy state.
•
• The difference in energy between the original state and
this new state leads to a shift in the emitted photon's
frequency away from the excitation wavelength.
• The Raman effect, which is a light scattering
phenomenon, should not be confused with absorption
(as with fluorescence) where the molecule is excited to
a discrete (not virtual) energy level.

8. • If the
, then the
emitted photon will be shifted to a lower
frequency in order for the total energy of the
system to remain balanced. This shift in
frequency is designated as a .
• If the
then the emitted photon
will be shifted to a higher frequency, and this is
designated as an
• Raman scattering is an example of inelastic
scattering because of the energy transfer between
the photons and the molecules during their
interaction.

10. The different possibilities of visual light scattering: Rayleigh scattering (no
exchange of energy so the incident and emitted photons have the same
energy), Stokes scattering (the atom or molecule absorbs energy and the
emitted photon has less energy than the absorbed photon) and anti-Stokes
scattering (the atom or molecule loses energy and the emitted photon has
more energy than the absorbed photon)

12. Raman vs Infrared Spectra

13. Vibrational Spectroscopy
A molecule can be characterised (and identified) based on the position and
intensity of the spectral peaks by either FT/IR or Raman spectroscopy
Absorption
Scattering (“emission”)

14. Raman shift

16. TYPES OF RAMAN SPECTRA
• Several variations of Raman spectroscopy have been developed. The usual purpose is to
enhance the sensitivity (e.g., surface-enhanced Raman), to improve the spatial resolution
(Raman microscopy), or to acquire very specific information (resonance Raman).
• Surface Enhanced Raman Spectroscopy ( SERS) - Normally done in a silver or gold
colloid or a substrate containing silver or gold. 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 1011). This effect was originally observed by Martin Fleischmann
but the prevailing explanation was proposed by Van Duyne in 1977. A comprehensive theory
of the effect was given by Lombardi and Birke.
• 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 studying large molecules such as
polypeptides, which might show hundreds of bands in "conventional" Raman spectra. It is
also useful for associating normal modes with their observed frequency shifts.
• Surface-Enhanced Resonance Raman Spectroscopy (SERRS) - A combination of
SERS and resonance Raman spectroscopy that uses proximity to a surface to increase Raman
intensity, and excitation wavelength matched to the maximum absorbance of the molecule
being analysed.

17. • Angle Resolved Raman Spectroscopy - Not only are standard Raman
results recorded but also the angle with respect to the incident laser. If the
orientation of the sample is known then detailed information about the
phonon dispersion relation can also be gleamed from a single test.
• Hyper Raman - A non-linear effect in which the vibrational modes interact
with the second harmonic of the excitation beam. This requires very high
power, but allows the observation of vibrational modes that are normally
"silent". It frequently relies on SERS-type enhancement to boost the
sensitivity.
• Spontaneous Raman Spectroscopy (SRS) - Used to study the temperature
dependence of the Raman spectra of molecules.
• Optical Tweezers Raman Spectroscopy (OTRS) - Used to study
individual particles, and even biochemical processes in single cells trapped
by optical tweezers.
• Stimulated Raman Spectroscopy - A spatially coincident, two color pulse
(with polarization either parallel or perpendicular) transfers the population
from ground to a rovibrationally excited state, if the difference in energy
corresponds to an allowed Raman transition, and if neither frequency
corresponds to an electronic resonance. Two photon UV ionization, applied
after the population transfer but before relaxation, allows the intra-
molecular or inter-molecular Raman spectrum of a gas or molecular cluster
(indeed, a given conformation of molecular cluster) to be collected. This is
a useful molecular dynamics technique

18. • Spatially Offset Raman Spectroscopy(SORS) - The Raman scattering beneath an obscuring surface
is retrieved from a scaled subtraction of two spectra taken at two spatially offset points
• Coherent 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 means of a small difference
Transmission Raman- in the intensity of Raman scattering from chiral molecules in right- and left-
circularly polarized incident light or, equivalently, a small circularly polarized component in the
scattered light.
• Allows probing of a significant bulk of a turbid material, such as powders, capsules, living tissue, etc.
It was largely ignored following investigations in the late 1960s (Schrader and Bergmann, 1967) but
was rediscovered in 2006 as a means of rapid assay of pharmaceutical dosage forms.There are also
medical diagnostic applications.
• Inverse Raman spectroscopy.
• Tip-Enhanced Raman Spectroscopy(TERS) - Uses a metallic (usually silver-/gold-coated AFM or
STM) tip to enhance the Raman signals of molecules situated in its vicinity. The spatial resolution is
approximately the size of the tip apex (20-30 nm). TERS has been shown to have sensitivity down to
the single molecule level and holds some promise for bioanalysis applications.
• Surface plasmon polaritons enhanced Raman scattering (SPPERS) - This approach exploits
apertureless metallic conical tips for near field excitation of molecules. This technique differs from the
TERS approach due to its inherent capability of suppressing the background field. In fact, when an
appropriate laser source impinges on the base of the cone, a TM0 mode (polaritonic mode) can be
locally created, namely far away from the excitation spot (apex of the tip). The mode can propagate
along the tip without producing any radiation field up to the tip apex where it interacts with the
molecule. In this way, the focal plane is separated from the excitation plane by a distance given by the
tip length, and no background plays any role in the Raman excitation of the molecule

19. Selection rules
• The distortion of a molecule in an electric field, and
therefore the vibrational Raman cross section, is
determined by its polarizability.
• A Raman transition from one state to another, and
therefore a Raman shift, can be activated optically only
in the presence of non-zero polarizability derivative
with respect to the normal coordinate (that is, the
vibration or rotation):
• Raman-active vibrations/rotations can be identified by
using almost any textbook that treats quantum
mechanics or group theory for chemistry. Then, Raman-
active modes can be found for molecules or crystals
that show symmetry by using the appropriate character
table for that symmetry group.

20. Raman Spectroscopy
1 in 107 photons is scattered inelastically
Rotational Raman
Vibrational Raman
Electronic Raman

21. Classical Theory of Raman Effect
mind = aE
polarizability

22. max 0
max max 0
max max 0
( ) cos2
1
cos2 ( )
2
1
cos2 ( )
2
equil
z zz
zz
vib
zz
vib
t E t
d
r E t
dr
d
r E t
dr
m a n
a
 n n
a
 n n
 
  
 
When light interacts with a vibrating diatomic molecule, the induced
dipole moment has 3 components:
Photon-Molecule Interactions
Rayleigh scatter
Anti-Stokes Raman scatter
Stokes Raman scatter

24. max 0
max max 0
max max 0
( ) cos2
1
cos2 ( )
2
1
cos2 ( )
2
equil
z zz
zz
vib
zz
vib
t E t
d
r E t
dr
d
r E t
dr
m a n
a
 n n
a
 n n
 
  
 
Selection rule: v = ±1
Overtones: v = ±2, ±3, …
Raman Scattering
Must also have a change in polarizability
Classical Description does not suggest any difference
between Stokes and Anti-Stokes intensities
1
0
vibh
kT
N
e
N
n



25. Theory of atomic
vibrations and Raman
scattering

26. The simplest real vibrating system:
a diatomic molecule
 212
2
2
2
1
2
21
21
xxK
dt
xd
dt
xd
mm
mm










qK
dt
qd
2
2
m
Reduced mass displacement
x1 x2
m1 m2
K
 t2cosqq m0 n
m
n
K
2
1
mWhere:
Just like
Hooke’s
law: F=kX

27. Scattering of radiation from a diatomic molecule
 t2cosEE 00 n
 t2cosqq m0 n
?
 t2cosEEP 00 naaInduced dipole moment:
For a small amplitude of vibration, the
polarizability a is a linear function of q:







a
aa

q
q 0q
0
     
        t2cost2cosEq
q2
1
t2cosE
t2cosEt2cosq
q
t2cosEP
m0m000
0q
000
00m0
0q
000
nnnn






a
na
nn






a
na


Rayleigh
scattering
Stokes
scattering
Anti-Stokes
scattering


28. Example 1: the vibration modes of CO2
Raman
Active
IR Active
IR Active
        t2cost2cosEq
q2
1
t2cosEP m0m000
0q
000 nnnn






a
na


29. Example 2: the vibration modes of H2O
All the modes are both
Raman & IR Active

30. Normal vibrations of CH2Cl2
n = stretching
d = bending

31. Selection rules
dVxdVI 1i01i0i  m
 a dVxxdVI 1ji01ij0ij
mi ( i = x,y,z ) are the components of the dipole moment.
If one of the integrals Ii  0, than the transition is IR active
aij ( i,j = x,y,z ) are the components of the polarizability tensor.
If one of the integrals Iij  0, than the transition is Raman active
y0 and y1 are the wavefunctions of a ‘molecule’ before and after a
vibrational transition, respectively.




















aaa
aaa
aaa











z
y
x
zzzyzx
yzyyyx
xzxyxx
z
y
x
E
E
E
P
P
P

32. Presentation of Raman Spectra
lex = 1064 nm = 9399 cm-1
Breathing mode:
9399 – 992 = 8407 cm-1
Stretching mode:
9399 – 3063 = 6336 cm-1

33. Mutual Exclusion Principle
For molecules with a center of symmetry are not IR active
These transitions are Raman active.
Symmetric molecules
IR-active vibrations are not Raman-active.
Raman-active vibrations are not IR-active.
O = C = O O = C = O
Raman active Raman inactive
IR inactive IR active

34. Raman vs IR Spectra

35. Raman vs Infrared Spectra

37. Advantages of IR over Raman
• Simpler and cheaper instrumentation.
• Less instrument dependent than Raman spectra because
IR spectra are based on measurement of intensity ratio.
• Lower detection limit than (normal) Raman.
• Background fluorescence can overwhelm Raman.
• More suitable for vibrations of bonds with very low
polarizability (e.g. C–F).

38. Tunable Laser System Versatile Detection System

39. A Typical Raman System

40. Typical geometries for Raman scattring
90o scattering
180o scattering

41. Spectrographs for Raman
Spex 1877 triple
monochromator
Spex 1403/4 double
monochromator

42. Photo-Detectors
Photodiode array detector
Charge coupled device (CCD)

43. Have a good spectrograph!

45. APPLICATIONS OF RAMAN SPECTROSCOPY
Raman Spectra of Inorganic Species
The Raman technique is often superior to infrared for
spectroscopy investigating inorganic systems because
aqueous solutions can be employed. In addition, the
vibrational energies of metal-ligand bonds are
generally in the range of 100 to 700 cm-1, a region of
the infrared that is experimentally difficult to study.
These vibrations are frequently Raman active,
however, and peaks with n values in this range are
readily observed. Raman studies are potentially
useful sources of information concerning the
composition, structure, and stability of coordination

46. Raman Spectra of Organic Species
Raman spectra are similar to infrared spectra in that
they have regions that are useful for functional group
detection and fingerprint regions that permit the
identification of specific compounds. Raman spectra
yield more information about certain types of organic
compounds than do their infrared counterparts.
Biological Applications of Raman Spectroscopy
Raman spectroscopy has been applied widely for the
study of biological systems. The advantages of his
technique include the small sample requirement, the
minimal sensitivity toward interference by water, the
spectral detail, and the conformational and
environmental sensitivity.

47. Quantitative applications
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.
Despite these advantages, Raman spectroscopy
has not yet been exploited widely for
quantitative analysis. This lack of use has been
due largely to the rather high cost of Raman
spectrometers relative to that of absorption

48. Resonance Raman Spectroscopy
Resonance Raman scattering refers to a
phenomenon in which Raman line intensities
are greatly enhanced by excitation with
wavelengths that closely approach that of an
electronic absorption peak of an analyte.
Under this circumstance, the magnitudes of
Raman peaks associated with the most
symmetric vibrations are enhanced by a factor
of 102 to 106. As a consequence, resonance
Raman spectra have been obtained at analyte
concentrations as low as 10-8 M.

50. Resonance Raman Spectroscopy
The most important application of resonance Raman
spectroscopy has been to the study of biological
molecules under physiologically significant conditions;
that is , in the presence of water and at low to
moderate concentration levels. As an example, the
technique has been used to determine the oxidation
state and spin of iron atoms in hemoglobin and
cytochrome-c. In these molecules, the resonance
Raman bands are due solely to vibrational modes of
the tetrapyrrole chromophore. None of the other
bands associated with the protein is enhanced, and at
the concentrations normally used these bands do not

51. Surface-Enhanced Raman Spectroscopy (SERS)
Surface enhanced Raman spectroscopy involves
obtaining Raman spectra in the usual way on samples
that are adsorbed on the surface of colloidal metal
particles (usually silver, gold, or copper) or on roughened
surfaces of pieces of these metals. For reasons that are
not fully understood, the Raman lines of the adsorbed
molecule are often enhanced by a factor of 103 to 106.
When surface enhancement is combined with the
resonance enhancement technique discussed in the
previous section, the net increase in signal intensity is
roughly the product of the intensity produced by each of
the techniques. Consequently, detection limits in the 10-9
to 10-12 M range have been observed.