Raman Spectroscopy
Lecture Date: February 11th, 2013
The History of Raman Spectroscopy
 In 1928, C. V. Raman discovers that small
changes occur the frequency of a small
portion of the light scattered by
molecules. The changes reflect the
vibrational properties of the molecule.
 Raman was awarded the Nobel Prize in
Physics in 1930 for his discovery.
 In the 1970’s, lasers made Raman much
more practical. Near-IR lasers (1990’s)
allowed for avoidance of fluorescence in
many samples. New continuous-wave
(CW) and pulsed laser designs (2000’s)
have allowed for advances in Raman
microscopy and other modes of Raman
spectroscopy (such as CARS and UV
Raman).
C. V. Raman, K. S. Krishnan, Proc. Roy. Soc. London, 1929, 122, 23.
Sir Chandrasekhara Venkata Raman
(www.nobelprize.org)
Rayleigh and Raman Scattering
 Only objects whose dimensions are on the order of ~1-1.5
 will scatter EM radiation (molecules).
 Rayleigh scattering:
– occurs when incident EM radiation induces an oscillating dipole in
a molecule, which is re-radiated at the same frequency
 Raman scattering:
– occurs when monochromatic light is scattered by a molecule, and
the scattered light has been weakly modulated by the
characteristic frequencies of the molecule
 Raman spectroscopy measures the difference between
the wavelengths of the incident radiation and the
scattered radiation.
The Raman Effect
 Incident radiation excites “virtual states” (distorted or
polarized states) that persist for the short timescale of
the scattering process.
 Polarization changes
are necessary to form
the virtual state and
hence the Raman
effect
 This figure depicts
“normal” (spontaneous)
Raman effects
Virtual state
Virtual state
hv1
hv1
hv1 – hv2
Anti-Stokes line
hv1 – hv2
Stokes line
Excited state
(vibrational)
Scattering timescale ~10-14 sec
(fluorescence ~10-8 sec)
H. A. Strobel and W. R. Heineman, Chemical Instrumentation: A Systematic Approach, 3rd Ed. Wiley: 1989.
Ground state
(vibrational)
More on Raman Processes
 The Raman process:
inelastic scattering of a photon
when it is incident on the electrons in a molecule
– When inelastically-scattered, the photon loses some of its energy
to the molecule (Stokes process). It can then be experimentally
detected as a lower-energy scattered photon
– The photon can also gain energy from the molecule (anti-Stokes
process)
 Raman selection rules are based on the polarizability of
the molecule
 Polarizability: the “deformability” of a bond or a molecule
in response to an applied electric field. Closely related to
the concept of “hardness” in acid/base chemistry.
P. W. Atkins and R. S. Friedman, Molecular Quantum Mechanics, 3rd Ed. Oxford: 1997.
More on Raman Processes
 Consider the time variation of the dipole moment induced
by incident radiation (an EM field):
Induced dipole moment
 (t )   (t ) (t )
EM field
polarizability
 If the incident radiation has frequency  and the
polarizability of the molecule changes between min and
max at a frequency int as a result of this rotation/vibration:
 (t )    12  cos intt  0 cos t
mean polarizability
 = max - min
 Expanding this product yields:
 (t )  0 cos t  14 0 cos(  int )t  cos(  int )t 
Rayleigh line
Anti-Stokes line
P. W. Atkins and R. S. Friedman, Molecular Quantum Mechanics, 3rd Ed. Oxford: 1997.
Stokes line
The Raman Spectrum of CCl4
Rayleigh line
(elastic scattering)
0 = 20492 cm-1
0 = 488.0 nm
Stokes lines
(inelastic scattering)
Observed in
“typical”
Raman
experiments
Anti-Stokes lines
(inelastic scattering)
459
314
218
-218
-314
-459
400
200
0
-200
-400
Raman shift cm-1
0 = (s - 0)
Figure is redrawn from D. P. Strommen and K. Nakamoto, Amer. Lab., 1981, 43 (10), 72.
Raman-Active Vibrational Modes
 Vibrational modes that are more polarizable are more
Raman-active
 Examples:
– N2 (dinitrogen) symmetric stretch
 cause no change in dipole (IR-inactive)
 cause a change in the polarizability of the bond – as the bond gets
longer it is more easily deformed (Raman-active)
– CO2 asymmetric stretch
 cause a change in dipole (IR-active)
 Polarizability change of one C=O bond lengthening is cancelled by
the shortening of the other – no net polarizability (Raman-inactive)
 Some modes may be both IR and Raman-active, others
may be one or the other!
The Raman Depolarization Ratio
 Raman spectra are excited by linearly polarized radiation


(laser).
The scattered radiation is polarized differently depending
on the active vibration.
Using a polarizer to capture the two components leads to
the depolarization ratio p:
I
p
I
 The depolarization ratio p can be useful in interpreting the
actual vibration responsible for a Raman signal.
Raman Spectrometers
 The basic design dispersive Raman scattering system:
Sample
Wavelength
Selector
Detector
InGaAs or Ge
(90° angle)
Radiation
source
 Special considerations:
– Sources: lasers are generally the only source strong enough to
scatter lots of light and lead to detectable Raman scattering
– Lasers: He:Cd (441.6 nm), Ar ion (488.0 nm, 514.5 nm), He:Ne
(632.8 nm), Diode (785 or 830 nm), Nd:YAG (1064 nm)
Inteferometers for FT-IR and FT-Raman

The Michelson
interferometer, the
product of a famous
physics experiment:
Figures from Wikipedia.org

Produces
interference
patterns from
monochromatic
and white light
A Typical FT-Raman System
The Thermo Nicolet 960 FTRaman system
Horizontal stage for highthroughput, video controlled
micro and macro-sampling
Raman Sources: Lasers
 Lasers operate using the principle
of stimulated emission
– Stimulated emission is proportional to
the number of atoms in the excited
state (N2), the coefficient B21, and the
energy density E of radiation with
frequency 12
 Electronic population inversion is

required to achieve gain via
stimulated emission (before the
fluorescence lifetime is reached)
Population inversion is achieved by
“pumping” using lots of photons in a
variety of laser gain media
Lasers: The Nd:YAG System
 A typical laser system –
the neodymium-doped
yttrium aluminum garnet
or Nd3+:Y3Al3O12 system
(Nd:YAG)
– YAG is a cubic crystalline
material
 Crystal field splitting
causes electronic energy
level splitting
– 4F3/2 to 4I11/2 level emits
laser radiation
– The four-level system
achieves population
inversion more readily with
less pumping
Lasers and Non-linear Optics
 Non-linear optics (NLO):
at high light intensities, media
can behave such that their dielectric polarization is not
linear in response to the electric field of the light
 Second-harmonic generation (SHG):
two photons are
destroyed, and a single photon with twice the frequency is
created
– Example: a crystal potassium hydrogen phthalate (KHP) doubles
1064 nm laser radition (NIR) into 532 nm (green light)
Lasers in Raman Spectroscopy
 Common lasers used in Raman spectroscopy, plus a few
others of interest in chemistry (see Table 4.1 in Hooker
and Webb):
Laser
Wavelength
Nd:YAG
1064 nm (532 nm and 266 nm with frequency doubled
and quadrupuled systems)
He:Ne
633 nm
Argon ion
488 nm
GaAlAs diode
785 nm
CO2
10600 nm
Ti:sapphire
800 nm
 Key laser performance parameters include the
homogeneous and inhomogeneous linewidths, the
Einstein coefficient (A21), the peak gain cross-section, the
beam propagation factor (M2), …
Modern Raman Spectrometers
 FT-Raman spectrometers – also make use of Michelson
interferometers
– Use IR (1 m) lasers, almost no problem with fluorescence for
organic molecules
– Have many of the same advantages of FT-IR over dispersive
– But, there is much debate about the role of “shot noise” and
whether signal averaging is really effective
 CCD-Raman spectrometers – dispersive spectrometers
that use a CCD detector (like the ICP-OES system
described in the Optical Electronic lecture)
– Raman is detected at optical frequencies!
– Generally more sensitive, used for microscopy
– Usually more susceptible to fluorescence, also more complex
 Detectors - GaAs photomultiplier tubes, diode arrays, in
addition to the above.
Basic Applications of Raman Spectroscopy
 Raman can be used to study aqueous-phase samples
– IR is normally obscured by H2O modes, these happen to be less
intense in Raman
– However, the water can absorb the scattered Raman light and
will damp the spectrum, and lower its sensitivity
 Raman has several problems:
– Susceptible to fluorescence, choice of laser important
– When used to analyze samples at temperatures greater than
250C, suffers from black-body radiation interference (so does
IR)
– When applied to darkly-colored samples (e.g. black), the Raman
laser will heat the sample, can cause decomposition and/or
more black-body radiation
Applications of Raman Spectroscopy
 Biochemistry:


water is not strongly detected in Raman
experiments, so aqueous systems can be studied.
Sensitive to e.g. protein conformation.
Inorganic chemistry: also often aqueous systems.
Raman also can study lower wavenumbers without
interferences.
Other unique examples:
– Resonance Raman spectroscopy: strong enhancement (102 –
106 times) of Raman lines by using an excitation frequency close
to an electronic transition (Can detect umol or nmol of analytes).
– Surface-enhanced Raman (SERS): an enhancement obtained
for samples adsorbed on colloidal metal particles.
– Coherent anti-Stokes Raman (CARS): a non-linear technique
using two lasers to observe third-order Raman scattering – used
for studies of gaseous systems like flames since it avoids both
fluorescence and luminescence issues.
Comparison of IR and Raman Spectroscopy
 Advantages of Raman over IR:
– Avoids many interferences from solvents, cells and sample
preparation methods
– Better selectivity, peaks tend to be narrow
– Depolarization studies possible, enhanced effects in some cases
– Can detect IR-inactive vibrational modes
 Advantages of IR over Raman:
– Raman can suffer from laser-induced fluorescence and
degradation
– Raman lines are weaker, the Rayleigh line is also present
– Raman instruments can be more costly (especially lab systems)
– Spectra are spread over many um in the IR but are compressed
into several nm (20-50 nm) in the Raman
 Final conclusion – they are complementary techniques!
IR and Raman Spectra of an Organic Compound
O
OH
The ATR FTIR and FT-Raman (1064
nm laser) spectra of flufenamic acid (an
analgesic/anti-inflammatory drug):
CF3
FT -IR Fl u fena m ic ac id A ldric h a s re c d
0 .30
0 .25
Abs
0 .20
0 .15
0 .10
0 .05
FT -Ra m a n Fl ufen am ic a c id A ldric h as rec d
60
50
Int
40
30
20
10
0
3 500
3 000
2 500
2 000
Ra m a n s h ift (c m -1)
1 500
1 000
5 00
IR and Raman Spectra of an Organic Compound
O
The ATR FTIR and FT-Raman (1064
nm laser) spectra of flufenamic acid (an
analgesic/anti-inflammatory drug):
OH
CF3
FT -IR Fl u fena m i c ac i d A l dri c h a s re c d
0 .30
0 .25
Note – materials
usually limit IR
in this region
Abs
0 .20
0 .15
0 .10
0 .05
FT -Ra m a n Fl ufen am i c a c i d A l dri c h as rec d
60
50
Int
40
30
20
10
0
1 600
1 400
1 200
1 000
8 00
W ave num bers (c m -1 )
6 00
4 00
2 00
IR and Raman Spectra of an Organic Compound
The ATR FTIR (blue) and FT-Raman (red, 1064 nm laser) spectra of a
crystalline polymorph of the drug tranilast:
ATR FTIR Tr a n ila s t For m I
FT- R a ma n Tr a n ila s t Fo r m I
500
450
O3
C4
O
C5
C3
C6
C2
400
N1
C15
C18
350
C14
H3C
C16
C11
C10
C8
C9
C1
N
H
C12
C13
C7
O
O
O5
300
O2
C17
Int
H3C
O
OH
O1
O4
250
200
150
100
50
3500
3000
2500
2000
Raman s hift (c m-1)
1500
1000
500
Confocal Raman Microscopy Instrumentation
 Combines a confocal microscope (discussed later in
class) with a Raman spectrometer
Am. Pharm. Rev., 13, 58-65 (2010).
Confocal Raman Microscopy Instrumentation
 Multiple lasers and laser switching systems are common
on confocal Raman microscope systems
Mapping a Drug Tablet with Confocal Raman
Microscopy
levoflaxacin
microcrystalline cellulose
Am. Pharm. Rev., 2010, 13, 58-65.
Mapping a Cross-sectioned Drug-coated Sphere
(a)
-800
-700
-140
-120
-100
-600
-80
-60
-500
-40
-400
Y (µm)
-20
-300
-200
0
20
40
Y (µm)
-100
60
0
80
100
100
120
200
140
10 µm
160
300
400
-100
-50
0
X (µm)
50
100
150
-100
-50
0
X (µm)
50
100
150
10
20
30
40
50
60
500
(b)
600
700
-140
-120
-100
50 µm
800
-1 000
-800
-600
-400
-200
0
200
400
600
800
-80
-60
1 000
X (µm)
-40
Y (µm)
-20
0
20
40
60
80
100
rPTH(1-31)NH2 API
120
140
10 µm
160
(c)
10
Dried enteric coating
(Eudragit L30-D55)
Points
20
30
40
Sucrose sphere
50
60
3500
3000
2500
2000
Raman shift
1500
1000
500
(cm-1)
Points
Anal. Chem. 2012, 84, 4357-4372.
Mapping with Confocal Raman Microscopy
Polymer (outer)
z = -50 mm
z = -25 mm
Drug layer
z = 0 mm
z = 25 mm
z = 50 mm
Sucrose core
1600
1400
1200
1000
800
-1
Raman shift (cm )
Anal. Chem. 2012, 84, 4357-4372.
600
400
Hand-held Raman Spectrometers
 Handheld Raman instruments
are useful for the identification
of chemicals
 Designed for safe for use in
manufacturing plant
environment, for military and
chemical weapons
applications, etc…
Hand-held Raman Spectrometers
 Identification of diisopropylethylamine, a commercial
chemical and synthetic reagent
UV and Resonance Raman Spectroscopy
 UV lasers allow for better Raman performance, because
of the 1/4 dependence of scattering, but fluorescence is
a problem
 With lasers in the 245-266 nm region, the Raman
spectrum can be “fit” in the region above the laser but
below the normal Stokes-shifted fluorescence spectrum
UV and Resonance Raman Spectroscopy
 Resonance Raman scattering excites an electronic

transition (e.g. using a UV laser in the 240-270 nm range)
Transitions can achieve 1000x increase in signal
Raman
Resonance Raman
Surface Enhanced Raman Spectroscopy (SERS)
 SERS is a form of Raman spectroscopy that involves a molecule

adsorbed to the surface of a nanostructured metal surface which can
support local surface plasmon resonance (LSPR) excitations
The Raman scattering intensity depends on the product of the
polarizability of the molecule and the intensity of the incident beam; the
LSPR amplifies the beam intensity when the beam is in resonance with
plasmon energy levels – leads to signal enhancements of >106
– Single-molecule detection with SERS has been demonstrated
R. A. Halverson, P. J. Vikesland, Environ. Sci. Technol. 2010, 44, 7749–7755, http://dx.doi.org/10.1021/es101228z
Coherent Anti-Stokes Raman Spectroscopy
(CARS)
 In CARS, the sample is

excited by a probe beam with
frequency pump, a Stokes
beam (Stokes) and a probe
beam (probe)
CARS uses tightly focused
beams delivered via a
microscope to achieve a
phase matching condition
necessary for the coherent
process
 Scanning a sample using a given vibrational resonance
frequency can be used to determine the spatial distribution a
Raman-active vibrational transitions at this frequency
CARS Applications
 CARS is commonly used
to perform rapid chemical
imaging of biological
materials for these
components
– DNA (phosphate
stretching vibration)
– Protein (amide I stretch)
– Water (OH stretch)
– Lipids (CH vibrations –
stretching, bending, etc…)
 Video-rate imaging of
cells has been
demonstrated
C. L. Evans, X. S. Xie, Annu. Rev. Anal. Chem. 2008, 1, 883- 909, http://dx.doi.org/10.1146/annurev.anchem.1.031207.112754
Raman Optical Activity (ROA)
 ROA is a technique that employs circularly polarized



radiation to study chiral molecules
ROA comes in two flavors, scattered circular polarization
(SCP) and incident circular polarization (ICP)
Both right-angle and backscattered configurations are used
Main applications are to chiral analysis and molecular
conformation (including biomolecules)
L. D. Barron, A. D. Buckingham, Chem. Phys. Lett. 2010, 492, 199-213.
Further Reading
Optional but recommended:
J. Cazes, Ed. Ewing’s Analytical Instrumentation Handbook, 3rd Ed., Marcel Dekker, 2005,
Chapter 7.
Optional:
http://www.spectroscopynow.com/raman/details/education/sepspec13199education/Introdu
ction-to-Raman-Spectroscopy-from-HORIBA-Jobin-Yvon.html
D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of Instrumental Analysis, 6th Edition,
Brooks-Cole, 2006, Chapter 18.
D. A. Long, The Raman Effect, Wiley, 2002.
S. Hooker, C. Webb, Laser Physics, Oxford, 2010.
P. W. Atkins and R. S. Friedman, Molecular Quantum Mechanics, 3rd. Ed., Oxford, 1997.
http://www.rp-photonics.com/yag_lasers.html