Lecture Date: February 11th, 2013
Raman Spectroscopy
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
Polarization changes are necessary to form the virtual state and hence the Raman effect
This figure depicts “normal” (spontaneous) Raman effects
H. A. Strobel and W. R. Heineman, Chemical Instrumentation: A Systematic Approach, 3rd Ed. Wiley: 1989.
hv1
Scattering timescale ~10-14 sec(fluorescence ~10-8 sec)
Virtual state
Virtual state
hv1
Ground state(vibrational)
Incident radiation excites “virtual states” (distorted or polarized states) that persist for the short timescale of the scattering process.
Excited state(vibrational)
hv1 – hv2
Stokes line
hv1 – hv2
Anti-Stokes line
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):
)()()( ttt
P. W. Atkins and R. S. Friedman, Molecular Quantum Mechanics, 3rd Ed. Oxford: 1997.
EM fieldInduced dipole moment
Expanding this product yields:
tttt )cos()cos(cos)( intint041
0
Rayleigh line Anti-Stokes line Stokes line
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:
ttt coscos)( 0int21
mean polarizability = max - min
The Raman Spectrum of CCl4
Figure is redrawn from D. P. Strommen and K. Nakamoto, Amer. Lab., 1981, 43 (10), 72.
Observed in “typical” Raman
experiments
0 = 20492 cm-1
0 = 488.0 nm
Anti-Stokes lines(inelastic scattering)
-218
Raman shift cm-1 0 = (s - 0)
-200
Stokes lines(inelastic scattering)
-400400 200
218314
-314
-459
459
0
Rayleigh line(elastic scattering)
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:
IIp
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:
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)
Sample WavelengthSelector
DetectorInGaAs or Ge
Radiationsource
(90° angle)
Inteferometers for FT-IR and FT-Raman
The Michelson interferometer, the product of a famous physics experiment:
Produces interference patterns from monochromatic and white light
Figures from Wikipedia.org
A Typical FT-Raman System
Horizontal stage for high-throughput, video controlled micro and macro-sampling
The Thermo Nicolet 960 FT-Raman system
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
250C, 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 CompoundThe ATR FTIR and FT-Raman (1064 nm laser) spectra of flufenamic acid (an analgesic/anti-inflammatory drug):
CF3
O OH
FT-IR Flufenamic acid Aldrich as recd
0.05
0.10
0.15
0.20
0.25
0.30
Abs
FT-Raman Flufenamic acid Aldrich as recd
0
10
20
30
40
50
60
Int
500 1000 1500 2000 2500 3000 3500 Raman shift (cm-1)
IR and Raman Spectra of an Organic CompoundThe ATR FTIR and FT-Raman (1064 nm laser) spectra of flufenamic acid (an analgesic/anti-inflammatory drug):
CF3
O OH
FT-IR Flufenamic acid Aldrich as recd
0.05
0.10
0.15
0.20
0.25
0.30
Abs
FT-Raman Flufenamic acid Aldrich as recd
0
10
20
30
40
50
60
Int
200 400 600 800 1000 1200 1400 1600 Wavenumbers (cm-1)
Note – materials usually limit IR
in this region
IR and Raman Spectra of an Organic CompoundThe ATR FTIR (blue) and FT-Raman (red, 1064 nm laser) spectra of a
crystalline polymorph of the drug tranilast:ATR FTIR Tranilast Form IFT-Raman Tranilast Form I
50
100
150
200
250
300
350
400
450
500
Int
500 1000 1500 2000 2500 3000 3500 Raman shift (cm-1)
O
O
NH
O
OHO
C1C6C2
C3C4
C5
C7
N1C8
C9C10
C11
C12C13
C14
C15C16
C17
C18
H3C
H3C
O4
O5
O3
O2 O1
Confocal Raman Microscopy Instrumentation
Am. Pharm. Rev., 13, 58-65 (2010).
Combines a confocal microscope (discussed later in class) with a Raman spectrometer
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
Am. Pharm. Rev., 2010, 13, 58-65.
levoflaxacin microcrystalline cellulose
Mapping a Cross-sectioned Drug-coated Sphere-120
-100
-80
-60
-40
-20
020
40
60
80
100
120
140
160
Y (µ
m)
-100 -50 0 50 100 150X (µm)
10 µm
-120
-100
-80
-60
-40
-20
0
20
40
60
80
100
120
140
160
Y (µ
m)
-100 -50 0 50 100 150X (µm)
10 µm
-140
-140
10 20 30 40 50 60Points
Poi
nts
10
20
30
40
50
60
(a)
(b)
-800
-700
-600
-500
-400
-300
-200
-100
0
100
200
300
400
500
600
700
800
Y (µ
m)
-1 000 -800 -600 -400 -200 0 200 400 600 800 1 000X (µm)
50 µm
Dried enteric coating(Eudragit L30-D55)
Sucrose sphere
rPTH(1-31)NH2 API
3500 3000 2500 2000 1500 1000 500
Raman shift (cm-1
Anal. Chem. 2012, 84, 4357-4372.
Mapping with Confocal Raman Microscopy
z = -50m
z = -25m
z = 0m
z = 25m
z = 50m
1000 800Raman shift (cm-1)
600 400120014001600
Anal. Chem. 2012, 84, 4357-4372.
Polymer (outer)
Drug layer
Sucrose core
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/Introduction-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
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