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