Raman spectroscopy

tion almost universally employs notch or edge lters for

laser rejection and spectrographs either axial transmissive (AT), CzernyTurner (CT) monochromator, or FT
(Fourier transform spectroscopy based), and CCD detectors.

Virtual
energy
states

Vibrational
energy states

Infrared
absorption

Rayleigh
scattering

There are a number of advanced types of Raman spectroscopy, including surface-enhanced Raman, resonance
Raman, tip-enhanced Raman, polarised Raman, stimulated Raman (analogous to stimulated emission), transmission Raman, spatially oset Raman, and hyper Raman.

4
3
2
1
0
Stokes
Anti-Stokes
Raman
Raman
scattering scattering

1 Theoretical basis

Energy-level diagram showing the states involved in Raman signal. The line thickness is roughly proportional to the signal
strength from the dierent transitions.

The Raman eect occurs when electromagnetic radiation impinges on a molecule and interacts with the polarizable electron density and the bonds of the molecule
in the phase (solid, liquid or gaseous) and environment
in which the molecule nds itself. For the spontaneous
Raman eect, which is a form of inelastic light scattering, a photon (electromagnetic radiation of a specic
wavelength) excites (interacts with) the molecule in either the ground rovibronic state (lowest rotational and vibrational energy level of the ground electronic state) or
an excited rovibronic state. This results in the molecule
being in a so-called virtual energy state for a short period of time before an inelastically scattered photon results. The resulting inelastically scattered photon which
is emitted"/"scattered can be of either lower (Stokes)
or higher (anti-Stokes) energy than the incoming photon. In Raman scattering the resulting rovibronic state of
the molecule is a dierent rotational or vibrational state
than the one in which the molecule was originally, before interacting with the incoming photon (electromagnetic radiation). The dierence in energy between the
original rovibronic state and this resulting rovibronic state
leads to a shift in the emitted photons frequency away
from the excitation wavelength, the so-called Rayleigh
line. The Raman eect is due to inelastic scattering and
should not be confused with emission (uorescence or
phosphorescence) where a molecule in an excited electronic state emits a photon of energy and returns to the
ground electronic state, in many cases to a vibrationally
excited state on the ground electronic state potential energy surface.

Raman spectroscopy (/rmn/; named after Sir C. V.

Raman) is a spectroscopic technique used to observe vibrational, rotational, and other low-frequency modes in
a system.[1] Raman spectroscopy is commonly used in
chemistry to provide a ngerprint by which molecules can
be identied.
It relies on inelastic scattering, or Raman scattering, of
monochromatic light, usually from a laser in the visible,
near infrared, or near ultraviolet range. The laser light
interacts with molecular vibrations, phonons or other excitations in the system, resulting in the energy of the laser
photons being shifted up or down. The shift in energy
gives information about the vibrational modes in the system. Infrared spectroscopy yields similar, but complementary, information.
Typically, a sample is illuminated with a laser beam.
Electromagnetic radiation from the illuminated spot is
collected with a lens and sent through a monochromator.
Elastic scattered radiation at the wavelength corresponding to the laser line (Rayleigh scattering) is ltered out,
while the rest of the collected light is dispersed onto a
detector by either a notch lter or a band pass lter.

Spontaneous Raman scattering is typically very weak, and

as a result the main diculty of Raman spectroscopy is
separating the weak inelastically scattered light from the
intense Rayleigh scattered laser light. Historically, Raman spectrometers used holographic gratings and multiple dispersion stages to achieve a high degree of laser rejection. In the past, photomultipliers were the detectors If the nal vibrational state of the molecule is more enerof choice for dispersive Raman setups, which resulted in getic than the initial state, the inelastically scattered pholong acquisition times. However, modern instrumenta- ton will be shifted to a lower frequency for the total energy
1

of the system to remain balanced. This shift in frequency

is designated as a Stokes shift. If the nal vibrational state
is less energetic than the initial state, then the inelastically
scattered photon will be shifted to a higher frequency, and
this is designated as an anti-Stokes shift. Raman scattering is an example of inelastic scattering because of the
energy and momentum transfer between the photons and
the molecules during the interaction. Rayleigh scattering
is an example of elastic scattering, the energy of the scattered Rayleigh scattering is of the same frequency (wavelength) as the incoming electromagnetic radiation.
A change in the molecular electric dipole-electric polarizability with respect to the vibrational coordinate corresponding to the rovibronic state is required for a molecule
to exhibit a Raman eect. The intensity of the Raman
scattering is proportional to the electric dipole-electric
dipole polarizability change. The Raman spectra (Raman
scattering intensity as a function of the Stokes and antiStokes frequency shifts) is dependent on the rovibronic
(rotational and vibrational energy levels of the ground
electronic state) states of the sample. This dependence
on the electric dipole-electric dipole polarizability derivative diers from infrared spectroscopy where the interaction between the molecule and light is determined by the
electric dipole moment derivative, the so-called atomic
polar tensor (APT); this contrasting feature allows one
to analyze transitions that might not be IR active via Raman spectroscopy, as exemplied by the rule of mutual
exclusion in centrosymmetric molecules. Bands which
have large Raman intensities in many cases have weak
infrared intensities and vice versa. For very symmetric
molecules, certain vibrations may be both infrared and
Raman inactive (within the harmonic approximation). In
those instances, one can use a techniquie inelastic incoherent neutron scattering to determine the vibrational frequencies. The selection rules for inelastic incoherent neutron scattering (IINS) are dierent from those of both
infrared and Raman scattering. Hence the three types of
vibrational spectroscopy are complementary, all giving in
theory the same frequency for a given vibrational transion, but the relative intensities giving dierent information due to the types of interaction between the molecule
and the electromagnetic radiation for infrared and Raman
spectroscopy and with the neutron beam for IINS.

RAMAN SHIFT

lter to create monochromatic light, and a "crossed lter" to block this monochromatic light. He found that a
small amount of light had changed frequency and passed
through the crossed lter.
Systematic pioneering theory of the Raman eect was
developed by Czechoslovak physicist George Placzek between 1930 and 1934.[3] The mercury arc became the
principal light source, rst with photographic detection
and then with spectrophotometric detection.
In the years following its discovery, Raman spectroscopy
was used to provide the rst catalog of molecular vibrational frequencies. Originally, heroic measures were required to obtain Raman spectra due to the low sensitivity of the technique. Typically, the sample was held in
a long tube and illuminated along its length with a beam
of ltered monochromatic light generated by a gas discharge lamp. The photons that were scattered by the sample were collected through an optical at at the end of
the tube. To maximize the sensitivity, the sample was
highly concentrated (1 M or more) and relatively large
volumes (5 mL or more) were used. Consequently, the
use of Raman spectroscopy dwindled when commercial
IR spectrophotometers became available in the 1940s.
However, the advent of the laser in the 1960s resulted
in simplied Raman spectroscopy instruments and also
boosted the sensitivity of the technique. This has revived
the use of Raman spectroscopy as a common analytical
technique.

3 Raman shift
Raman shifts are typically reported in wavenumbers,
which have units of inverse length, as this value is directly
related to energy. In order to convert between spectral
wavelength and wavenumbers of shift in the Raman spectrum, the following formula can be used:

(
w =

1
1

0
1

)
,

where w is the Raman shift expressed in wavenumber,

0 is the excitation wavelength, and 1 is the Raman spectrum wavelength. Most commonly, the unit chosen for
Although the inelastic scattering of light was predicted expressing wavenumber in Raman spectra is inverse cenby Adolf Smekal in 1923,[2] it was not until 1928 that it timeters (cm1 ). Since wavelength is often expressed in
was observed in practice. The Raman eect was named units of nanometers (nm), the formula above can scale for
after one of its discoverers, the Indian scientist Sir C. this unit conversion explicitly, giving
V. Raman who observed the eect by means of sunlight
(1928, together with K. S. Krishnan and independently by
Grigory Landsberg and Leonid Mandelstam).[1] Raman
)
(
won the Nobel Prize in Physics in 1930 for this discovery
1
(107 nm)
1
1

.
w(cm
)
=
accomplished using sunlight, a narrow band photographic
0 (nm) 1 (nm)
(cm)

History

Applications

Raman spectroscopy is commonly used in chemistry,

since vibrational information is specic to the chemical
bonds and symmetry of molecules. Therefore, it provides
a ngerprint by which the molecule can be identied.
For instance, the vibrational frequencies of SiO, Si2 O2 ,
and Si3 O3 were identied and assigned on the basis of
normal coordinate analyses using infrared and Raman
spectra.[4] The ngerprint region of organic molecules is
in the (wavenumber) range 5002000 cm1 . Another way
that the technique is used is to study changes in chemical
bonding, as when a substrate is added to an enzyme.

brational modes that show a shift in Raman frequency

with applied stress. Polypropylene bers also exhibit similar shifts. The radial breathing mode is a commonly used
technique to evaluate the diameter of carbon nanotubes.
In nanotechnology, a Raman microscope can be used to
analyze nanowires to better understand the composition
of the structures.

Spatially oset Raman spectroscopy (SORS), which is

less sensitive to surface layers than conventional Raman,
can be used to discover counterfeit drugs without opening their packaging, and for non-invasive monitoring of
biological tissue.[5] Raman spectroscopy can be used to
investigate the chemical composition of historical documents such as the Book of Kells and contribute to knowlRaman gas analyzers have many practical applications. edge of the social and economic conditions at the time the
For instance, they are used in medicine for real-time mon- documents were produced.[6] This is especially helpful
itoring of anesthetic and respiratory gas mixtures during because Raman spectroscopy oers a non-invasive way to
determine the best course of preservation or conservation
surgery.
treatment for such materials.
In solid state chemistry and the bio-pharmaceutical industry, Raman spectroscopy can be used to not only iden- Several research projects demonstrated usage of Raman
tify (ID) active pharmaceutical ingredients (APIs), but in spectroscopy as a means to detect explosives using laser
the case of multiple polymorphic forms, it can also be beams from safe distance (Portendo, 2008,[7] TU Vienna,
used to identify the polymorphic form of the API. For 2012[8] ).[9]
example there are 4 dierent polymorphic forms of the Raman spectroscopy has also been used to conrm the
API (aztreonam) in Cayston, a drug marketed by Gilead prediction of existence of low-frequency phonons [10] in
Sciences for cystic brosis. Both infrared and Raman proteins and DNA (see, e.g., [11] [12] [13] [14] ) greatly stimspectroscopy can be used to identify and characterize the ulating the studies of low-frequency collective motion in
API which is used in the formulation of Cayston. In proteins and DNA and their biological functions.[15][16]
bio-pharmaceutical formulations, one must use not only
the correct molecule, but the correct polymorphic form, Raman reporter molecules with olen or alkyne moias dierent polymorphic forms have dierent physical eties are being developed to allow for tissue imaging with
[17]
Raman spectroscopy has
properties, for example, solubility, melting point, and Ra- SERS-labeled antibodies.
also been used as a noninvasive technique for real-time,
man/infrared spectra.
in situ biochemical characterization of healing wounds
In solid-state physics, spontaneous Raman spectroscopy and multivariate analysis of Raman spectra has enabled
is used to, among other things, characterize materials, a quantitative measure of wound healing progress.[18]
measure temperature, and nd the crystallographic orien- Raman spectroscopy has a wide usage in studies of
tation of a sample. As with single molecules, a given solid biominerals.[19]
material has characteristic phonon modes that can help
an experimenter identify it. In addition, Raman spectroscopy can be used to observe other low frequency excitations of the solid, such as plasmons, magnons, and 5 Microspectroscopy
superconducting gap excitations. The spontaneous Raman signal gives information on the population of a given Raman spectroscopy oers several advantages for
phonon mode in the ratio between the Stokes (down- microscopic analysis. Since it is a scattering technique,
shifted) intensity and anti-Stokes (upshifted) intensity.
specimens do not need to be xed or sectioned. Raman
Raman scattering by an anisotropic crystal gives infor- spectra can be collected from a very small volume (< 1
mation on the crystal orientation. The polarization of the m in diameter); these spectra allow the identication of
Raman scattered light with respect to the crystal and the species present in that volume. Water does not generpolarization of the laser light can be used to nd the orien- ally interfere with Raman spectral analysis. Thus, Raman
tation of the crystal, if the crystal structure (to be specic, spectroscopy is suitable for the microscopic examination
of minerals, materials such as polymers and ceramics,
its point group) is known.
cells, proteins and forensic trace evidence. A Raman miRaman spectroscopy is the basis for distributed temper- croscope begins with a standard optical microscope, and
ature sensing (DTS) along optical bers, which uses the adds an excitation laser, a monochromator, and a sensiRaman-shifted backscatter from laser pulses to determine tive detector (such as a charge-coupled device (CCD), or
the temperature along optical bers.
photomultiplier tube (PMT)). FT-Raman has also been
Raman active bers, such as aramid and carbon, have vi- used with microscopes. Ultraviolet microscopes and UV

7 VARIATIONS

enhanced optics must be used when a UV laser source is tors have become available, making the technique better
used for Raman microspectroscopy.
suited to general use. Raman microscopy of inorganic
can
In direct imaging, the whole eld of view is examined specimens, such as rocks and ceramics and polymers,
[23]
use
a
broader
range
of
excitation
wavelengths.
for scattering over a small range of wavenumbers (Raman shifts). For instance, a wavenumber characteristic
for cholesterol could be used to record the distribution of
cholesterol within a cell culture.

6 Polarized analysis

The other approach is hyperspectral imaging or chemical

imaging, in which thousands of Raman spectra are acquired from all over the eld of view. The data can
then be used to generate images showing the location and
amount of dierent components. Taking the cell culture
example, a hyperspectral image could show the distribution of cholesterol, as well as proteins, nucleic acids, and
fatty acids. Sophisticated signal- and image-processing
techniques can be used to ignore the presence of water,
culture media, buers, and other interference.

The polarization of the Raman scattered light also contains useful information. This property can be measured
using (plane) polarized laser excitation and a polarization
analyzer. Spectra acquired with the analyzer set at both
perpendicular and parallel to the excitation plane can be
used to calculate the depolarization ratio. Study of the
technique is useful in teaching the connections between
group theory, symmetry, Raman activity, and peaks
in the corresponding Raman spectra.[24] Polarized light
only gives access to some of the Raman active modes.
Raman microscopy, and in particular confocal mi- By rotating the polarization you can gain access to the
croscopy, has very high spatial resolution. For example, other modes. Each mode is separated according to its
the lateral and depth resolutions were 250 nm and 1.7 m, symmetry.[25]
respectively, using a confocal Raman microspectrometer
with the 632.8 nm line from a heliumneon laser with a The spectral information arising from this analysis gives
pinhole of 100 m diameter. Since the objective lenses insight into molecular orientation and vibrational symof microscopes focus the laser beam to several microme- metry. In essence, it allows the user to obtain valuable
tres in diameter, the resulting photon ux is much higher information relating to the molecular shape, for example
than achieved in conventional Raman setups. This has the in synthetic chemistry or polymorph analysis. It is often
in crystal
added benet of enhanced uorescence quenching. How- used to understand macromolecular orientation
[26]
lattices,
liquid
crystals
or
polymer
samples.
ever, the high photon ux can also cause sample degradation, and for this reason some setups require a thermally It is convenient in polarised Raman spectroscopy to deconducting substrate (which acts as a heat sink) in order scribe the propagation and polarisation directions using
to mitigate this process.
Portos notation,[27] described by and named after BrazilAnother approach called global Raman imaging[20] uses ian physicist Sergio Pereira da Silva Porto.
complete monochromatic images instead of reconstruction of images from acquired spectra. This technique is
being used for the characterization of large scale devices,
mapping of dierent compounds and dynamics study. It
has already been use for the characterization of graphene
layers,[21] J-aggregated dyes inside carbon nanotubes[22]
and multiple other 2D materials such as MoS2 and WSe2.
Since the excitation beam is dispersed over the whole eld
of view, those measurements can be done without damaging the sample.
By using Raman microspectroscopy, in vivo time- and
space-resolved Raman spectra of microscopic regions of
samples can be measured. As a result, the uorescence of
water, media, and buers can be removed. Consequently
in vivo time- and space-resolved Raman spectroscopy is
suitable to examine proteins, cells and organs.
Raman microscopy for biological and medical specimens
generally uses near-infrared (NIR) lasers (785 nm diodes
and 1064 nm Nd:YAG are especially common). This
reduces the risk of damaging the specimen by applying
higher energy wavelengths. However, the intensity of
NIR Raman is low (owing to the 4 dependence of Raman scattering intensity), and most detectors require very
long collection times. Recently, more sensitive detec-

7 Variations
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 specic 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 elds surrounding
the metal. Given that Raman intensities are proportional to the electric eld, there is large increase in
the measured signal (by up to 1011 ). This eect was
originally observed by Martin Fleischmann but the
prevailing explanation was proposed by Van Duyne
in 1977.[28] A comprehensive theory of the eect
was given by Lombardi and Birke.[29]
Resonance Raman spectroscopy The excitation wavelength is matched to an electronic transi-

5
tion 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.[30]
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.
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
gleaned from a single test.[31]
Hyper Raman A non-linear eect 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.[32]
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
dierence 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 intermolecular 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.
Spatially oset Raman spectroscopy (SORS)
The Raman scattering beneath an obscuring surface
is retrieved from a scaled subtraction of two spectra
taken at two spatially oset 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 dierence 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.[33]
Transmission Raman Allows probing of a signicant 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)[34] but was rediscovered in 2006
as a means of rapid assay of pharmaceutical dosage
forms.[35] There are medical diagnostic applications
particularly in the detection of cancer.[9][36][37]
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 (2030 nm).
TERS has been shown to have sensitivity down to
the single molecule level and holds some promise for
bioanalysis applications.[38]
Surface plasmon polariton enhanced Raman
scattering (SPPERS) This approach exploits
apertureless metallic conical tips for near eld excitation of molecules. This technique diers from
the TERS approach due to its inherent capability
of suppressing the background eld. In fact, when
an appropriate laser source impinges on the base of
the cone, a TM0 mode [39] (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
eld 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.[40][41][42][43]
Micro-Cavity Substrates A method that improves the detection limit of conventional Raman
spectra using micro-Raman in a micro-cavity coated
with reective Au or Ag. The micro-cavity has a
radius of several micrometers and enhances the entire Raman signal by providing multiple excitations
of the sample and couples the forward-scattered
Raman photons toward the collection optics in the
back-scattered Raman geometry.[44]
Stand-o Remote Raman In stando Raman,
the sample is measured at a distance from the Raman spectrometer, usually by using a telescope for
light collection. Remote Raman spectroscopy was
proposed in the 1960s [45] and initially developed
for the measurement of atmospheric gases.[46] The

9
technique was extended In 1992 by Angel et al. for
stando Raman detection of hazardous inorganic
and organic compounds.[47] Stando Raman detection oers a fast-Raman mode of analyzing large
areas such as a football eld in minutes. A pulsed
laser source and gated detector allow Raman spectra measurements in the daylight [48] and reduces
the long-lived uorescent background generated by
transition ions and rare earth ions. Another way
to avoid uorescence, rst demonstrated by Sandy
Asher in 1984, is to use a UV laser probe beam. At
wavelengths of 260 nm, there is eectively no uorescence interference and the UV signal is inherently
strong.[9][49][50] A 10X beam expander mounted in
front of the laser allows focusing of the beam and a
telescope is directly coupled through the camera lens
for signal collection. With the systems time-gating
capability it is possible to measure remote Raman of
your distant target and the atmosphere between the
laser and target.[9]

See also
Raman microscope

References

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Springer-Verlag. ISBN 978-0-387-50254-0.
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[3] Placzek G.: Rayleigh Streuung und Raman Eekt, In:
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[4] Khanna, R.K. (1981).
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[6] Irish classic is still a hit (in calfskin, not paperback) New
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[8] Finding explosives with laser beams, a TU Vienna pressrelease
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Porter, John N. et al. (August 13, 2012). Single-Pulse
Stando Raman Detection of Chemicals from 120 m Distance During Daytime. Applied Spectroscopy 66 (11).

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10

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10

External links

Raman Spectroelectrochemistry This application

note discusses Raman spectroscopy and its combination with electrochemical techniques.
DoITPoMS Teaching and Learning Package Raman Spectroscopy an introduction to Raman spectroscopy, aimed at undergraduate level.
An introduction to recent advances and current areas
of development in biomedical Raman spectroscopy
(including video-rate biomedical imaging). Raman
spectroscopy as a diagnostic tool, Analyst, 2013,
138, 3871-3884
Raman Spectroscopy Raman Spectroscopy News

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