Ramya.

Raman spectroscopy
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Energy-level diagram showing the states involved in Raman signal. The line thickness is roughly
proportional to the signal strength from the different transitions.
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]
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. 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.
Spontaneous Raman scattering is typically very weak, and as a result the main difficulty 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 of choice for dispersive Raman setups, which resulted in long acquisition times.
However, modern instrumentation almost universally employs notch or edge filters for laser
rejection and spectrographs either axial transmissive (AT), CzernyTurner (CT) monochromator,
or FT (Fourier transform spectroscopy based), and CCD detectors.
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 offset Raman, and hyper Raman.
Contents
1 Theoretical basis
2 History
3 Raman shift
4 Applications
5 Microspectroscopy
6 Polarized analysis
7 Variations
8 See also
9 References
10 External links
Theoretical basis[edit]
The Raman effect occurs when light impinges on 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. When
the molecule relaxes it emits a photon and it returns to a different rotational or vibrational 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.
If the final vibrational state of the molecule is more energetic than the initial state, the emitted
photon will be shifted to a lower frequency for the total energy of the system to remain balanced.
This shift in frequency is designated as a Stokes shift. If the final vibrational state is less
energetic than the initial state, then the emitted 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 transfer between the photons and the molecules during their interaction.
A change in the molecular polarization potentialor amount of deformation of the electron
cloudwith respect to the vibrational coordinate is required for a molecule to exhibit a Raman
effect. The amount of the polarizability change will determine the Raman scattering intensity.
The pattern of shifted frequencies is determined by the rotational and vibrational states of the
sample. This dependence on the polarizability differs from Infrared spectroscopy where the
interaction between the molecule and light is determined by the dipole moment; this contrasting
feature allows one to analyze transitions that might not be IR active via Raman spectroscopy, as
exemplified by the rule of mutual exclusion in centrosymmetric molecules.
History[edit]
Although the inelastic scattering of light was predicted by Adolf Smekal in 1923,
[2]
it is not until
1928 that it was observed in practice. The Raman effect was named after one of its discoverers,
the Indian scientist Sir C. V. Raman who observed the effect 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 accomplished
using sunlight, a narrow band photographic filter to create monochromatic light, and a "crossed
filter" to block this monochromatic light. He found that a small amount of light had changed
frequency and passed through the "crossed" filter.
Systematic pioneering theory of the Raman effect was developed by Czechoslovak physicist
George Placzek between 1930 and 1934.
[3]
The mercury arc became the principal light source,
first with photographic detection and then with spectrophotometric detection.
In the years following its discovery, Raman spectroscopy was used to provide the first 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 filtered monochromatic light generated by a gas
discharge lamp. The photons that were scattered by the sample were collected through an optical
flat 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 simplified Raman spectroscopy
instruments and also boosted the sensitivity of the technique. This has revived the use of Raman
spectroscopy as a common analytical technique.
Raman shift[edit]
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:

where 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 expressing wavenumber
in Raman spectra is inverse centimeters (cm
1
). Since wavelength is often expressed in units of
nanometers (nm), the formula above can scale for this unit conversion explicitly, giving

Applications[edit]
Raman spectroscopy is commonly used in chemistry, since vibrational information is specific to
the chemical bonds and symmetry of molecules. Therefore, it provides a fingerprint by which the
molecule can be identified. For instance, the vibrational frequencies of SiO, Si
2
O
2
, and Si
3
O
3

were identified and assigned on the basis of normal coordinate analyses using infrared and
Raman spectra.
[4]
The fingerprint region of organic molecules is in the (wavenumber) range 500
2000 cm
1
. Another way that the technique is used is to study changes in chemical bonding, as
when a substrate is added to an enzyme.
Raman gas analyzers have many practical applications. For instance, they are used in medicine
for real-time monitoring of anaesthetic and respiratory gas mixtures during surgery.
In solid-state physics, spontaneous Raman spectroscopy is used to, among other things,
characterize materials, measure temperature, and find the crystallographic orientation of a
sample. As with single molecules, a given solid 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 superconducting
gap excitations. The spontaneous Raman signal gives information on the population of a given
phonon mode in the ratio between the Stokes (downshifted) intensity and anti-Stokes (upshifted)
intensity.
Raman scattering by an anisotropic crystal gives information on the crystal orientation. The
polarization of the Raman scattered light with respect to the crystal and the polarization of the
laser light can be used to find the orientation of the crystal, if the crystal structure (to be specific,
its point group) is known.
Raman spectroscopy is the basis for Distributed Temperature Sensing (DTS) along optical fibers,
which uses the Raman-shifted backscatter from laser pulses to determine the temperature along
optical fibers.
Raman active fibers, such as aramid and carbon, have vibrational modes that show a shift in
Raman frequency with applied stress. Polypropylene fibers 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 offset 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 knowledge of the social and economic conditions at the time the documents were
produced.
[6]
This is especially helpful because Raman spectroscopy offers a non-invasive way to
determine the best course of preservation or conservation treatment for such materials.
Several research projects demonstrated usage of Raman spectroscopy as a means to detect
explosives using laser beams from safe distance (Portendo, 2008,
[7]
TU Vienna, 2012
[8]
).
Raman spectroscopy has also been used to confirm the prediction of existence of low-frequency
phonons
[9]
in proteins and DNA (see, e.g.,
[10]

[11]

[12]

[13]
) greatly stimulating the studies of low-
frequency collective motion in proteins and DNA and their biological functions.
[14][15]

Raman reporter molecules with olefin or alkyne moieties are being developed to allow for tissue
imaging with SERS-labeled antibodies.
[16]

Microspectroscopy[edit]

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Raman spectroscopy offers several advantages for microscopic analysis. Since it is a scattering
technique, specimens do not need to be fixed or sectioned. Raman spectra can be collected from
a very small volume (< 1 m in diameter); these spectra allow the identification of species
present in that volume. Water does not generally interfere with Raman spectral analysis. Thus,
Raman spectroscopy is suitable for the microscopic examination of minerals, materials such as
polymers and ceramics, cells, proteins and forensic trace evidence. A Raman microscope begins
with a standard optical microscope, and adds an excitation laser, a monochromator, and a
sensitive detector (such as a charge-coupled device (CCD), or photomultiplier tube (PMT)). FT-
Raman has also been used with microscopes. Ultraviolet microscopes and UV enhanced optics
must be used when a UV laser source is used for Raman microspectroscopy.
In direct imaging, the whole field of view is examined 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.
The other approach is hyperspectral imaging or chemical imaging, in which thousands of Raman
spectra are acquired from all over the field of view. The data can then be used to generate images
showing the location and amount of different 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, buffers, and other interferents.
Raman microscopy, and in particular confocal microscopy, has very high spatial resolution. For
example, the lateral and depth resolutions were 250 nm and 1.7 m, respectively, using a
confocal Raman microspectrometer with the 632.8 nm line from a heliumneon laser with a
pinhole of 100 m diameter. Since the objective lenses of microscopes focus the laser beam to
several micrometres in diameter, the resulting photon flux is much higher than achieved in
conventional Raman setups. This has the added benefit of enhanced fluorescence quenching.
However, the high photon flux can also cause sample degradation, and for this reason some
setups require a thermally conducting substrate (which acts as a heat sink) in order to mitigate
this process.
By using Raman microspectroscopy, in vivo time- and space-resolved Raman spectra of
microscopic regions of samples can be measured. As a result, the fluorescence of water, media,
and buffers 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 detectors have become available,
making the technique better suited to general use. Raman microscopy of inorganic specimens,
such as rocks and ceramics and polymers, can use a broader range of excitation wavelengths.
[17]

Polarized analysis[edit]

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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.
The spectral information arising from this analysis gives insight into molecular orientation and
vibrational symmetry. In essence, it allows the user to obtain valuable information relating to the
molecular shape, for example in synthetic chemistry or polymorph analysis. It is often used to
understand macromolecular orientation in crystal lattices, liquid crystals or polymer samples.
[18]

It is convenient in polarised Raman spectroscopy to describe the propagation and polarisation
directions using Porto notation, described by and named after Brazilian physicist Sergio Pereira
da Silva Porto.
Variations[edit]
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 10
11
). This effect was originally observed by Martin
Fleischmann but the prevailing explanation was proposed by Van Duyne in 1977.
[19]
A
comprehensive theory of the effect was given by Lombardi and Birke.
[20]

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.
[21]

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.
[22]

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.
[23]

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.
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 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.
[24]

Transmission Raman 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)
[25]
but was rediscovered in 2006 as a
means of rapid assay of pharmaceutical dosage forms.
[26]
There are also medical
diagnostic applications.
[27]

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.
[28]

Surface plasmon polariton 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
[29]
(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.