RAMAN

SPECTROSCOPY

Dr. R. D. Senta
Raman Spectroscopy
✓ Introduction
✓ Normal Raman
✓ Resonance Raman
✓ FT – Raman
✓ Dispersive Spectrophotometers
✓ Fourier Transform Spectrometers
✓ Surface – enhanced Raman Spectroscopy (SERS)
✓ Raman Microprobe
✓ Remote Raman Analysis
✓ Raman Depolarization Ratios
✓ Analytical Information
✓ Applications
 Introduction
❑ The vibrational spectroscopies have been largely
employed in the determination of the molecular
structure and played a central role in many areas, from
fundamental chemistry to materials engineering.
❑ Among these techniques, the Raman spectroscopy is
essential today in the study of nanostructured materials
and biological systems in in-situ and in-vivo conditions.
❑ In addition, by intensification process, very small
quantities can be analysed.
❑ Detailed information about the molecular structure can
be obtained, because each vibration of atoms shows a
characteristic position and intensity and are influenced
by the chemical environment or in other words by
chemical bonds, inter and intramolecular forces.
❑ Raman spectroscopy is a molecular spectroscopic technique
that utilizes the interaction of light with matter to gain
insight into a material's make up or characteristics, like FTIR.
❑ The information provided by Raman spectroscopy results from
a light scattering process, whereas IR spectroscopy relies on
absorption of light.
❑ Raman spectroscopy yields information about intra and inter-
molecular vibrations and can provide additional
understanding about a reaction.
❑ Both Raman and FTIR spectroscopy provide a spectrum
characteristic of the specific vibrations of a molecule
("molecular fingerprint') and are valuable for identifying a
substance.
❑ However, Raman spectroscopy can give additional information
about lower frequency modes, and vibrations that give insight
into crystal lattice and molecular backbone structure.
❑ In 1928, Sir C.V. Raman documented the phenomenon
of inelastic light scattering.
❑ Radiation, scattered by molecules, contains photons
with the same frequency as that of the incident
radiation, but may also contain a very small number of
photons with a changed or shifted frequency.
❑ The spectroscopic process of measuring these shifted
photons was later named after Sir Raman, with the
shifting of frequency referred to as the Raman effect
and frequently shifted light as Raman radiation.
❑ By the end of the 1930s, Raman spectroscopy had
become the principle method of non-destructive
chemical analysis.
 Principle
 Raman spectroscopy is a molecular spectroscopy, which is
observed as inelastically scattered light and enables the
interrogation and identification of vibrational (phonon)
states of molecules.
 Therefore, Raman spectroscopy is insensitive to aqueous
absorption bands and requires little to no sample
preparation.
 This characteristic of Raman spectroscopy enables the
measurement of gases, liquids, and solids not only directly,
but also through transparent containers such as plastic,
glass, and quartz.
 When light interacts with molecules in a gas, liquid, or solid,
the vast majority of the photons are dispersed or scattered
at the same energy as the incident photons. This is
described as elastic scattering, or Rayleigh scattering.
❑ When light is irradiated on molecules, the light is
scattered by molecules.
❑ Most of scattered light have the same frequency with
incident light but some fraction of light have different
frequency due to interaction between oscillation of
light (Rhythmic, repetitive change in the intensity
and direction of electric and magnetic field that
constitute a light wave) and molecular vibration.
❑ The phenomenon that the light is scattered with
frequency change is called Raman scattering.
❑ As a result, Raman spectroscopy provides an
invaluable analytical tool for molecular fingerprinting
as well as monitoring changes in molecular bond
structure.
Raman Scattering Process
 The Raman Scattering Process, as described by quantum
mechanics, is when photons interact with a molecule, the
molecule may be advanced to a higher energy, virtual state.
From this higher energy state, there may be a few different
outcomes.
 The difference between the energy of the incident photon and
the energy of the scattered photon is the called the Raman
shift. When the change in energy of the scattered photon is
less than the incident photon, the scattering is called Stokes
scatter.
 Some molecules may begin in a vibrationally excited state
and when they are advanced to the higher energy virtual
state, they may relax to a final energy state that is lower
than that of the initial excited state. This scattering is called
anti-Stokes.
 Two Types of Raman Scattering

Elastic: Collision between energy photon and molecule

results in no change in energy

Inelastic: Collision between energy photon and

molecule results in a net change in energy
✓ Probability of Emission
✓ Observed Intensity
✓ Raleigh scattering >> Stokes >> anti-Stokes
✓ Difference in population of energy levels of vibrational transitions
✓ Intensity of Raman lines are 0.001% intensity of the source
Pure Rotational Raman Spectra

For Linear Molecules

EJ = BJ(J+1) & ∆𝑱 = 𝟎 𝒐𝒓 ∓ 𝟐
EJ = BJ (J+1) - DJ2 (J+1)2 cm-1
IF ∆𝐽 = +2 &J=J+2 then,
EJ = BJ(J+1) -----------------------1
EJ+2 = B(J+2)(J+2+1)
= B(J+2)(J+3)
= B[J2+3J+2J+6] ----------------------2
∆𝑬 = EJ+2 - EJ
= B(J +3J+2J+6)- BJ(J+1)
2

= B[J2+3J+2J+6- J2-J]
= B (4J +6) Cm-1 ----------------------3
J = 0, ∆E0 → 2 = B(4× 0 + 6) =6B
J = 1, ∆E1→ 3 = B(4× 1 + 6) =10B
J = 2, ∆E2 → 4 = B(4× 2 + 6) =14B
J = 3, ∆E3→ 5 = B(4× 3 + 6) =18B
For vibrational Raman spectra
1 1
E = (1 + ) ̅e - (1 + )2 ̅e Xe (= 0, 1, 2,…)
2 2
𝑰𝒇 𝒕𝒓𝒂𝒏𝒔𝒊𝒕𝒊𝒐𝒏 𝒊𝒔 𝒇𝒓𝒐𝒎  = 𝟎 →  = 𝟏, then
∆𝑬 = 𝑬𝟏 − 𝑬𝟎
1 1 2 1 1 2
= (1 + ) ̅e - (1 + ) ̅e Xe - (0 + ) ̅e - (0 + ) ̅e Xe
2 2 2 2
= ̅e (1-2Xe)
 = 𝟎 →  = 𝟐,
∆𝑬 = 𝑬𝟐 − 𝑬𝟎
= 2̅e (1-3 Xe)
 = 𝟎 →  = 𝟑,
∆𝑬 = 𝑬𝟑 − 𝑬𝟎
= 3̅e (1-4Xe)
𝟏 →  = 𝟐,
∆𝑬 = 𝑬𝟐 − 𝑬𝟏
1 1 1 1
= (2 + ) ̅e - (2 + )2 ̅e Xe - (1 + ) ̅e - (1 + )2 ̅e Xe
2 2 2 2
= ̅e (1-4Xe )
 INSTRUMENTATION

Block diagram of a Raman spectrometer.

❑ A Raman spectrometer includes three main components
such as the laser, the sampling interface, and the
spectrometer itself.
❑ A typical Raman laser will consists of different
characteristics, such as a small form factor, low power
consumption, narrow linewidth, a stable power output, and
a stable wavelength output.
❑ In case Raman measurements are conducted using a
785nm source, it is important to ensure that the source is
only emitting 785nm.
❑ The second component includes the sampling interface.
❑ In many Raman spectrometers, fiber-optic probe is
typically used which offers an extremely flexible sampling
interface.
❑ These fiber-optic probes can be easily adapted to a range of
optical microscopes, gas flow cells, liquid flow cells, and
other sampling chambers.
➢ One critical aspect of a fiber-optic probe is a high-optical-
density Raman cut-off.
➢ This means, when users are looking at the Raman
spectrum, they need to ensure that the laser wavelength is
blocked as much as possible so that the Raman shift can
be observed.
➢ It is extremely important that the Raman shift is observed
very close to the laser line since many materials have vital
spectral features very near to the line.
➢ The third component is the spectrometer. Here,
important performance factors are small form factor, high
resolution, low power consumption, and low noise.
➢ An appropriate detector is very important and must be
utilized depending on which excitation laser is being used.
➢ For visible excitation, a standard CCD is selected; for UV
excitation, a photomultiplier tube (PMT) or CCD is
typically chosen; and for NIR excitation, an indium
gallium arsenide (InGaAs) array is normally employed.
Dispersive Raman spectrometer
❑ Dispersive Raman Spectrometer has become the
largest and most widely used of the Raman systems.
The instrument will typically consist of –

❑ Various laser sources- ranging from UV to NIR.

❑ Monochromator (spectrometer or spectrograph),
❑ Grating
❑ Charge transfer devices (CCDs) Detector and
❑ A microscope is often used to enable micron scale
analysis to be undertaken.
To observe the Raman spectrum, it is necessary to
separate the collected Raman scattered light into
individual wavelengths.
In dispersive Raman instruments, this is
accomplished by focusing the Raman signal on a
grating, which spatially separates the different
wavelengths. This spatially dispersed beam is
directed to a CCD.

Light Source
Dispersive Raman usually employs visible laser
radiation.
Typical laser wavelengths are 780 nm, 633 nm,
532 nm, and 473 nm although others are common.
 One advantage of using shorter wavelength lasers is
the enhancement in the Raman signal that occurs at
shorter wavelengths.
 The efficiency of Raman scatter is proportional to 1/λ4,
so there is a strong enhancement as the excitation
laser wavelength becomes shorter.
 This would suggest that all Raman should be done
with the shortest wavelength lasers available.
 However, one factor hindering the practice of Raman
as a routine tool is the unpredictable fluorescence that
often occurs.
 Fluorescence is a very efficient emission several orders
of magnitude stronger than the Raman signal, so
minor fluorescence can overwhelm the desired Raman
measurement.
 Fluorescence occurs when the virtual energy level
overlaps an upper electronic level, so as the energy of
the laser gets higher (shorter wavelength), the
likelihood of fluorescence increases.
➢ A laser source, a sample illumination system
and a suitable spectrometer.
➢ The sources used in modern Raman
spectrometry are nearly always lasers
because their high intensity is necessary to
produce Raman scattering of sufficient
intensity to be measured with a reasonable
signal-to-noise ratio.
➢ Because the intensity of Raman scattering
varies as the fourth power of the frequency,
argon and krypton ion sources that emit in
the blue and green region of the spectrum
have and advantage over the other sources.
➢ Generally a laser to get required intensity of light for
reasonable S/N
➢ Raman scattering is only 0.001% of light source
➢ Doesn’t have to be in IR region, since look at changes
around central peak.
➢ visible source used because of high intensity
➢ Allows use of glass/quartz sample cells & optics.
➢ UV/Vis type detectors (photomultiplier tubes)
Sample Illumination System

➢ Sample handling for Raman spectroscopic

measurements is simpler than for infrared
spectroscopy because glass can be used for windows,
lenses, and other optical components instead of the
more fragile and atmospherically less stable
crystalline halides.
➢ In addition, the laser source is easily focused on a
small sample area and the emitted radiation
efficiently focused on a slit.
➢ Consequently, very small samples can be investigated.
➢ A common sample holder for non absorbing liquid
samples is an ordinary glass melting-point capillary.
Liquid Samples (Fig. b & c):
➢ A liquid can be sealed in ampoules, glass tubes or capillaries.
➢ Capillaries can be as small as 0.5-0.1mmbore and 1mm long.
➢ The spectra of nanoliter volume of sample can be obtained with
capillary cells.
➢ A large cylindrical cell can be used to reduce local heating,
particularly for absorbing samples.
➢ The laser beam is focused to an area near the wall minimize
absorption of the incident beam.
➢ Further reduction of localized heating is often achieved by
orating the cell.
➢ A major advantage of sample handling in Raman spectroscopy
compared with infrared arises because water is a weak Raman
scatterer but a strong absorber of infrared radiation.
➢ Thus, aqueous solutions can be studied by Raman spectroscopy
but not by infrared.
➢ This advantage is particularly important for biological and
inorganic systems and in studies dealing with water pollution
problems.
 Solid Samples (Fig. d):
➢ Raman spectra of solid samples are often acquired by
filling a small cavity with the sample after it has been
ground to a fine powder.
➢ Polymers can usually be examined directly with no sample
pretreatment.
➢ In some cases KBr pellets similar to those used in IR
spectroscopy are employed.
Gas samples (Fig. a):
➢ Gas are normally contained in glass tubes 1-2cm in
diameter and about 1mm thick.
➢ Gases can also be sealed in a small capillary tube.
➢ For weak scatterer an external multiple pass set up with
mirror can be used.
Grating
 The grating has a strong influence on spectral resolution and
instrument throughput.
 Gratings have many lines or grooves “blazed” into the surface,
which disperse the incoming light. The higher the number of
grooves on the grating, the wider the dispersion angle of the
exiting rays.
 It is necessary to have many grooves (for example, 1800 or 2400
lines/mm) for a higher solution spectrum, in which very closely
spaced wavelengths must be distinguished.
 A smaller number of lines are needed (300 or 600 lines/mm) for
a low-resolution spectrum.
 The higher the dispersion of the exiting rays, the larger the
area over which the different wavelengths will lie when
they reach the detector surface. With a fixed detector size,
there is a point (resolution) beyond which not all of the
Raman wavelengths fall on the detector.
In cases of higher dispersion (high resolution), it is
necessary to move either the grating or the detector
to collect sequential regions of the spectrum.
Grating response is also wavelength dependent, so
the dispersion (resolution) across the wavenumber
axis is not linear, but instead, the dispersion
becomes greater at higher wavenumbers (cm-1).
For this reason, spectral resolution must be stated
for a specific wavenumber and will vary across the
spectrum.
Finally, gratings are blazed for optimum
throughput over a relatively narrow wavelength
range, so a grating should be selected for the
desired resolution and for the correct laser
wavelength.
 Using a single grating for more than one laser wavelength
or more than one resolution requires compromises in both
instrument throughput and sensitivity.
 Ideally, gratings should be specifically matched to the laser
and experimental conditions of the experiment.
Detector
➢ The CCDs commonly used for dispersive Raman are silicon
devices with very high sensitivity.
➢ The detecting surface of the CCD is a two-dimensional
array of light-sensitive elements, called pixels (usually each
pixel is <30 μm).
➢ Each pixel acts as an individual detector, so each dispersed
wavelength is detected by a different pixel (or closely
spaced group of pixels).
➢ CCD detectors commonly have a large wavelength response
region, routinely extending from 400 nm up to
approximately 1000 nm.
 Specialized detectors extend the response up to approximately 1100
nm or down into the UV range.
 For dispersive Raman with a 780 nm laser, the 3000 cm-1 response
(corresponding to the C-H stretch region of the spectrum) results in
1018 nm radiation.
 Many common CCDs have very weak responses for the higher
wavenumber response of the NIR laser, and going any higher in laser
wavelength rapidly disqualifies the CCD as a viable detector.
 To get the most from your Raman spectrum, it is important to get the
full 100 – 3100 cm-1 spectrum at all laser wavelengths.
 Most of the current dispersive Raman set-ups are now equipped with
multichannel two-dimensional CCD detectors. The main advantages
of these detectors are:
 The high quantum efficiency.
 The extremely low level of thermal noise (when effectively cooled).
 Low read noise.
 The large spectral range available.
 Many CCD chips exist, but one of the most common
spectroscopy sensor formats is the 1024 x 256 pixel
array.
 TE cooled charge-coupled device (CCD) detector or
“Camera” that allows simultaneous collection of a
wide spectral wavelength range.
 A water cooling option allows -90º C operation.
Thermoelectric (TE) cooling is efficient,
maintenance-free and requires no liquid nitrogen
 TE cooling provides long-term stability at optimum
quantum efficiency
 Longer wavelengths can be detected more efficiently
at higher temperatures than liquid nitrogen cooling.
FT-RAMAN SPECTROMETER
FT-RAMAN is generally confined to routine bulk analysis in recent times - the
system will use a 1064nm laser, an interferometer and a LN2 cooled NIR
Michelson Interferometer
Interferogram
➢FT-Raman was developed to overcome some of
the problems occasionally encountered in
dispersive Raman spectroscopy.
➢One important advantage of FT-Raman lies in
the near lack of sample fluorescence.
➢An FT-Raman instrument typically employs a 1
μm excitation laser, an interferometer and a
high-sensitivity near-infrared detector.
➢By using the longer wavelength excitation laser,
there is less energy supplied, so the virtual state
is lower and less likely to overlap an upper
electronic state.
➢This greatly reduces fluorescence interferences.
➢ Indium gallium arsenide (InGaAs) or liquid nitrogen-
cooled germanium (Ge) detectors are typically used for
FT-Raman spectroscopy.
➢ These detectors are very sensitive, but are still less
sensitive for near-infrared radiation than the silicon
CCD is for visible radiation.
➢ The advantages of the Fourier transform technique are
necessary to provide the sensitivity to extract functional
spectral information from this lower intensity signal.

➢ By virtue of the Fourier transform technique, FT-

Raman offers:
➢ High resolution with minimal throughput loss
➢ Measurement of all wavelengths at once
➢ Increased signal-to-noise by signal averaging
➢ Superior wavelength accuracy due to the internal
calibration inherent to an interferometer
➢ FT-Raman spectroscopy uses an interferometer to
produce an interferogram, which “encodes” the
unique frequencies of the Raman scattering into a
single signal.
➢ The signal is measured very quickly (usually in one
second), making signal averaging fast and accurate.
➢ The interferometer employs a beam splitter
optimized for near-infrared radiation, which divides
the incoming Raman scatter into two optical beams,
one transmitted and one reflected.
➢ The reflected beam travels to and reflects off a flat
mirror that is fixed in place.
➢ The transmitted beam travels to and reflects off a flat
mirror attached to a mechanism that allows the
mirror to move a short distance (typically a few
millimeters) away from the beamsplitter.
➢ The two beams recombine at the beam splitter where,
because they travelled different distances to and from the
mirrors, they constructively and destructively interfere with
each other.
➢ The moving mirror has a constant frequency and fixed
motion, so this interference is modulated.
➢ The resulting interferogram has the unique property that
every data point (a function of the moving mirror position)
has information about every frequency of the Raman scatter
collected from the sample.
➢ Vibrational spectra are typically presented as frequency
spectra (a plot of intensity at each individual frequency)
because the measured interferogram signal is not readily
interpreted.
➢ The individual frequencies are decoded using the well-
known mathematical technique called Fourier
transformation. The computer performs this transformation,
and the desired spectral information is presented.
Comparison of Raman or FTIR
Spectroscopy
❑ Although Raman and FTIR Spectroscopy give
complimentary information and are often interchangeable,
there are some practical differences that influence which
one will be optimal for a given experiment.
❑ Most molecular symmetry will allow for both Raman and
IR activity. One special case is if the molecule contains a
centre of inversion.
❑ In a molecule that contains a centre of inversion, Raman
bands and IR bands are mutually exclusive, i.e. the bond
will either be Raman active or it will be IR active but it will
not be both.
❑ One general rule is that functional groups that have large
changes in dipoles are strong in the IR, whereas functional
groups that have weak dipole changes or have a high
degree of symmetry will be better seen in Raman spectra.
Choose Raman Spectroscopy when:
❑ Investigating carbon bonds in aliphatic and aromatic rings
are of primary interest
❑ Bonds that are difficult to see in FTIR (i.e., 0-0, S-H, C=S,
N=N, C=C etc.)
❑ Examination of particles in solution is important, e.g.
polymorphism
❑ Lower frequency modes are important (e.g. Inorganic-
Oxides)
❑ Reactions in aqueous media are investigated
❑ Reactions in which observation through a reaction window
is easier and safer (e.g. high pressure catalytic reactions,
polymerizations)
❑ Investigating lower frequency lattice modes is of interest
❑ Investigation of reaction initiation, endpoint, and product
stability of biphasic and colloidal reactions
Choose FTIR Spectroscopy when:

❑ Studying liquid-phase reactions

❑ Reactions in which reactants, reagents, solvents
and reaction species fluoresce
❑ Bonds with strong dipole changes are important
(e.g. C=O, O-H, N=O)
❑ Reactions in which reagents and reactants are at
low concentration
❑ Reactions in which solvent bands are strong in
Raman and can swamp key species signal
❑ Reactions in which intermediates that form are IR
active
Depolarization In Raman Spectroscopy
➢ In Raman spectroscopy, the depolarization ratio is the intensity ratio
between the perpendicular component and the parallel component of
Raman scattered light.
➢ Early work in this field was carried out by George Placzek, who
developed the theoretical treatment of bond polarizability.
➢ The Raman scattered light is emitted by the stimulation of the electric
field of the incident light. Therefore, the direction of the vibration of the
electric field or polarization direction of the scattered light might be
expected to be the same as that of the incident light. In reality,
however, some fraction of the Raman scattered light has a polarization
direction that is perpendicular to that of the incident light. This
component is called the perpendicular component.
➢ Naturally, the component of the Raman scattered light whose
polarization direction is parallel to that of the incident light is called
the parallel component and the Raman scattered light consists of the
parallel component and the perpendicular component.
➢ The ratio of the peak intensity of the parallel and perpendicular
component is known as the depolarization ratio (ρ), defined in equation
as  = Iperpendicular/Iparallel &  = Idepolarised/Ipolarised
➢ For example, a spectral band with a peak of intensity 10
units when the polarizers are parallel, and intensity 1 unit
when the polarizers are perpendicular, would have a
depolarization ratio of  = 1/10 = 0.1, which corresponds
to a highly polarized band.
➢ The value of the depolarization ratio of a Raman band
depends on the symmetry of the molecule and the normal
vibrational mode, in other words, the point group of the
molecule and its irreducible representation to which
the normal mode belongs.
➢ Under Placzek’s polarizability approximation, it is known
that the depolarization ratio of a totally symmetric
vibrational mode is less than 0.75, and that of the other
modes equals 0.75.
➢ A Raman band whose depolarization ratio is less than 0.75
is called a polarized band, and a band with a
depolarization ratio equal to or greater than 0.75 is called
a depolarized band.
In rotational Raman spectroscopy, the moment of inertia (I)
of a molecule can be determined from the rotational constant
(B), which is found from the spacing between spectral lines.
The moment of inertia is a measure of a molecule's
resistance to rotational motion, and its value is inversely
proportional to the rotational constant: 𝒉
𝑩=
𝟖𝝅𝟐𝒄𝑰
For a diatomic molecule, I is calculated as the product of the
reduced mass μ and the square of the bond length r0.
I = μ𝑟02
The reduced mass μ is calculated from the individual masses
of the two atoms m1 and m2 using the formula:
𝑚1𝑚2
μ=
𝑚1+𝑚2
Determine the rotational constant (B): The spacing between
consecutive Stokes or anti-Stokes lines is approximately (6B)
for the first lines and (4B) for subsequent lines.
➢ For a spherical top molecule like CH4, CCl4 etc. in which
all three axes are equivalent, symmetric vibrations have
Raman spectral bands which are completely polarized (ρ =
0).
➢ An example is the symmetric stretching mode
of methane (CH4) in which all 4 C–H bonds vibrate in
phase.
➢ However for the asymmetric mode in which one C–H bond
stretches while the other three contract, the Raman
scattered radiation is depolarized.
➢ For molecules of lower symmetry (symmetric tops C6H6,
NH3, C4H4 etc. or asymmetric tops H2O, NO2 etc. ), a
vibration with the full symmetry of the molecule leads to a
polarized or partially polarized Raman band (ρ < 0.75),
while a less symmetric vibration yields a depolarized band
(ρ ≥ 0.75).
Resonance Raman Spectroscopy

➢ It has grater sensitivity than non resonance technique.

➢ Resonance Raman technique in which the laser
excitation frequency is chosen to be close to the
frequency of an electronic transition of the sample.
➢ Raman scattering refers to a phenomenon in which
Raman line intensities are greatly enhanced by
excitation with wavelengths that closely approach that
of an electronic absorption peak of an analyte.
➢ Under this circumstances, the magnitudes of Raman
peaks associated with the most symmetric vibrations
are enhanced by a factor of 102 to 106.
➢ As a consequence, resonance Raman spectra can be
obtained at analyte concentrations as low as 10-8 M.
➢ The most important application of resonance
Raman spectroscopy has been to the study of
biological molecules under physiologically
significant conditions; that is , in the presence
of water and at low to moderate concentration
levels.
➢ As an example, the technique has been used to
determine the oxidation state and spin of iron
atoms in hemoglobin and cytochrome-c.
➢ In these molecules, the resonance Raman bands
are due solely to vibrational modes of the
tetrapyrrole chromophore.
➢ None of the other bands associated with the
protein is enhanced, and at the concentrations
normally used these bands do not interfere as a
consequence.
Surface-Enhanced Raman Spectroscopy
(SERS)

➢ Surface enhanced Raman spectroscopy involves obtaining

Raman spectra in the usual way on samples that are
adsorbed on the surface of colloidal metal particles (usually
silver, gold, or copper) or on roughened surfaces of pieces of
these metals.
➢ For reasons that are not fully understood, the Raman lines
of the adsorbed molecule are often enhanced by a factor of
103 to 106.
➢ When surface enhancement is combined with the resonance
enhancement technique discussed in the previous section,
the net increase in signal intensity is roughly the product of
the intensity produced by each of the techniques.
➢ Consequently, detection limits in the 10-9 to 10-12 M range
have been observed.
✓ Formula used to calculate enhancement factor is as follows:
✓ Ef=（Is/In）×（Cn/Cs）
✓ Where, In is the normal Raman intensity of peak while Is is the
SERS intensity.
✓ Cn and Cs are the concentrate of molecule for normal and SERS
spectra, respectively.
✓ The EF of NNPs was determined by computing the ratio of
SERS to normal Raman scattering.
✓ EF = (ISERS × CNormal)/(INormal × CSERS),
✓ where ISERS and Inormal, CSERS was calculated using the equation.
✓ CSERS = N × CAu,
✓ where N is the number of Sample molecules in the NNPs.
Raman Microscopy/Raman Microprobe
➢ It is advantageous to couple the strength and flexibility of
Raman spectroscopy with a microscope that allows
analysis of very small samples.
➢ The goal of microscopy is to analyze the smallest samples
possible and to distinguish the substance of interest from
its surroundings.
➢ This is known as spatial resolution, and in microscopy,
the highest spatial resolution is attained using small
pinholes or “apertures” somewhere in the microscope.
➢ To reach higher resolution, it is necessary to use smaller
apertures. As light passes through these smaller
apertures, diffraction becomes the limiting factor.
Therefore, the limit to spatial resolution is diffraction,
which is wavelength dependent according to the equation:
1.22 λ
D = . where n.a. is the numerical aperture of the
n.a.
collection optics.
➢ Shorter excitation wavelengths provide the highest
spatial resolution, so dispersive Raman microscopy offers
excellent spatial resolution (<1 μm).
➢ In addition, by placing a sufficiently small aperture in the
focal plane of the microscope, it is possible to perform
confocal microscopy, in which light rays from surrounding
regions of the sample are blocked by the aperture and
only rays from the optical focal point pass to the detector.
➢ This is a useful technique for non-destructively probing
the depths of the sample without cross-sectioning.
➢ Confocal microscopy is done best by dispersive Raman
microscopy with short wavelengths because diffraction at
longer wavelengths limits how small the confocal aperture
can be, thus limiting the z-axis resolution.
➢ The technique is quite useful for analysing polymer
laminates, stacked structures and inclusions, as long as
fluorescence is not an issue.
Confocal Raman Microscopy

✓ Raman microscopy can be used for chemical or

molecular analysis of unknown compounds in a
small area, down to less than a micron.
✓ Combined with a moving stage, Raman
microscopy can map areas of a sample, useful for
samples with patterns or two different regions.
✓ Raman can perform depth profiling, allowing the
user to see layers of a sample without sample
preparation.
✓ Raman microscopy is a useful tool for the
identification and mapping of multilayer samples,
minerals, particulate identification, and chemical
distribution to name a few applications.
Confocal Raman Microscopy Principles
Figure 1 depicts a general layout for Confocal Raman
microscopy. Raman utilizes a laser (a) as a source due to
weak Raman scattering (stokes and anti-stokes shifts),
which is about a million times weaker than typical
absorption spectroscopy. A beamsplitter (b) splits the light,
with the beam path focused onto the sample (c) through an
objective lens (d). The beam can be split between a camera
(e) and the spectrograph (f) for observation or measurement
respectively, and after passing through a rejection filter to
remove the Rayleigh scatter, the stokes (and anti-stokes)
wavelengths are diffracted by a grating (g) onto a detector
(h), either a CCD or an InGaAs array.
Lasers
Selection of the laser wavelength is important in obtaining
meaningful Raman data; the excitation wavelength can be
selected from the UV though visible to near IR, with a
range of power options depending on the application.
Multiple lasers with a variety of wavelengths can often be
installed in a Raman microscope to allow data to be
measured for different types of sample (Figure 5). A
popular laser wavelength is 532 nm because it offers
relatively high Raman excitation energy with low
fluorescence. However, for greater reduction in
fluorescence, longer wavelength lasers (which cause less
electronic excitation) such as 785 nm and 1064 nm are
often used. Less frequently required UV lasers offer the
additional benefit of “resonance Raman”, an excellent
choice for carbonaceous materials.
Rejection Filters

Rejection filters are used to remove or reduce the

Rayleigh scatter of the probe laser’s wavelength
before it reaches the spectrograph. There are three
major types of filters: edge, notch and low-E filters
(Figure 6). Edge, the most commonly used, rejects
both the Rayleigh scatter as well as the entire anti-
Stokes region. The intensity is typically >92 % and
the cut off is around 100 cm-1. If measurement of
the anti-Stokes region is also desired, then a notch
filter may be used at the cost of intensity (>80 %)
and cut off (150 cm-1). The Low-E filter is for studies
requiring low wavenumber studies (5-50 cm-1 cut
off) and also have a lower intensity (>80 %).
Detectors
Detectors are a key part of a Raman microspectrometer.
Detectors convert photons into a usable electronic signature. A
Charge-Coupled Device (CCD) Is a lattice of pixels (Figure X, a)
that convert light to electronic signals. These pixels are then
binned in a horizontal then vertical registers and the data is sent
to a computer. The CCD is the most common detector used in
Raman spectroscopy. An Electron Multiplied Charge Coupled
Device (EMCCD) works similarly to a regular CCD except during
the vertical data transfer the signal is multiplied as it passes
through a special material (Figure X, b). The consequence of this
is a 50x multiplication of gain allowing for faster scans (shorter
accumulation times). InGaAs detectors use a linear array (as
opposed to the grid used in CCDs) of indium gallium arsenide to
detect light in the near-infrared region. These detectors are
dedicated to Raman spectroscopy using a 1064 nm laser.
Multilayer Samples
Confocal Raman microscopy can be used to
determine layers in a multi-layer sample. Firstly, a
cross-section of a sample can be made and the
sample mapped across the sections (Figure 9). For
very thin layers (<3 μm), confocal microscopy is very
effective due to its high spatial resolution in the XY
directions. Thin layers (<30 μm) can be probed
without sample preparation with depth profiling by
confocal microscopy. Since scattered light in the z
dimension is filtered out, a map in the z-direction
can be obtained simply by moving the stage in the z
direction.
Remote Raman Analysis
➢ Raman spectroscopy is typically envisioned as an in
situ analysis technique as it requires a strong
monochromatic exciting source (now invariably a laser),
and highly efficient spectral measurement apparatus.
➢ However, Raman analysis of minerals can be performed at
ranges of tens of meters using low-power lasers similar to
those which have already flown on planetary missions,
using modest receiver diameters (10 cm) and modern
efficient spectral measurement hardware.
➢ This capability enables rapid and model-independent
mineralogical analysis of rocks and soils in the vicinity of a
lander or a large rover.
➢ This capability does not replace passive remote spectral
analysis which can survey much larger areas in less time,
but does offer an entirely new ability to make less
ambiguous mineralogical identification.
Standoff System Setup and Parameters.
➢ The components of a standoff Raman system are similar to
those of a conventional Raman system, comprising a laser,
collection optics, spectrometer, detector, and optical filters.
➢ The main distinction for standoff systems is the
incorporation of a telescope for collection of scattered
radiation, allowing analysis of a target meters away from
the system.
➢ Continuous wave and pulsed lasers have both been
implemented, with pulsed lasers the preferred choice for
their ability to be coupled with gated detectors for
measurements under ambient light conditions.
➢ A charge-coupled device (CCD) or intensified CCD (ICCD)
array is the most common detector, with ICCDs used
extensively with pulsed lasers.
➢ Sharma et al. stated that small f-number spectrometers
perform better than larger f-number spectrometers for
standoff systems.
APPLICATIONS OF RAMAN SPECTROSCOPY
Raman Spectra of Inorganic Species
The Raman technique is often superior to infrared
for spectroscopy investigating inorganic systems
because aqueous solutions can be employed. In
addition, the vibrational energies of metal-ligand
bonds are generally in the range of 100 to 700 cm-
1, a region of the infrared that is experimentally

difficult to study. These vibrations are frequently

Raman active, however, and peaks with  values
in this range are readily observed. Raman studies
are potentially useful sources of information
concerning the composition, structure, and
stability of coordination compounds.
Raman Spectra of Organic Species
Raman spectra are similar to infrared spectra in that
they have regions that are useful for functional
group detection and fingerprint regions that permit
the identification of specific compounds. Raman
spectra yield more information about certain types of
organic compounds than do their infrared
counterparts.

Biological Applications of Raman Spectroscopy

Raman spectroscopy has been applied widely for the
study of biological systems. The advantages of his
technique include the small sample requirement, the
minimal sensitivity toward interference by water,
the spectral detail, and the conformational and
environmental sensitivity.
Geology and mineralogy

Identification of various minerals and

precious/semiprecious stones
Analyzing fluid inclusions
Studying the distribution of minerals and phases
within a section of rock
Analyzing phase transitions
Studying the behaviour of minerals under harsh
conditions
Identifying chondrite or achondrite meteorites
Carbon materials
 Study of single-walled carbon nanotubes (SWCNTs)
 Analyzing the purity and measuring the electrical
properties of carbon nanotubes (CNTs)
 Study of sp2 and sp3 structure in carbon materials
 Testing hard disk drives
 Analysis of the coating properties of diamond- like
carbon (DLC)
 Detection of defects or disorder in carbon materials
 Testing diamond quality and place of origin
 Measuring the electrical properties and the number of
layers of 2D materials like graphene.
Semiconductors
 Identifying and describing inherent strain and stress of
the molecular or chemical structure
 Assaying the purity
 Analysis of the composition of an alloy
 Identifying contaminants
 Studying the structure of a superlattice
 Analyzing defects in a structure
 Characterization of heterostructures
 Study of doping effects
 Microanalysis of photoluminescence
 Studying the electrical properties of 2D transition metal
dichalcogenides such as MoS, WSe, WSe, and
phosphorene
Life Sciences

 Analysis of biocompatibility of a material

 Analysis of nucleic acids
 Study of interactions between drugs and cells
 Photodynamic therapy (PDT)
 Analyzing metabolic accumulations of a substance or
compounds
 Diagnosis of disease
 Analysis of individual cells
 Cell sorting applications
 Analyzing the features of biomolecules
 Study of bone structure
Applications of Raman spectroscopy
 It can be used to identify explosives such as RDX, PETN, TNT, etc.
and also the binding agents inside explosive materials.
 The technique is also effective for identifying toxic solvents in
biowarfare agents and for studying other potentially harmful agents.
 In the pharmaceutical industry, one major application for Raman is
the detection and substantiation of incoming raw materials.
 The technique is employed for the analysis of liquids, tablets, and gel
caps.
 Raman is also an essential tool in counterfeit-drug analysis. Figure 2
illustrates the generic form of the carbamazepine drug and the
spectra of Tegretol.
 This analysis was performed without opening the blister packs when
looking at these chemicals.
 Although the variations in the spectra are not visually clear, the
BWID software package can easily differentiate the two chemicals.
 Besides this, Raman technique is used in carbon analysis and
bioscience and medical diagnosis.
Pharmaceutical agents and cosmetic products
 The distribution of a compound within a tablet
 The uniformity of a blend
 High throughput screening
 The concentration of an API
 Testing the content and purity of a powder
 Verifying raw material quality
 Detecting polymorphism
 Analysis of crystalline structure
 Identification of contaminants
 Conducting in vivo analysis
 Skin depth studies
 Assaying the dose and uniformity of content
 Studying chemical combinations
❑ Raman spectroscopy is used in industry for a
variety of applications, including:
❑ Crystallization Processes
❑ Polymorphism Identification
❑ Polymerization Reactions
❑ Hydrogenation Reactions
❑ Chemical Synthesis
❑ Biocatalysts and Enzymatic Catalysis
❑ Flow Chemistry
❑ Bioprocess Monitoring
❑ Synthesis Reactions
 Quantitative applications

➢ Raman spectra tend to be less cluttered with peaks

than infrared spectra.
➢ As a consequence, peak overlap in mixtures is less
likely, and quantitative measurements are simpler.
➢ In addition, Raman sampling devices are not subject
to attack by moisture, and small amounts of water
in a sample do not interfere.
➢ Despite these advantages, Raman spectroscopy has
not yet been exploited widely for quantitative
analysis.
➢ This lack of use has been due largely to the rather
high cost of Raman spectrometers relative to that of
absorption instrumentation.
Raman spectrum of phenol/aniline benzoxazine monomer.
Raman spectrum of m-cresol/methylamine monomer.
Raman spectrum of bisphenol A/m-toluidine benzoxazine monomer.
Comparison of Raman and IR Spectra