HAWASSA UNIVERSITY INSTISTUTE OF TECHNOLOGY

School of Electrical & Computer

Engineering
Post graduate program in Control & Instrumentation Engineering

Course title: Analytical Instrumentation

Assignment -1

Title: Raman spectrophotometry

Submitted by ID No.
YESHIWAS FETENE ……………………………….. PGConR/009/10

Submitted to: Mr. Dessie Fentaw

Submitted on: JAN 27/2019 G.C.
Hawassa, Ethiopia

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Contents Page

1. Introduction ............................................................................................................................. 1
2. History of the Origin of Raman Spectroscopy ........................................................................ 2
3. Principle of operations of Raman spectrophotometry ............................................................. 4
3.1. Basic Raman theory ......................................................................................................... 4
3.2. Raman instrumentation: major components ..................................................................... 7
4. Application of Raman Spectrophotometer ............................................................................ 11
5. Advantages and weakness of using Raman spectroscopy ..................................................... 12
6. Summary................................................................................................................................ 12

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 1. Introduction
The Raman effect occurs when electromagnetic radiation affects molecules and interacts with
electron densities polarized and molecular bonds in phases (solid, liquid or gas) and the
environment. For the spontaneous Raman effect, which is a form of inelastic light scattering,
photons (electromagnetic radiation from a certain wavelength) interact with molecules produced
scattered inelastic photons which are "emitted" can be either (anti-Stokes) or (Stokes).

Figure 1: Raman Spectrophotometer

Radiation decomposition by the atom or molecule goes in all directions in length waves and
intensities that are affected by molecular particle size. If the media transparent contains only
particles with atomic dimensions (surface 0.01 𝐴2 ) there will be radiation decomposition with very
weak intensity. Radiation the decomposition does not appear because the wavelength is in the
ultraviolet region. The scattering radiation is known as Rayleigh scattering. As well as which
occurs in molecules of large diameter or aggregated as example of suspension or colloidal
molecules. Scattering in suspension solutions and colloidal systems its wavelength is close to the
size of a molecular particle of suspension or a colloidal system that is. The scattering radiation is
known as Tyndal scattering or scattering of noodles which gave birth to the turbidimetric method.
Different scattering with the original radiation (radiation source) different in terms of wavelength,

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the frequency and intensity is known as "feeble fluorescence" or Raman scattering. The Raman
scattering gives a Raman line with an intensity of no more than 0.001% of the spectral source of
the radiation line.

This Raman effect is very weak and becomes a reality after the discovery of laser techniques 40
years later. Laser light is not the same as light coming from the sun or from a light bulb, because
laser light has all one wavelength. By therefore, if the light hits the object, all the light will be
scattered back by the object that is, where the wavelength of light that comes is the same as that
emitted turn back. Not only that, the light waves are lined up neatly in the same direction
(polarization). Thus, the laser can put many photons on a small spot. There are many photons that
pound the sample, one in a million, increase the signal so become strong enough to be detected.
Because all photons have wavelengths the same, then all will interact in the same way on the
molecule the type is the same, thus strengthening the effect that occurs.

Since the discovery of the Raman effect in 1982, many Raman spectroscopy used as a solution to
various technological needs, especially in industry laboratory. Raman spectroscopy is a
spectroscopic technique based on inelastic scattering from monochromatic light which usually
comes from a laser beam so that resulting in molecular deformation by the electric field E
determined by ability of α molecular polarization. The Raman effect is the frequency of the
photons retransmitted can be raised or lowered against the original frequency of light
monochromatic. This change provides information about vibration, rotation, and transition other
low frequencies on the molecule. Raman spectroscopy can be used for study solid, liquid and gas
materials.

2. History of the Origin of Raman Spectroscopy

A difficult study with very meaningful findings, in physics was carried out by Chandra Venkrama
Raman, an Indian physicist, at in 1928. He was the first to be interested in the phenomenon of
inelastic light emission. Radiation emitted by bombed molecules containing photons of the same
frequency as those of incident radiation, but also photons of different frequencies. This effect is
very small - approximately 1 photon on 1 million (0.0001%) will be emitted with a wavelength
slightly different from the incident wavelength. This process was then called after the name of his

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discoverer, and the change of frequency is called Raman effect. At the end of the 1930s, Raman
spectroscopy had become the main non-destructive method of analysis chemicals.

After the Second World War, infrared spectroscopy became the most more widespread, mainly
because it is much easier to use the development of very sensitive infrared sensors and advances
in electronics. Measurements by infrared spectroscopy became a pure routine, so that Raman
spectroscopy required highly skilled operators and dark rooms.

Despite the development of lasers in the 1960s, Raman spectroscopy remained largely confined in
the labs because always requiring very skilled people to obtain very simple spectra. The system
needed to be constantly monitored and calibrated because the wavelength scales used depended
largely on the ambient conditions, reliable analyzes and bibliographic data not being all prevalent
at the time. In addition, Raman spectroscopy suffered greatly from fluorescence phenomenon.
Fluorescence is a strong light emission coming from the sample and interferes with - and often
completely drowns - the weak Raman effect. However, the risk that an unknown sample is
fluorescent is highly dependent on the wavelength of the laser used for excitation. Recent
publications have shown that more than 40% of natural samples suffer from fluorescence, even
with modern using red light (about 800 nm) for excitation. It's only by approximating the
wavelength of the infrared (wavelengths of about 1 micron) that the risk of fluorescence drops to
a few percent.

In 1986, a commercial infrared / Fourier transform interferometer and excitation source close to
the infrared have been combined to obtain a Raman spectrum. This has given many positive points:

✓ Near infrared laser excitation greatly reduces the number of samples that can give rise to
fluorescence and allows the use of lasers more powerful without photo decomposition.
✓ The database of interferometric obtained by Fourier transform and Raman spectroscopy
is now comparable to that obtained by infrared. This reduces dramatically the search speed
and makes the identification of a sample trivial.
✓ Accurate spectrum libraries and quantitative analysis are available for the
Raman spectroscopy.

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 3. Principle of operations of Raman spectrophotometry
3.1. Basic Raman theory
When a laser is used to probe a sample, light impinges upon a molecule and interacts the electron
cloud of the bonds of that molecule. When light is scattered from a molecule most photons are
elastically scattered (Rayleigh scatter), meaning that the scattered photons have the same energy
(frequency) and, therefore, wavelength, as the incident photons and no change in the molecular
status of the molecule is observed. However, a small fraction of light (approximately 1 in 107
photons) is scattered at optical frequencies different from, and usually lower than, the frequency
of the incident photons. The process leading to this inelastic scatter is the termed the Raman effect.
In quantum mechanics the scattering is described as an excitation to a virtual state lower in energy
than a real electronic transition with nearly coincident de-excitation and a change in vibrational
energy. The scattering event occurs in 10−14 seconds or less. The difference in the energy level
between the excited and ground level are shown in figure 2 below.

Figure 2: Energy-level diagram showing the states involved in Raman spectra

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At room temperature the thermal population of vibrational excited states is low, although not zero.
Therefore, the initial state is the ground state, and the scattered photon will have lower energy
(longer wavelength) than the exciting photon. Since more photon undergo Stokes scatter, this
Stokes shift is what is usually observed in Raman spectroscopy.

According to the law of conservation of energy, the change in energy between the incident photon
(ℎ𝑣0 ) and the scattered photon (ℎ𝑣𝑠 ) corresponds to the energy change of the molecule, which
refers to the transition between two vibrational states, the initial (ℎ𝑣𝑖 ) and the final excited state
(ℎ𝑣𝑓 ) according to the equation:

ℎ𝑣0 − ℎ𝑣𝑠 = ℎ𝑣𝑓 − ℎ𝑣𝑖 (1)

where ℎ is Planck’s constant and 𝜈 is the frequency of the electromagnetic wave.

Therefore, the change in the vibrational status of the molecule (𝛥𝐸𝑓−𝑖 ) can be expressed as:

𝛥𝐸𝑓−𝑖 = ℎ(𝑣0 − 𝑣𝑠 ) (2)

If in (2) we substitute the definition of frequency with the number of waves in the distance light
travels in one second, where c is the velocity of light and λ is the wavelength of the radiation, we
find that

𝑐 𝑐
𝛥𝐸𝑓−𝑖 = ℎ(𝜆 − 𝜆 ) (3)
0 𝑓

and since c is constant and h is constant, the change in the difference of energy can be expressed
in terms of the reciprocal of the wavelength, whose dimension is 𝑐𝑚−1, and it is denominated the
wave number. This is the normal unit used both in Raman and Infrared spectroscopy.

If we want to study the Raman scattering in ∆ classical terms, the interaction between the
electromagnetic radiation and the sample can be viewed as a perturbation of the molecule’s electric
field. A simple classical electromagnetic field description of Raman spectroscopy can be used to
explain many of the important features of Raman band intensities. The dipole moment, P, induced
in a molecule by an external electric field, E, is proportional to the field as shown in equation 4

𝑃 = 𝛼𝐸 (4)

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The proportionality constant α is the polarizability of the molecule. The polarizability measures
the ease with which the electron cloud around a molecule can be distorted. If we substitute the
equation which describes the normal fluctuation with time (t) of the electromagnetic field of the
electromagnetic wave (the laser beam), where 𝐸0 is the vibrational amplitude and 𝜈0 is the
frequency of the laser, we obtain

𝑃 = 𝛼𝐸 = 𝛼𝐸0 𝑐𝑜𝑠(2𝜋𝑣0 𝑡) (5)

When a diatomic molecule vibrates at a frequency 𝑣𝑚 , assuming a simple harmonic motion, its
internuclear distance (q) can be written in the form:

𝑞 = 𝑞0 𝑐𝑜𝑠(2𝜋𝑣𝑚 𝑡) (6)

Where 𝑞0 is the amplitude of vibration. The polarizability, which in this case is a scalar quantity,
can be expanded as a Taylor series as a linear function of q

ⅆ𝛼
𝛼 = 𝛼0 + ( ⅆ𝑞 ) 0 𝑞0 + ⋯ (7)

ⅆ𝛼
where 𝛼0 is the polarizability at the equilibrium position and (ⅆ𝑞 ) 0 is the rate of change of 𝛼 with

respect to the change in 𝑞. Combining (5) and (6) and (7), we obtain

𝑃 = 𝛼𝐸0 𝑐𝑜𝑠(2𝜋𝑣0 𝑡)

ⅆ𝛼
= 𝛼0 𝐸0 𝑐𝑜𝑠(2𝜋𝑣0 𝑡) + (ⅆ𝑞 ) 0𝑞𝐸0 𝑐𝑜𝑠(2𝜋𝑣0 𝑡)

ⅆ𝛼
= 𝛼0 𝐸0 𝑐𝑜𝑠(2𝜋𝑣0 𝑡) + (ⅆ𝑞 ) 0𝑞0 𝐸0 𝑐𝑜𝑠(2𝜋𝑣0 𝑡) 𝑐𝑜𝑠(2𝜋𝑣𝑚 𝑡)

1 ⅆ𝛼
= 𝛼0 𝐸0 𝑐𝑜𝑠(2𝜋𝑣0 𝑡) + 2 (ⅆ𝑞 ) 0𝑞0 𝐸0 {𝑐𝑜𝑠[2𝜋(𝑣0 + 𝑣𝑚 )]𝑡 + 𝑐𝑜𝑠[2𝜋(𝑣0 − 𝑣𝑚 )]𝑡} (8)

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The first term of equation (8) represents an oscillating dipole that radiates light of frequency 𝜈0
(Rayleigh scatter), that is the same of the incident laser, while the second term correspond to the
Raman scattering of frequency 𝜈0 + 𝜈𝑚 (anti-Stokes) and 𝜈0 − 𝜈𝑚 (Stokes), as shown in Figure 1
ⅆ𝛼
above. It is important to note that the second term of the equation contains the factor (ⅆ𝑞 ) 0 ,

which, if zero, yields no Raman scattering. Therefore, the selection rule for a molecule to be Raman
active is that the rate of change in polarizability with vibration must be non-zero. If a vibration
does not greatly change the polarizability of the molecule, then the polarizability derivative will
be near zero, and the intensity of the Raman band will be low. The vibrations of a highly polar
moiety, such as the O-H bond, are usually weak. An external electric field cannot induce a large
change in the dipole moment and stretching or bending the bond does

not change this. For this reason, water is only weakly Raman active and therefore does not interfere
with Raman spectroscopy of biological samples. Typical strong Raman scatterers are moieties with
distributed electron clouds, such as carbon-carbon double bonds. The pi-electron cloud of the
double bond is easily distorted in an external electric field. Bending or stretching the bond changes
the distribution of electron density substantially, and causes a large change in induced dipole
moment.

3.2. Raman instrumentation: major components

Commercially available Raman spectrometers consists of four major components:

a) Excitation source, usually a laser

b) Sample illumination and collection system
c) Wavelength selectors
d) Detection and computer control system.

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 Figure 3: Components of Raman Spectrophotometry

a) The excitation sources

Lasers became the excitation source in Raman instrumentation set-ups after the 1960s. The main
advantages of using lasers are that their beams are highly monochromatic, usually with a small
diameter that can be further reduced by using lens systems and thus focused on small samples (
~1 𝑚𝑚3 ), which in case of Raman microscopy can be regions as small as 2 µm in diameter.

Different kinds of lasers are available:

✓ gas state lasers such as 𝐴𝑟 + , 𝑘𝑟 + , He-Ne

✓ solid state lasers such as Neodymium -YAG

The latter having its primary lasing wavelength at 1064nm which is especially suitable for FT-
Raman system operating in the infrared region.

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 ✓ Diode lasers can be obtained at specific wavelength in the blue or the infrared regions.
✓ Others such as dye lasers, excimer and nitrogen laser have been used for Raman
spectroscopy.

b) Sample illumination and collection system

Since Raman scattering is weak, the laser beam must be properly focused onto the sample and the
scatter radiation collected efficiently. In principle, excitation and collection from the sample can
be accomplished in any geometry. More frequently, the 90° or the 180° (the back scattered)
configuration are used, the latter being the one depicted in Figure 3 below.

Figure 4: Collection system in 900

c) Wavelength selectors

Wavelength selectors can be classified in different categories, the simplest one being the
dichroitic filter. Prisms, grating monochromators and spectrographs have been used in
Raman instrumentation. Coupling of two or three monochromators put in series was
developed to reduce stray or extraneous light that can overlap the weak Raman light.
Recently holographic notch filters (filters that pass all frequencies except those in a stop

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 band centered on a selected frequency) have been used to suppress the Rayleigh light,
before grating diffraction.
d) Detection and computer control system
Early on, detection of Raman light was done with photographic films, using long time
exposures. Subsequently, in Raman spectrometer equipped with monochromators, the light
exiting through the slits was focused onto a photomultiplier tube, which converts photons
into an electrical signal. In the former Raman spectrometers, the detection of the Raman
signal was done for each frequency, but this time consuming single-channel technique was
substituted by multiple channel detection and recently by Charge-Coupled Devices (CCDs)
which are silicon-based semiconductors arranged as an array of photosensitive elements,
each one generating photoelectrons and storing them as an electrical charge. Charges are
stored on each individual pixel as a function of the number of photons striking that pixel
and then read by an analogue-to-digital converter. Finally data is manipulated, stored and
displayed to the observer by a digital instrument usually we call it computer.

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 Figure 5: Representation of modern Raman spectrometer

4. Application of Raman Spectrophotometer

a) In the field of biological Micro trace

Micro trace samples sometimes reveal tiny blood particles, one of which genetic profile could
be exploited. To authenticate the presence of blood by avoiding consuming of biological
material by specific tests, Raman spectroscopy allows rapid identification of the characteristic
signature of hemoglobin. Furthermore, the application of chemometric techniques to Raman
spectra highlights subtle differences that could help to tie the human blood to the canine or
feline blood.

b) In the field of explosives and risk substances

The use of fiber optic probes and the design of portable Raman spectrometers has It has also
been possible to measure various explosives in situ or in glass containers or plastic maintaining
an acceptable spectral resolution. These studies have shown that various explosives, chemical
weapons agents, precursors or solvents are correctly identified in Raman spectroscopy without
encountering false positives.

c) In the field of drugs

Pure drugs are good Raman diffusers that lead to a spectrum rich in information and therefore
to rapid identification. However, the presence of additives (cutting agents, excipients) and
especially fluorescence that they generate can sometimes deteriorate the quality of the spectra
obtained on real samples. The use of a near-infrared excitation source and techniques
Chemometrics can help identify the active ingredient of the narcotic.

d) In the field of colored substances

Colored substances are one of the favorite fields of Raman spectroscopy. Indeed, these organic
molecules are usually built around a highly conjugated chromophore and generate an intense

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Raman signal. Moreover, the coloring is ubiquitous in our society and intervenes in various forms
in the field forensics. Raman spectroscopy is particularly useful when the substance colored is
either in the form of pigment and that thin layer chromatography (TLC) is not applicable, or that
it is trapped in a translucent matrix.

5. Advantages and weakness of using Raman spectroscopy

Here are some reasons why someone would prefer to use Raman Spectroscopy.

✓ Non-destructive to samples (minimal sample prep)

✓ Higher temperature studies possible (don’t care about IR radiation)
✓ Easily examine low wavenumber region: 100 cm-1 readily achieved.
✓ Better microscopy; using visible light so can focus more tightly.
✓ Easy sample prep: water is an excellent solvent for Raman. Can probe sample through
transparent containers (glass or plastic bag).

Weakness:

✓ The spectral spectrum of the powder sample or heterogeneous tablet cannot inform the
composition of the sample as a whole.
✓ Raman spectroscopy often creates interference such as fluorescence.

6. Summary
Raman spectroscopy is a vibrational spectroscopy that capable of providing chemical component
information with high spectral resolution. It is of a great potential in a broad range of applications,
from pharmaceutical, material science to gem identifications to forensics. Traditionally Raman
applications were limited by low cross sections and diffraction limits. But, developments in those
areas have brought a bright future and enormous opportunities.

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