1. SPECTROSCOPY

2. Theory of light
• Light is electromagnetic radiation that is visible to
the human eye, and is responsible for the sense of sight.
• Visible light has a wavelength in the range of about
380 nm, or 380×10−9 m, to about 740 nanometres –
between the invisible infrared with longer wavelengths
and the invisible ultraviolet with shorter wavelengths.
• Light is emitted and absorbed in tiny "packets"
called photons, and exhibits properties of both
waves and particles. This property is referred to as
the ”wave–particle duality”.
• The study of light, known as optics.

3. Electromagnetic spectrum

4. What are the different theories of Light?
1.Corpuscular Theory by Sir Isaac Newton
2.Wave Theory
3.Electromagnetic Theory
4.Photoelectric Theory
5.Dual Property
6.Quantum Theory by Max Planck

5. What are the basic properties of
light?
• Light travels in straight lines
• Light can be reflected
• Light can be refracted ~ bent
• Light is a form of energy
• Light can be dispersed
• Light can be diffracted

6. Spectroscopy
• Spectroscopy pertains to the dispersion of an object's light
into its component colours (i.e. energies).
• By performing this dissection and analysis of an object's light,
physical properties of object such as temperature, mass,
luminosity and composition can be inferred.
• Spectroscopy is the study of the interaction between matter
and radiated energy.
• Applications:
1. To determine the molecular structure
2.To estimate the energy levels of the ions and complexes in a chemical
system along with the compositions.
3. To understand the structure making and structure breaking processes
in solutions
4, To get an idea regarding absorption and emission details of the
specimen
5. To understand the intrinsic configuration and relative association and
chemical shifts

7. Spectroscopy – Radiation Terminology
• Wavelength (λ) -- length between two equivalent points on
successive waves
• Wavenumber (n)–– the number of waves in a unit of length or
distance per cycle -- reciprocal of the wavelength
• Frequency (ν) –– is the number of oscillations of the field per
second (Hz)
• Velocity (c) –– independent of wavelength –– in vacuum is
3.00 x 1010 cm/s (3.00 x 108m/s)
• Photon (quanta) –– quantum mechanics ~ the minimum
amount of any physical entity involved in an interaction

8. Chromophore& Auxochrome
• Chromophore : A Chromophore is a group of atoms within a
molecule which are responsible for the color of the molecule.
• A Chromophore adds color to a molecule because of the
nature of the atoms involved and the way they are bonded
with each other.
• These specialized moieties are also present in atoms inside
cells which have a color-related function, including photo
pigments and chromatophores.
• A classic example of a photo pigment can be found in the
human eye, where sensitized cells respond to visible light to
provide a picture of the visible world.
• e.g. chlorophyll's porphyrin ring, or an azo dye's benzene
rings joined by a N=N double bond.

9. Chromophore& Auxochrome
• Auxochrome: functional group that does not absorb radiation
itself in the UV range but has a shifting effect on main
chromophore peaks to longer wavelength as well as increasing
their intensity.
OR
Auxochromes are functional groups attached to chromophore
that alter the shade of colour it emits.
• Example: --OH and ––NH2 on benzene chromophore

10. Quantification terms
• Transmittance : The transmittance of a sample is the ratio of
the intensity of the light that has passed through the sample
to the intensity of the light when it entered the sample
T = I₀/I
where,
I₀= Light passed out
I = Light entered
Absorbance: the measure of the quantity of light that a sample
neither transmits nor reflects (= absorbs) and is proportional to
the concentration of a substance in a solution.
Absorbance = -log (percent transmittance/100)
A = log(1/T) = - log T = log I/ I₀

11. Beer-Lambert’s Law
• The absorbance of light is directly proportional to the
thickness of the media through which the light is being
transmitted multiplied by the concentration of absorbing
chromophore.
A = εbc
where,
A = absorbance
ε =molar extinction coefficient
b = thickness of the solution
c = concentration

12. Types of Spectroscopy
 There are as many different types of spectroscopy as there are energy
sources. Few of them are as follows :
Astronomical Spectroscopy
Electron Paramagnetic Spectroscopy
Electron Spectroscopy
Infrared Spectroscopy
Mass Spectroscopy
Laser Spectroscopy
Raman Spectroscopy
NMR Spectroscopy
Fluorescence spectroscopy
Gamma-ray Spectroscopy

13. LASER-Raman Spectroscopy
 Introduction:
Raman spectroscopy is a spectroscopic technique based on
“inelastic scattering” of monochromatic light, usually from a laser
source.
 Inelastic scattering means that the frequency of photons in
monochromatic light changes upon interaction with a sample
 Photons of the laser light are absorbed by the sample and then
reemitted. Frequency of the reemitted photons is shifted up or down
in comparison with original monochromatic frequency, which is
called the Raman effect.
 This shift provides information about vibrational, rotational and other
low frequency transitions in molecules of solid, liquid and gaseous
samples.

14. Origins of Raman Spectroscopy
 The Raman effect is based on molecular deformations in electric
field E determined by molecular polarizability α. The laser beam can
be considered as an oscillating electromagnetic wave with electrical
vector E. Upon interaction with the sample it induces electric dipole
moment P = αE which deforms molecules. Because of periodical
deformation, molecules start vibrating with characteristic frequency
υm.
 Amplitude of vibration is called a nuclear displacement. In other
words, monochromatic laser light with frequency υ0 excites
molecules and transforms them into oscillating dipoles. Such
oscillating dipoles emit light of three different frequencies

15. Energy level diagram showing the
states involved in Raman signal.

16. A molecule with no Raman-active modes absorbs a photon with the
frequency υo.
The excited molecule returns back to the same basic vibrational state and
emits light with the same frequency υo as an excitation source. This type
of interaction is called an elastic Rayleigh scattering.
A photon with frequency υo is absorbed by Raman-active molecule
which at the time of interaction is in the basic vibrational state. Part of
the photon’s energy is transferred to the Raman-active mode with
frequency υm and the resulting
frequency of scattered light is reduced to υo- υm. This Raman frequency
is called Stokes frequency or just “Stokes”.
A photon with frequency υo is absorbed by a Raman-active
molecule, which, at the time of interaction, is already in the excited
vibrational state. Excessive energy of excited Raman active mode is
released, molecule returns to the basic vibrational state and the
resulting frequency of scattered light goes up to υo+ υm. This
Raman frequency is called Anti-Stokes frequency, or just “Anti-
Stokes”.

17. Instrumentation
 A Raman system typically consists of four
major components:
1. Excitation source (Laser)
2. Sample illumination system and light collection
optics.
3. Wavelength selector (Filter or Spectrophotometer).
4. Detector (Photodiode array, CCD or PMT).

19. Source:
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 of argon and krypton ion sources that emit
in the blue and green region of the spectrum have an
advantage over the other sources.

20. Common laser sources for Raman
LASER type Wave lengths in nm
Argon ion 488.0 – 514.5
Krypton ion 530.9 – 647.1
Helium-neon 632.8
Diode 785 – 830
Nd-YAG 1064

21. Sample system:
 Sample handling for Raman spectroscopic
measurements is simple 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.

22. Liquid Samples: A major advantage of sample handling in
Raman spectroscopy 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: 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.

23. Spectrophotometer/Filters
 Since spontaneous Raman scattering is very weak the main
difficulty of Raman spectroscopy is separating it from the intense
Rayleigh scattering. As rayleigh scattering may greatly exceed the
intensity of the useful Raman signal in the close proximity to the
laser wavelength.
 In many cases the problem is resolved by simply cutting off the
spectral range close to the laser line where the stray light has the
most prominent effect. Commercially available, interference (notch)
filters which cut-off spectral range of ± 80-120 cm-1 from the laser
line. This method is efficient in stray light elimination but it does not
allow detection of low-frequency Raman modes in the range below
100 cm-1.
 Raman spectrometers typically use holographic gratings which
normally have much less manufacturing defects in their structure
then the ruled ones.

24. Detectors:
 In earlier times people primarily used single-point
detectors such as photon-counting Photomultiplier
Tubes (PMT). However, a single Raman spectrum
obtained with a PMT detector in wave number scanning
mode was taking substantial period of time, slowing
down any research or industrial activity based on
Raman analytical technique.
 Nowadays, more and more often researchers use multi-
channel detectors like Photodiode Arrays (PDA) or,
more commonly, a Charge-Coupled Devices (CCD) to
detect the Raman scattered light. Sensitivity and
performance of modern CCD detectors are rapidly
improving. In many cases CCD is becoming the detector
of choice for Raman spectroscopy.

25. Raman Strength & Limitations
 Easy identification of
chemical structures
 Widely applicable for
various materials
 Samples can be
solid/aqueous
 Little or no sample
treatment
 Remote control with fiber
optics
 When incidence light goes
to blue, Fluorescence is
required
 Expensive due to cost of
laser sources
 Low excitation probability
 Spatially refrained by
optical limits
Strength Limitations

26. Applications
 Pharmaceutical and Bio-medical Applications
 Material science and Nano-technology
 Forensic labs, also used by Anti-terror squad
 Gemology, Geology, Mineralogy
 Archaeology, Art, Heritage

27. FLUORESCENCE
SPECTROSCOPY
 Fluorescence is the emission of light by a substance that
has absorbed light or other electromagnetic radiation at a
longer wavelength than absorbed.
 Emission Spectroscopy: A spectroscopic technique that
examines the wavelength of photons emitted by atoms or
molecules during their transition from an excited state to
lower energy state.
 Luminescence: Luminescence is emission of light by a
substance not resulting from heat; it is thus a form of cold
body radiation. It is caused by chemical reactions, electrical
energy, subatomic motions, or stress on a crystal.

28.  Luminescence is basically divided into two categories:
Phosphorescence
Fluorescence
 Emission rates of Fluorescence is typically 10^8 s-1 ;
while emission rates of Phosphorescence are slow
i.e., 10^3 to 100 s-1.
 Life time of a phosphorescence molecule is milliseconds
to seconds, while that of a fluorescence is nanoseconds.

29.  Fluorescence is a spectrochemical method of analysis
where the molecules of the analyte are excited by
irradiation at a certain wavelength and emit radiation of a
different wavelength.
 The emission spectrum provides information for both
qualitative and quantitative analysis.
 when light of an appropriate wavelength is absorbed by
a molecule (i.e., excitation), the electronic state of the
molecule changes from the ground state to one of many
vibrational levels in one of the excited electronic states.

30. The excited electronic state is usually the first excited singlet state, Once the
molecule is in this excited state, relaxation can occur via several processes.
Fluorescence is one of these processes and results in the emission of
Light.
Following absorption, a number of vibrational levels of the excited state
are populated. Molecules in these higher vibrational levels then relax to
the lowest vibrational level of the excited state (vibrational relaxation).
From the lowest vibrational level, several processes can cause the
molecule to relax to its ground state.
An overall energy balance for the fluorescence process could be
written as:
Efluor = Eabs − Evib − Esolv.relax
Efluor is the energy of the emitted light,
Eabs is the energy of the light absorbed by the molecule during
excitation, Evib is the energy lost by the molecule from vibrational
relaxation.
Esolv.relax term arises from the need for the solvent cage of the molecule
to reorient itself in the excited state and then again when the molecule
relaxes to the ground state.

31. Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a
type of electromagnetic spectroscopy which analyses fluorescence from
a sample. It involves using a beam of light, usually ultraviolet light, that
excites the electrons in molecules of certain compounds and causes
them to emit light
Devices that measure fluorescence are called fluorometers or
fluorimeters.
The difference between these two wavelengths is known as Stokes
Shift

32.  The light from an excitation source passes through a
filter or a monochromator and strikes the sample, Xenon
lamps are widely used as a light source.
 A portion of the incident light is absorbed by the sample
and some by the fluorescent molecules. The fluorescent
light is emitted in all directions, some of this light passes
through the second filter/ monochromator and reaches
the detectors.
 These detectors are placed at 90º to the incident light
beam to minimize the risk of transmitted or reflected
incident light reaching the detector. Commonly diffraction
grating is used.

35.  Determination of fluorescent drugs in low dose formulations
 In carrying out limit-tests where Fluorescent compounds are treated
as impurities as Fluorescent probes
 Useful for studying binding of drugs to component in complex
formulations.
 Widely used in Bio-analysis for measuring components present in
low concentrations
 Enzyme assays and kinetic analysis
 Fluorescence recovery after bleaching (FRAP)~ Used to study the
movement and working of a biological membrane.
 Fluorescence resonance energy transfer (FRET) ~ Used to deduce
the distance between Protein molecules
 Fluorescence activated cell sorter (FACS)~ used in Fluorescent
immunoassays.

36. Thank You . . .
Archa Dave
M.Sc MB
Sem II
12031G1901