Detailed description of spectroscopic techniques.

1. INSTITUTE OF BIOMEDICAL SCIENCES,
BUNDELKHAND UNIVERSITY, JHANSI
SPECTROSCOPIC TECHNIQUES
PRESENTED BY- Ms. PALAK AGRAWAL

2. INTRODUCTION
‘Spectroscopy’ is a branch of science that
deals with the study of interaction of matter
with light, using electromagnetic radiation
as an investigation to obtain information
about atoms and molecules that are too
small to see. It is an analytical, structural
elucidation technique which helps us to “see
the unseeable”.

3. ELECTROMAGNETIC RADIATION
Electromagnetic radiation consists of
particles representing a quantum of
light or discrete packages of energy
known as ‘photons’. A photon carries
energy proportional to the radiation
frequency but has zero rest mass.
It consists of an oscillating electric field
(E) and an oscillating magnetic field
(B), which are perpendicular to each
other.

4. ILLUSTRATIONOF ELECTROMAGNETIC WAVES

6. The relationship between wavelength & frequency can
be written as:
c = ν λ
As photon is subjected to energy, so
E = h ν = h c / λ
 The electromagnetic spectrum is the range of all possible
wavelengths of electromagnetic radiation, ranging from
high energy gamma rays through visible light and down to
low energy radio waves.
 Various astronomical phenomenon can only be
observed via specific wavelengths different from visible
light.

8.  On passage through a glass prism, the white light is
separated into its component colors - red, orange, yellow,
green, blue, indigo and violet. The separation of
visible light into its different colors is known as ’Dispersion
of Light’.

9. SPECTROSCOPY
ABSORPTION EMISSION
ATOMIC
MOLECULAR
FLOURESCENCE

10. Law of Absorption : THE BEER-LAMBERT LAW
SOURCE OF
RADIATION
BEAM
SELECTOR
BEAM
SPLITTER
WAVELENGTH
SELECTOR
MONOCHROMATIC
RADIATION
MIRROR
REFERENCE CUVETTE
SAMPLE CUVETTE
ELECTRON
PHOTOPLATE
ELECTRON
CAPTURE
CIRCUIT
A
Io
Io
I
I
WATER
ABSORBING MEDIA

11. BEER’S LAW  A ∝ c
LAMBERT’S LAW  A ∝ l
On combining the two laws, we get the BEER-LAMBERT
LAW as follows :
Log10 Io/I = Ɛ.l.c or A = Ɛ.l.c
where, Io = Intensity of incident light
I = Intensity of transmitted light
A = Absorbance
l = Path length of absorbing media (in cm)
c = Concentration (in moles/litre)
Log10 Io/I = Absorbance or optical density
Ɛ = Molar extinction coefficient or Molar
absorptivity constant

12. UV-VIS ABSORPTION SPECTROSCOPY or
ELECTRONIC EXCITATION SPECTROSCOPY
This spectroscopy is based on the transitions of electron
from ground state molecular orbital to excited state due to
the absorption of electromagnetic radiation from UV and
visible region.

13. ORGANIC ABSORPTION AND ELECTRONIC
TRANSITONS
GRAPHICAL REPRESENTATION : The Energy Level Diagram
These are
normally empty
Contain lone pair
These contain
normal bonding
pair of electron

14. • σ → σ* transition1
• π → π* transition
2 • n → σ* transition
3
• n → π* transition4
TYPES OF ELECTRONIC
TRANSITIONS

15. λmax

16. (μl/ml)

17. UV-VIS
SPECTROPHOTOMETER
ABSORBANCE SCREEN WAVELENGTH
SELECTOR
METHYLENE BLUE SAMPLES WITH
DIFFERENT CONCENTRATIONS
CUVETTES CUVETTE HOLDER

18. USES OF UV-VIS SPECTROSCOPY :
 Quantitative measurement.
 Structural elucidation for presence of n electrons
(heteroatom).
 Identify the degree of unsaturation.
 Determination of λmax.
LIMITATIONS OF UV-VIS SPECTROSCOPY :
 It is measured ideally in micromole(μmol)
concentration and shows inappropriate results beyond
it.

19. INFRARED SPECTROSCOPY / VIBRATIONAL
SPECTROSCOPY / FUNCTIONAL GROUP
SPECTROSCOPY
‘IR Spectroscopy’ is capitalized on the concept that
functional groups absorb specific frequencies of energy
based on their structure. So basically, it measures the
vibrations of atoms which make it possible to determine
which functional group is present.
The radiations used in IR spectroscopy are low energy
infrared radiation and microwaves.
Infrared radiation is commonly divided into three sub-
regions:
(1) Near – IR or NIR
(2) Mid – IR or MIR
(3) Far – IR or FIR

20. TYPES OF VIBRATIONS :
(1) STRETCHING (2) BENDING
(3) ROTATIONAL

21. INSTRUMENT

22. USES OF IR SPECTROSCOPY :
 It is used to identify the presence or absence of
a heteroatom in a molecule.
 Various parameters of a molecule like bond length,
bond angle and bond energy are also identified from
this technique.
LIMITATIONS OF IR SPECTROSCOPY :
 Poor sensitivity to molecular units with small oscillatory
dipoles during a vibrational transition since these
modes
do not absorb strongly in the infrared.
 It does not provide information about the relative
location

23. NUCLEAR MAGNETIC RESONANCE
 ‘NMR’ is a spectroscopic technique that involves
change in
nuclear spin energy in the presence of an external
magnetic field.
 It is based on the absorption of electromagnetic
radiation
in the radio-frequency/ radiowave region.
 The radiowaves flip the nucleus from lower energy state
to
higher energy state. The nucleus now wants to return to
the lower energy state and when it does so, the energy

25. INSTRUMENT

26. USES OF NMR SPECTROSCOPY :
 It is one of the most advanced spectroscopic techniques
used to identify the position of heteroatom in molecular
form.
 A very important diagnostic tool in medicine that is
based on the principle of NMR is a technique known as
Magnetic Resonance Imaging (MRI). It uses strong
magnetic fields and radiowaves to form images of the
body. It is used for the diagnosis and evaluation of
diseases.
LIMITATIONS OF NMR SPECTROSCOPY :
 Long duration of analysis.
 High cost.

27. ELECTRON SPIN
RESONANCEESR is a method for studying materials
with unpaired electrons (radical study).
The basic concept of ESR are analogous to
those of NMR but it is electron spins that
are excited instead of the spins of atomic
nuclei.
ESR is particularly useful for studying
metal complexes or organic radicals.

28. INSTRUMENT

29. OPTICAL ROTATORY DISPERSION /
CIRCULAR DICHROISM
Most of the biological compounds are optically active,
and show optical rotation. The technique of ORD
measures the ability of optically active compounds to
rotate the PPL as a function of wavelength.
 When PPL is passed through a solution that contains an
optically active compound, there is net rotation of the
PPL. The light is rotated either clockwise (dextrorotatory)
or counterclockwise (laevorotatory) by an angle that
depends on the molecular structure and concentration of
the compound, the pathlength and wavelength of light.

30. NORMAL
UNPOLARISED
LIGHT
POLARISER
PLANE
POLARISED
LIGHT
OPTICALLY ACTIVE
COMPOUND IN SAMPLE
TUBE
ANALYSER
ROTATION OF
PLANE POLARISED
LIGHT
RAY DIAGRAM OF A SPECTROPOLARIMETER

31.  In CD, circularly polarised light is used, which is
obtained by superimposing two PPL’s of same
wavelengths and amplitudes which are polarised in
two perpendicular planes, but there is a phase
difference of 90° between them.
POLARISER PLANE
POLARISED
LIGHT
PHASE
CHANGER
SUPERIMPOSED RADIATION
(CIRCULARLY POLARISED)

32. INSTRUMENT

33. USES OF ORD/CD SPECTROSCOPY
Excellent method for the study of conformations
adopted by proteins and nucleic acids due to various
interactions in a solution.
Comparison of secondary and tertiary structure of wild
type and mutant proteins.
Used in determining direction and angle of deflection of
chiral compounds (D or L).
LIMITATIONS OF ORD/CD SPECTROSCOPY
It only provides qualitative analysis of data and does not
provide atomic level structural analysis.
The observed spectrum is not enough for claiming one
and only possible structure.

34. X-RAY DIFFRACTION (XRD)
“ Every crystalline substance gives a pattern; the same
substance always gives the same pattern; and in a mixture
of substances each produces its pattern independently of
the
others”
 The X-Ray diffraction pattern of a pure substance is
like a fingerprint of the substance. It is based on the
scattering of X-Rays by crystals.
 The atomic planes of a crystal cause an incident beam
of X-rays to interfere with one another as they leave the
crystal. This phenomenon is called X-Ray diffraction.

35. K Shell
L shell
M shell
X-Rays
PRODUCTION OF X-RAYS :

36.  When an incident X-Ray beam hits a scatterer,
scattered X-Rays are emitted in all directions. Interference
occurs among the waves scattered by the atoms when
crystalline solids are exposed to X-Rays. There are 2 types
of interference patterns depending on how the waves
overlap one another :

37. The constructive interference from a diffracting
crystal is observed as a pattern of points on the
detector. The relative positions of these points
are related mathematically to the crystal’s unit
cell dimensions.

38. BRAGG’S LAW
(BY W.L. BRAGG & W.H. BRAGG)
The X-Ray diffracted from atoms in crystal planes obey
the laws of reflection.
The two rays reflected by successive planes will be in
phase if the extra distance travelled by the second ray is
an integral number of wavelengths.
BRAGG’S
EQUATION :
nλ = 2d sinθ
where, n = Integer
d = Lattice spacing
θ = Angle of incidence
λ = Wavelength of incident X-Rays

39. INSTRUMENT
Picture : INNOVATION CENTRE, BU
Model : RIGAKU

40. USES OF X-RAY CRYSTALLOGRAPHY
:
To find the structure of an unknown material.
To determine the atomic arrangement of a crystal.
To measure the thickness of thin films and multilayers.
The powder XRD pattern may be thought of as finger
print of the single crystal structure, and it may be used
to conduct qualitative and quantitative analysis.
LIMITATIONS OF X-RAY
CRYSTALLOGRAPHY:
High cost.
Time consuming.
The technique ionizes the sample, thereby, the sample
cannot be used again.

41. MASS SPECTROMETRY
 Mass Spectrometry (MS) is an analytical technique that
measures the mass-to-charge ratio of ions. The results
are typically presented as a mass spectrum, a plot of
intensity as a function of the mass-to-charge ratio.
 It is based on the absorption of electromagnetic
radiation
in the gamma wave region.
 This technique basically studies the effect of ionizing
energy on molecules.
 In the past few years, ongoing technological
developments have contributed substantially to

42. COMPONENTS OF MS : General Block Diagram

43. MASS SPECTROMETRY APPARATUS

44. TYPES OF IONISATION TECHNIQUES
:
 VOLATILE SAMPLES
 Electron ionisation
 Chemical ionisation
 NON-VOLATILE SAMPLES
 Fast atom bombardment
 Thermospray
 Matrix Assisted Laser Desorption Ionisation
(MALDI)
 Electrospray ionisation
 Atmospheric pressure chemical ionisation.

45. INSTRUMENT
Picture : INNOVATION CENTRE, BU
Model : CLARUS SQ 8 C

46. USES OF MS :
 The mass spectra are used to determine the elemental
or isotopic signature of a sample, the masses of particles
and of molecules, and to elucidate the chemical identity
or structure of molecules and other chemical
compounds.
 The high sensitivity, detection selectivity, qualitative
capability and mass accuracy of the instrument makes
its use in drug testing and discovery, food
contamination detection, pesticide residue analysis,
isotope ratio determination, protein identification and
carbon dating.LIMITATION OF MS :
 If the isomers of a compound have the same m/z
ratio,
they will not be distinguished by the MS.

47. EMISSION SPECTROSCOPY
 Atoms or molecules that are excited to high energy
levels can decay to lower levels by emitting radiation.
The substance first absorbs energy and then emits this
energy as light.
 Emissions can be induced by sources of energy such as
flames or EMR. For atoms excited by high temperature,
the light emission is commonly called atomic emission
(emission spectroscopy) and for atoms excited with
EMR, the light emission is called atomic flourescence
(flourometry).

48. FLOURESCENCE SPECTROSCOPY
Flourescence is an emission phenomenon and is
observed when after excitation by the absorption of a
photon, an electron returns from the first excited state to
the ground state.
When photons are incident with original intensity Io,
they induce some energy to the sample, thereby, exciting
the electrons of the same energy level. These particles
then emit radiations in all directions.

49. INSTRUMENT

50. USES OF FLOURESCENCE
SPECTROSCOPY : Used for heavy metal detection.
 Used in flourescent solar collector.
 Diagnostic and research tool in medical field.
 Quantitative measurement up to femtomoles.
 Allows real-time labelling of molecules of interest.
LIMITATIONS OF FLOURESCENCE
SPECTROSCOPY :
All molecules are not flourescent hence, only a few can
be detected.
Loss of recognition capability and photostability.
Susceptible to auto-flourescence.

51. ‘SCIENCE’ is simply the
word we use to describe
a method of organizing
our ‘CURIOSITY’
- TIM MINCHIN
Thank You!