for B.PHARM, 5TH SEMSETER

1. BRIEF REVIEW OF EMR,
UV-VISIBLE SPECTROSCOPY
AND CHROMOPHORE
CONCEPT

2. INTRODUCTION
What is Spectroscopy?
- Spectroscopic techniques involve the study of
interaction of matter with electromagnetic
radiations.
- Electromagnetic radiation, which is made up of
photons, have both wave as well as particle
characteristics.
- Matter/atoms/molecules shows unique
characters by releasing or absorbing the
radiation.
 When electromagnetic radiation travels
through a medium containing atoms, there are
three possibilities-
1. There is no absorption of light
2. There maybe reflection, refraction, or
scattering of light
3. There maybe absorption of energy.

3. In case of absorption,
The atoms are excited to their excited
state, and then they return to the
ground state, which is a more
stable state, with emission of
energy.
UV-Visible Spectroscopic techniques
utilize the measurement of this
emitted energy, for analytical
purposes.

4. ELECTRO MAGNETIC RADIATION
EMR is a form of energy that
is transmitted through space
at an enormous velocity.
 EMR is made up of discrete
particles called photons.

5.  EMR have both wave characteristic
and particle characteristics.
Waves or Particles
• Electromagnetic radiation has properties of
waves but also can be thought of as stream of
particles.
Example: Light
• Light as a wave: Light behaves as a
transverse wave which we can filter using
polarized lenses.
• Light as particles (photons).

6. A photon is a tiny
particle that comprises
waves of electromagnetic
radiation.
As shown by
Maxwell, photons are just
electric fields traveling
through
space. Photons have no
charge, no resting mass,
and travel at the speed of
light.
A photon

7. Representation of an Electromagnetic wave

8. FREQUENCY point in unit time.
Units- Hz (Hertz)
Number of complete wavelength units passing through
a given) or cps (cycles per second).
WAVE LENGTH
Distance between the two peaks.
Units- metres, centimetres, millimetres, nanometers
WAVE NUMBER
Number of waves per cm.
Expressed in cm-1

12. Type of Electro Magnetic Radiation
• Cosmic rays
• Gama rays
• X- rays
• UV radiation
• Visible radiation
• IR radiation
• Microwaves
• Radio waves

15. The energy of EMR can be given
by the equation
E = h‫ט‬
Where E = Energy of radiation
h= Plank’s constant ( 6.624 X 10 -34
JSec)
‫ט‬ = Frequency of radiation

16. velocity of light in vacuum/
wavelength
Or
3 X 108 msec-1 / wavelength
Hence E = h‫ט‬ = hc/ λ = hc‫ט‬

17. UV – VISIBLE SPECTROSCOPY
 The wavelength of UV- Visible ranges from 200nm – 800nm
 Wavelength for UV ranges from 200nm-400nm
 Wavelength for visible ranges from 400nm-800nm.
Types of spectroscopy:
Absorption
Emission
Scattering

18. THE ELECTROMAGNETIC SPECTRUM
Ultraviolet region : 180-400 nm ( 200 – 380 nm used for analytical purposes,
also called as the near ultraviolet region.)
Vacuum ultraviolet region : 1 – 180 nm
Visible region : 400 – 750 nm
Infrared region : 780 nm – 300 

19. VISIBLE SPECTRUM

20. Used for quantitative analysis and serve as an
useful auxiliary tool for structural elucidation.
UNITS USE IN UV-VISIBLE SPECTOSCOPY
For the measurement of wavelength in UV-Visible
spectroscopy following units are used,
nm (nanometer)
Ǻ (angstrom)
cm-1(centimeter inverse)
1 nm=10 Ǻ=10-7cm

21. ELECTRONIC TRANSITIONS
 Absorption of ultraviolet and visible
radiation in organic molecules is restricted to
certain functional groups (chromophores) that
contain valence electrons of low excitation
energy.
This is because the superposition of
rotational and vibrational transitions on the
electronic transitions gives a combination of
overlapping lines.

24. CHROMOPHORE
The functional group gives colour
to a compound. Example nitro
group.
Exhibits absorption of
electromagnetic radiations in the
visible or ultraviolet region.

25. Chromophores -one of two forms:
Conjugated pi systems
Common examples include retinal, various food
colourings, lycopene, β-carotene and
anthocyanins, fabric dyes (azo compounds), etc.

26. METAL COMPLEXES
 Chlorophyll
 Haemoglobin

27. Their interaction with EMR
 Straightens in response to a photon γ (light), of the
correct wavelength

28. AUXOCHROMES
Group of atoms attached to a chromophore
which modifies their ability to absorb light
Example: -OH , - NH2 , Aldehydes
A functional group of atoms with non-bonded
electrons which is attached to a chromophore
This occurs due to the presence of at least one
lone pair of electrons

29. Benzene no colour (chromophore
absent)
Nitrobenzene (pale yellow colour)
nitro group.
Para-hydroxynitrobenzene exhibits a deep
yellow color. Here an auxochrome (-OH) is
conjugated with the chromophore -NO2.

30. There are mainly two types of
auxochromes-
1. Acidic -COOH, -OH, -SO3H
2. Basic -NHR, -NR2, -NH2

32.  Lambert’s Law Vs Beer’s Law

33. DEVIATION FROM BEER’S LAW
 In presence of impurities.
 If monochromatic light is not used.
 If the width of slit is not proper.
 If undesirable radiations fall on the detector.
 If solution species under goes polymerization.
 Beer’s law can’t be applied to suspension.

34. Instrumentation
1. Colorimeters ( for the Visible
region )
2. Spectrophotometers ( for the
Ultraviolet region)

35. Colorimeters
 These are instruments used to measure the
absorbance of light in the visible region.
 Measurement of the wavelength and the intensity
of electromagnetic radiation in the visible
region of the spectrum.
 It is used extensively for identification and
determination of concentrations of substances
that absorb light.
 They are comparatively inexpensive and less
accurate.
 The range of wavelength used is small.
E.g. 400-700nm

37. Spectrophotometer
 Spectrophotometers are instruments used
for the quantitative measurement of
reflection or transmission properties of a
material as a function of wavelength.
 They can be used over a wider
wavelength range e.g.. 300-900 nm or
sometimes up to 1000nm and are very
accurate.

39. Instrumentation
Colorimeters
 Source of light
 Filters
 Sample Cells
 Detectors
Spectrophotometers
 Source of light
 Monochromator
s
 Sample Cells
 Detectors
 Recorders

40. Source of light
Colorimeters
 The visible
spectrum ranges
from 400-800 nm.
 Common sources
of light-
 Tungsten Filament
Lamp
 Carbon Arc Lamp
Spectrophotometers
 The Ultraviolet region
ranges from 180-
400nm.
 Sources of light -
 Hydrogen discharge
Lamp
 Deuterium Lamp
 Xenon Discharge
Lamp
 Mercury Arc Lamp

41. Filters and Monochromators
 Filters and Monochromators act as
wavelength selectors.
 Colorimeters commonly use filters while
monochromators are seen in
spectrophotometers.
 Filters are comparatively less accurate as
wavelength selectors than
monochromators.

42. Filters
 There are two types of filters-
Absorption Filters
Interference filters

43. Absorption Filters
 Chiefly made of glass which are coated
with pigments
 Or they contain colored gelatin sheets
sandwiched between two glass sheets.
 These filters absorb the unwanted
radiation and transmit only the required
radiation.
 They are less accurate since their band
pass is more than 30nm.

44. Monochromators
 They are better and more efficient in
converting polychromatic light into
monochromatic light.
 Parts of a monochromator-
 Entrance slit
 Collimator
 Grating or Prism
 Exit slit

45. Sample cells
 These are cells or cuvettes used to hold the
sample solutions.
 A cuvette (French: cuvette = "little
vessel").
 The material of these cells do not absorb at
the wavelength being observed.
 Glass is used for the visible region, while for
the UV region cells made of quartz are used.
 Cells are mostly cylindrical or rectangular in
shape and of 1cm (short) to 10cm (long)
wavelength.
 Quartz exists in two forms, the normal α-
quartz and the high-temperature β-quartz,
both of which are chiral. The transformation
from α-quartz to β-quartz takes place
abruptly at 573 °C (846 K; 1,063 °F).

47. Detectors
 Detectors used in UV-Visible
Spectrophotometers can be called as
Photometric detectors.
 Principle –
 When a particular wavelength of radiation
passes through a sample solution, a part
of it is absorbed and some part of it is
transmitted. The amount of transmitted
radiation is measured by a detector.

48. Different types of detectors
1. Barrier Layer cell or Photo Voltaic Cell
2. Photo tubes or Photo emissive cells
3. Photo multiplier tubes

49. Applications
1. Quality control and purity – Colorimeters can
be used to detect impurities.
2. Quantitative analysis
3. Determination of Ligand-metal ratio
4. Structure ellucidation of organic compunds
5. Determination of pka values of indicators
6. Determination of molecular weight of amines
7. Determination of elements, ions or functional
groups.
8. Determination of organic substances and
pharmaceuticals.
9. Chemical Kinetics.
10. Proteins estimation
11. Enzymes Assay
12. DNA/RNA estimation

50. REFERENCES
 Anjaneyulu Y, Chandrasekhar K, Manickam Valli, A Textbook of Analytical Chemistry,
Pharma Book Syndicate, Hyderabad, 2006 edition, Page No 351.
 Willard Hobart H, Marritt Lynne L, Dean John A, Settle Frank A, Instrumental
methods of analysis, CBS Publishers and Distributors, New Delhi, seventh edition, Page
No 97,100.
Khopkar S.M. ,Basic concept of analytical chemistry, New Age International (P)
Limited, Publishers, New Delhi, third edition 2008Page No 245.
Instrumental methods of analysis ,seventh edition by Hobart H.
Willard,Lynne L. Merritt,John A. Dean,Frank A. Settle,pp-118
Instrumental methods of chemical analysis fifth edition by Gurdeep R.
Chatwal,Sham K. Anand pp-2.149
A textbook Analytical Chemistry by Y.Anjaneyulu,K.Chandrasekhar,Valli
Manickan,pp-362.
Introduction of instrumental analysis,third edition by Robert D. Braun,pp-
261-263.