3. • It is the branch of science that deals with the study of
interaction of matter with light.
OR
• It is the branch of science that deals with the study of
interaction of electromagnetic radiation with matter.
3
4. Electromagnetic radiation consist of discrete
packages of energy which are called as photons.
A photon consists of an oscillating electric field
(E) & an oscillating magnetic field (M) which are
perpendicular to each other
4
6. The principle is based on the measurement of
spectrum of a sample containing atoms /molecules.
Spectrum is a graph of intensity of absorbed or
emitted radiation by sample verses frequency
(ν) or wavelength (λ).
Spectrometer is an instrument design to measure
the spectrum of a compound.
6
7. 1. Absorption Spectroscopy:
• An analytical technique which concerns with the
measurement of absorption of electromagnetic radiation.
• e.g. UV (185 - 400 nm) / Visible (400 - 800 nm)
Spectroscopy, IR Spectroscopy (0.76 - 15 μm)
2. Emission Spectroscopy:
• An analytical technique in which emission (of a particle or
radiation) is dispersed according to some property of the
emission & the amount of dispersion is measured.
• e.g. Mass Spectroscopy
7
8. 8
Its is the absorption spectroscopy in which sample solution
is placed in light path the sample will absorb the particular
wavelength which is characteristic to the functional group
present in the light path. The excitation of electron takes
place from higher energy level to lower energy level.The
spectrum will recorded which is the graph consist of
absorption against wavelength.
9. • Sources
• Collimating system.
• monochromator system.
• sample holder or container to hold sample.
• detector system of collecting transmitted
radiation.
• suitable amplifier or readout device.
Spectrophotometer:
o Components of optical instruments
9
11. 11
1- Sources of light
Sources used in UV-Vis Spectrophotometers are continuous sources.
• Continuous sources emit radiation of all wavelengths within the
spectral region for which they are to be used.
• Sources of radiation should also be stable and of high intensity.
Continuous
Sources
Visible and
near IR
radiation
Tungsten
Lamp
320-2500 nm
Ultraviolet
radiation
Deuterium
Lamp
200-400 nm
12. 12
• Filters permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass
through.
• The simplest kind of filter is absorption filters , the most common of this
type of filters is colored glass filters.
• They are used in the visible region.
• The colored glass absorbs a broad portion of the spectrum (complementary
color) and transmits other portions (its color).
Disadvantage
• They are not very good wavelength selectors and can’t be used in
instruments utilized in research.
• This is because they allow the passage of a broad bandwidth which gives
a chance for deviations from Beer’s law.
• They absorb a significant fraction of the desired radiation.
i- Filters
13. ii- Monochromators
They are used for spectral scanning (varying the wavelength of
radiation over a considerable range ).
They can be used for UV/Vis region.
All monochromators are similar in mechanical construction.
All monochromators employ slits, mirrors, lenses, gratings or
prisms.
13
14. 14
1- Prism monochromators
Dispersion by prism depends
on refraction of light which
is wavelength dependent
Violet color with higher
energy (shorter wavelength)
are diffracted or bent most
While red light with lower
energy (longer wavelength
are diffracted or bent least
As a result, the poly-
chromatic white light is
dispersed to its individual
colors.
15. 15
3- Sample compartment (cells)
For Visible and UV spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.
Glass is suitable for visible but not for UV spectroscopy because it
absorbs UV radiation. Quartz can be used in UV as well as in visible
spectroscopy
1 cm 1 cm
Opaque
Face
Transparent
Face Long pathlength
Short pathlength (b)
1 cm pathlength cuvet
16. 16
4- Detectors
The detectors are devices that convert radiant energy into electrical
signal.
A Detector should be sensitive, and has a fast response over a
considerable range of wavelengths.
In addition, the electrical signal produced by the detector must be
directly proportional to the transmitted intensity (linear response).
h
e-
-V
Photosensitive cathode
amplifier
i- Phototube
anode
Phototube emits electrons
from a photosensitive,
negatively charged cathode
when struck by visible or
UV radiation
The electrons flow through
vacuum to an anode to
produce current which is
proportional to radiation
intensity.
17. 17
ii. Photomultiplier tube
It is a very sensitive device in which electrons emitted from the photosensitive
cathode strike a second surface called dynode which is positive with respect to
the original cathode.
Electrons are thus accelerated and can knock out more than one electrons from
the dynode.
If the above process is repeated several times, so more than 106 electrons are
finally collected for each photon striking the first cathode.
18. Double Beam Spectrophotometer
Double beam instrument is the one in which two beams are formed
in the space by a U shaped mirror called as beam splitter or beam
chopper .
Chopper is a device consisting of a circular disc. One third of the
disc is opaque and one third is transparent, remaining one third is
mirrored. It splits the monochromatic beam of light into two beams
of equal intensities.
18
20. 20
Advantages of double beam instruments over single beam
instruments
Single beam spectrophotometer is inconvenient because
1. The sample and blank must be placed alternately in the light path.
2. For measurements at multiple wavelengths, the blank must be run at each
wavelength.
In double beam instruments
1. The absorption in the sample is automatically corrected for the absorption
occurring in the blank, since the readout of the instrument is log the
difference between the sample beam and the blank beam.
2. Automatic correction for changes of the source intensity and changes in the
detector response with time or wavelength because the two beams are
compared and measured at the same time.
3. Automatic scanning and continuous recording of spectrum (absorbance
versus wavelength).
22. UV absorption spectroscopy is one of the best methods
for determination of impurities in organic molecules.
Additional peaks can be observed due to impurities in the
sample and it can be compared with that of standard raw
material. By also measuring the absorbance at specific
wavelength, the impurities can be detected.
22
23. UV spectroscopy is useful in the structure elucidation
of organic molecules, the presence or absence of
unsaturation, the presence of hetero atoms.
From the location of peaks and combination of peaks,
it can be concluded that whether the compound is
saturated or unsaturated, hetero atoms are present or not
etc.
23
24. UV absorption spectroscopy can be used for the
quantitative determination of compounds that absorb UV
radiation. This
determination is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where :
ε -is extinction co-efficient,
c- is concentration, and
b- is the length of the cell used
in UV spectrophotometer.
24
25. UV absorption spectroscopy can characterize those types
of compounds which absorbs UV radiation.Identification
is done by comparing the absorption spectrum with the
spectra of known compounds
25
26. This technique is used to detect the presence or absence
of functional group in the compound
Absence of a band at particular wavelength regarded as an
evidence for absence of particular group
TOLUENE
26
27. 1. Sharma. Y.R. Elementary Organic Spectroscopy.
First edition .S.Chand Publisher; 2010.
2. Chatwal G.R. Instrumental methods of chemical analysis.
First edition. Himalaya Publisher; 2010
.
3.Textbook: Principles of instrumental analysis, Skoog
et al., 5th edition, chapter 7, 13.
4.Quantitative chemical analysis, Daniel C. Harris, 6th
edition , chapter 20.
5. Ultra-violet Visible Spectroscopy by Alain Martelli .
27
28. 28
6. M. Hesse, H. Meier, B. Zeeh, Spektroskopische
Methoden in der organischen Chemie, 7th ed., Georg
Thieme Verlag, Stuttgart, 2005.
7. P. W. Atkins, J. D. Paula, Physikalische Chemie, 5th
ed., Wiley - VCH, Weinheim, 2013
