by HARVINDAR SINGH .M.PHARM PHARMACEUTICS

1. Submitted To
Mrs. Neetu Sachan
Asst. Prof. IFTMU
Submitted By
Mr. HARVINDAR
M.PHARM 1st SEM

2.  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.  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.

4.  U V Spectroscopy is concerned with the study of
absorption of uv radiation. Which ranges from 200 nm
to 400 nm.
 Compound which are coloured, absorb radiation from
400 nm to 800nm. But compound which are colorless
absorbed radiation in UV region.
 In both UV as well as Visible spectroscopy only the
valence electrons absorb the energy there by the
molecules undergoes transitions from ground state to
exited state.

5.  This absorption is characteristic and depends on the
nature of electron present.
 The intensity of absorption is characteristic and
depends on the concentration and path length as given
by BEER’s and LAMBERT’s law.

6.  Any molecule has either n, pie or sigma or a
combination of these electrons. These bonding (sigma
& pie ) and non bonding (n) electrons absorb the
characteristic radiation and under goes transition from
ground state to exited state .
 By the characteristic absorption peak, the nature of
electrons present and hence the molecular structure can
be elucidated.

7.  When a monochromatic radiation is passed through a
solution, the decrease in the intensity of radiation with
thickness of the solution is directly proportional to the
intensity of the incident light.

8. When a monochromatic radiation is passed
through a solution, the decrease in the
intensity of radiation with thickness of the
solution is directly proportional to the
intensity of the incident light as well as
concentration of the solution.
Generally the main use of Beer’s Law is to
determine the concentration of various
solutions
& Lambert’s Law

9.  When a monochromatic radiation is passed through a
solution, the decrease in the intensity of radiation with
thickness of the solution is directly proportional to the
intensity of the incident light.
 Generally the main use of Beer’s Law is to
determine the concentration of various
solutions

10.  Intensity of beam of parallel monochromatic
radiation decrease expontially with the no. of
absorbing molecules. More simply…
“Absorbance directly proportional to
concentration)”.
A = log(IO/IT)=k”C/2.303 .
Where's,
A = log(IO/IT)-absorbance.
C-concentration
k’/2.303-absorptivity

11.  Beer-Lambert Law states about the linear
relationship between absorbance and concentration of
an absorbing species.
A = abc
A is the absorbance
“a” is molar absorptivity in L/[(mole)(cm)]
“b” is the path length in cm
“c” is the concentration of the analyte
(sample) in mol/L

12. From A Working Curve – one can
determine the concentration of an
unknown sample by knowing the
absorption.

13.  The Beer-Lambert Law is rigorously obeyed when a
single species is present at relatively low
concentrations.
 The Beer-Lambert Law is not obeyed: High
concentrations Solute and solvent form complexes
Thermal equilibrium exist between the ground state and
the excited state Fluorescent compounds are present in
solution

14.  The UV radiation region extends from 10 nm to 400
nm and the visible radiation region extends from 400
nm to 800 nm. Near UV Region: 200 nm to 400 nm Far
UV Region: below 200 nm
 Far UV spectroscopy is studied under vacuum
condition.
 The common solvent used for preparing sample to be
analyzed is either ethyl alcohol or hexane.

15.  σ electron from orbital is excited to corresponding anti-
bonding orbital σ*.
 The energy required is large for this transition.
 e.g. Methane (CH4) has C-H bond only and can
undergo σ → σ* transition and shows n absorbance
maxima at 125 nm.

17.  π electron in a bonding orbital is excited to
corresponding anti-bonding orbital π*.
 Compounds containing multiple bonds like alkenes,
alkynes, carbonyl, nitriles, aromatic compounds, etc
undergo π → π* transitions.
 e.g. Alkenes generally absorb in the region 170 to 205
nm.

18.  • Saturated compounds containing atoms with lone pair
of electrons like O, N, S and halogens are capable of n
→ σ* transition.
 • These transitions usually requires less energy than σ
→ σ* transitions.
 • The number of organic functional groups with n → σ*
peaks in UV region is small (150 – 250 nm).

19.  • An electron from non-bonding orbital is promoted to
anti-bonding π* orbital.
 • Compounds containing double bond involving hetero
atoms (C=O, C≡N, N=O) undergo such transitions.
 • n → π* transitions require minimum energy and show
absorption at longer wavelength around 300 nm.

20. •These electronic transitions are forbidden transitions &
are only theoretically possible.
•Thus, n → π* & π → π* electronic transitions show
absorption in region above 200 nm which is accessible
to UV-visible spectrophotometer.
•The UV spectrum is of only a few broad of absorption.

23.  In U.V spectrometer, the most commonly used
radiation source are:
 Tungsten lamp
 Hydroger or Deuterium lamp
 Xenon discharge lamp
 Mercury arc…

24.  Functioning same as an electric light bulb. The
tungsten filament lamp is commonly employed as a
source of visible light. This type of lamp is used in the
wavelength range of 350 - 2500 nm.
 The energy emitted by a tungsten filament lamp is
proportional to the fourth power of the operating
voltage. This means that for the energy output to be
stable, the voltage to the lamp must be very stable
indeed. Electronic voltage regulators or constant
voltage transformers are used to ensure this stability.

25.  Grating monochrometors are used. Filters and prism
monochrometors are not used because of low
resolution.
 The mirror gratings etc. are made up of quartz. Since
glass absorb UV radiation from 200nm to 300nm
mirrors are front surfaced to prevent absorption of
radiation.

27.  CUVETTES: Virtually all UV spectra can be recorded
in the solution phase and the samples are placed in cells
or cuvettes. Cells may be made of glass, plastic or
quartz. Quartz is transparent in all (200- 700nm) ranges
and is normally used in UV region. Plastic and glass
are only suitable for visible spectra.

28.  Signal from detector have to be proportional to the
amount of radiation Response of detector have to be
linear Detector should have wide linear response range
 DETECTORS: The four common types of detectors
are:
1.Barrier layer cell or photovoltaic cell
2.Phototube or photocell or photo-emissive tubes
3.Photomultiplier tubes(PMT)
4. The linear photodiode array
5. Charge-Coupled Devices (CCDs)

29.  The Ultra-violet spectroscopy has been mainly applied
for the detection of functional group, the extent of
conjugation, detection of polynuclear compound by
comparison etc.
 The wavelengths of absorption peaks can be correlated
with the types of bonds in a given molecule and are
valuable in determining the functional groups within a
molecule.

30. Some important application is as follow:
1. Extent of conjugation: Conjugation may be between two or
more C=C or triple bond, between C-C or C-O bond,
between double bond in aromatic rings.
2. Identification of unknown compound: Unknown compound
can be identified by their characteristic absorption peaks.
3. Detection of functional group.
4. Distinction of conjugation and non conjugation compound.
5. Examination of poly nuclear carbon atom.
6. Elucidation of the structure of Vit A and Vit K.
7. Preference over two Tautomer form.
8. Detection of impurity.