UV Visible Spectroscopy involves the study of interaction between electromagnetic radiation and matter. It is used to measure absorption or transmission of light passing through a sample. The technique utilizes wavelengths in the UV and visible range from 200-800 nm. Key aspects covered include Beer's law, electronic transitions involved, instrumentation components, and applications such as determining impurities, functional groups, and drug assay.

1. UV Visible Spectroscopy
Presented By
Ankush Awatale
First Year M.Pharm
Pharmaceutics
Institute of Pharmacy and Research

2. • Introduction
• Common Definitions
• Principle
• Electronic Transition
• Beer’s Lambert’s Law and its Deviation
• Instrumentation
• Choice of Solvent and Solvent Effect
• Applications
Contents

3.  Spectroscopy is the brand of science dealing with the study of interaction
of electromagnetic radiation & matter.
 Electromagnetic radiation is a form of energy that is generated when
electrically charged particles move through matter or vacuum.
 UV Visible spectroscopy is an analytical technique which is used to
measure absorption or transmission of light that passes through medium.
 It is suitable for large spectrum of organic compounds and some
inorganic species.
 Region of UV- Visible :
Ultraviolet – 200 to 400 nm
Visible – 400 to 800 nm
Introduction

4. Wavelength :-
It is distance between two successive maxima or
minima on an electromagnetic wave.
 It is denoted by λmax.
 Unit is nm
Frequency :-
No. of wavelength passing through given point in unit
time is called as frequency.
 It is denoted by v (nu) or f.
 Unit is Hz.
Common Definitions

5.  UV – Visible is an absorption technique which is used for
measurement of attenuation of beam of light which
passes through sample solution containing transparent
medium or after reflection from sample surface.
Absorption measurement can be single λmax or over an
extented spectral range.
Principle

6.  Electronic Transition is defined as electromagnetic radiation which
causes electron to be excited which result in promotion from a
bonding or non-bonding orbitals.
 Energy absorbed by UV- Visible region by molecule causes change
in electronic energy resulting in transition of electron from ground
state to excited state.
 4 types of electronic transition are mostly used :
1. σ → σ*
2. n → σ*
3. π → π*
4. n → π*
Electronic Transition

7. 1.σ → σ*:
This transition involves compound in which all
electrons are involved in single bond.
A transition of electron from bonding sigma orbital to higher
energy antibonding sigma orbital .
Higher energy is required.
Very short wavelength.
Eg : CH4
2. n → σ* :
This transition involves saturated compounds with
atoms containing lone pair of electrons.
It requires lesser energy than the σ → σ* transition.
Eg : NH3

8. 3. π → π* :
This transition involves excitation of electron from
bonding π orbital to an antibonding π* orbital.
It is considered as non-polar transition and it is solvent
independent.
It requires lesser energy than n → σ* transition.
Eg : Alkene,Alkyne.
4. n → π* :
This transition involves excitation of lone pair
electron to the π* antibonding orbital.
It requires least energy and higher wavelength.
This transition is observed in acidic solution in which bond is
formed between proton and lone pair on the heteroatom.
Eg : C=O,C≡N.

9. σ → σ*>n → σ*>π → π*>n → π*
WAVELENGTH
ENERGY

10.  Beer’s law :
When a beam of light is passed through transparent
medium the intensity of incident light decreases exponentially as the
concentration of absorbing medium increases.
 Lambert’s law :
When a beam of light is passed through transparent
medium the rate of intensity of incident light decreases as thickness
of medium increases.
dI/ db = KI ………1
Where, I is intensity of light
b is thickness of medium
K is proportionality constant
Beer’s Lambert’s Law

11. Integrating the equation 1 and putting I=Io when b=0
It = I0 e-kb ………2
Where, I0 is intensity of incident light
It is intensity of transmitted light
k is absorption coefficient
Converting eq 2 from natural log to base 10 we get,
It = I0 10-kb ………3
Similarly from Beer’s law we get,
It = I0 e-k’c ..…..…4
Converting eq 4 from natural log to base 10 we get,
It = I0 10-k’c ………5
Combining two law i.e. eq 3 and 5 we get,
It = I0 10Ecb ………6
Where, k and k’ is E i.e Molar absorptivity which means constant which
is dependent upon λmax of incident radiation and nature of absorbing
material.

12. Taking log on both sides of eq 6 we get,
Log I0 / It = Ecb………7
The ratio of It / I0 is fraction of light transmitted through thickness of
medium i.e. called as Transmittance.
Reciprocal of Log Io / It is called as absorbance which is denoted by A.
Therefore we get,
A = Ecb
There are 3 types of deviation in Beer’s Lambert’s Law,
1. Real Deviation occurs due to changes in refractive index of substance.
2. Chemical Deviation occurs due to substance undergoing ionization,
dissociation or even may react with solvent.
3. Instrumental Deviation occurs due to changes in the instrument like
fluctuation,sensitivity,errors.

13. Instrumentation
The instrumentation of UV – Visible Spectroscopy has following basic
components :
1. Radiation Source
2. Collimating System
3. Monochromator
4. Sample Holder
5. Detector
6. Read out System

14. 1) Source :
Source are those which emits the radiation. In all sources excitation is done by
passing electron through gas which result in electronic, vibrational, and rotational
excitation of gas molecules.
There are 5 types of sources :
1. Tungsten lamp
2. Hydrogen discharge lamp
3. Deuterium lamp
4. Xeon discharge lamp
5. Mercury arc lamp
2) Collimating System :
This is a device which produces a beam of parallel rays or form distant virtual
image that can be viewed without parallax.
The system contains-
1. Lens which must be transparent to the radiation being used.
2. Mirror which is used to reflect, focus, collimate light beam in
spectrophotometer.
3. Slit is an important device in resolving polychromatic radiation into
monochromatic radiation.

15. 3) Monochromator :
Monochromator is the device which gives continuous spectrum of narrow band of
wavelength and allow light of only required wavelength and absorb other
wavelength.
It consists :
1. Filter – It is used for isolation of narrow band of radiant energy of desired
spectral region.
There are many types of filter like glass, gelatin, interference, absorption filter.
2. Prism – It is made up of glass, quartz or fused silica. It is used to disperse UV
light, Visible, IR radiation.
3. Grating – It consists of parallel lines or grooves placed on highly polished
surface such as alumina. When light falls on this surface it spread out in different
direction and acts as a scattering center.
4) Sample Holder :
The sample holder used in UV Visible are known as cuvette and it is made up of
quartz or fused silica. It should be transparent and allow to pass the wavelength
from the holding sample. It is rectangular cylindrical with flat end. Mostly 1 cm
path length cuvette are used but may vary in path length i.e. 0.1 to 10 cm.

16. 5) Detector :
Detector used in UV Visible spectrophotometer help to convert the EMR into
current which can be visualize in read out system.
It should be highly sensitive and have fast response time.
It should provide quantitative response in wide spectrum of radiation.
It should generate sufficient signal or electric current which can be measured.
There are different types of Detector used in UV Visible Spectroscopy but 3 are
mostly used which are :
1. Barrier Layer Cell [ Photovoltaic Cell]
2. Photoemmissive cell [ Phototube]
3. Photomultiplier [ Vacuum Phototube]
6) Read Out System :
The read out system is gives the readings of the radiation in visual form on the
display.

17.  There are different types of solvents used in UV – Visible Spectroscopy
like water and some organic solvents like ethanol, acetonitrile,
cyclohexane, dimethylformamide, dimethylsulfoxide.
 Some solvents like acetone, isopropyl alcohol, Toluene are also used.
 95% ethanol is the best solvent as it is cheap and provide proper result
because its absorbance cut off wavelength region is very low i.e 205nm.
 Hexane and hydrocarbon can also be used as they are less polar and least
interact with molecules under investigation.
 The Effect of solvent on absorption spectra is that it shift peak to shorter
or longer wavelength. It depends on nature of interaction of particular
solvent with environment of chromophore in excited state.
Choice of Solvent and Solvent Effects

18.  Chromophores are the functional group if present in the compound are
responsible for colour to the compound.
 Eg. Nitro group if present gives yellow colour to the compound.
 Auxochromes are the groups which itself does not act as chromophore
but when attached to a chromophore it shift absorption maxima towards
longer wavelength with increase in intensity of absorption.
 Two shifts take place because of Auxochrome :
1. Bathochromic shift :
It is also called as Red shift. It shifts absorption maxima towards longer
wavelength because of presence of certain group such as OH and NH2 or by
change in solvent.
2. Hypsochromic shift :
It is also called as Blue shift . It shifts absorption maxima towards shorter
wavelength or maybe caused by removal of conjugation or by change in
solvent.

19.  Two effects which also take place because of Auxochrome :
1. Hyperchromic effect :
This effect involve an increase in intensity of absorption and is
usually occurs due to introduction of an auxochrome.
2. Hypochromic effect :
This effect involve an decrease in intensity of absorption and is
usually occurs due to groups which are able to distort the geometry
of molecules.

20.  It is used to determine the impurities present in the organic
molecules.
 It is also used in quantitative determination of compounds that
absorb UV radiation.
 This technique is used to detect the presence and absence of
functional groups.
 The assay of many drugs which are in form of raw material or
formulation in suitable solution can be done.
 This technique is also used in determination of metal contaminants.
 Structural elucidation of organic compounds can be done like
presence of double bonds or hetero atoms like O, N, S.
 It can be also used in determination of Molecular weight of the
compound.
Applications

21. THANK
YOU