UV-Visible spectroscopy, a versatile analytical technique used to study the interaction of molecules with light within the ultraviolet and visible regions of the electromagnetic spectrum. It is a fundamental tool in chemistry, biochemistry, and related fields for analyzing the electronic structure of molecules, determining their concentrations, and studying their behavior. 1. Principle of UV-Visible Spectroscopy: UV-Vis spectroscopy is based on the principle that molecules absorb specific wavelengths of light due to electronic transitions. When molecules absorb light in the UV or visible range, they move from a ground state to an excited state. 2. Instrumentation: UV-Vis spectrophotometer is the key instrument used for this technique. Components include a light source, sample holder, monochromator, and a detector. The sample is placed in a cuvette, and the spectrophotometer measures the absorbance of light passing through the sample. 3. Beer-Lambert Law: The Beer-Lambert law relates the concentration of a solution, the path length (distance that light travels through the solution), and the absorbance of light by the solution. A = ε * c * l, where A is absorbance, ε is the molar absorptivity (a constant for a specific compound and wavelength), c is the concentration, and l is the path length. 4. Absorbance Spectra: UV-Vis spectroscopy generates absorbance spectra, which are plots of absorbance versus wavelength. Peaks in the spectra indicate the wavelengths of light that are absorbed by the sample, providing information about the electronic structure of the molecules. 5. Applications: Quantitative Analysis: UV-Vis is widely used for quantitative analysis of compounds by measuring the absorbance of a sample and comparing it to a standard curve. Identification of Compounds: The unique absorbance spectra can be used to identify compounds. Kinetics: UV-Vis can monitor reaction kinetics by following the change in absorbance over time. Pharmaceutical Analysis: It is crucial in quality control for pharmaceuticals. Environmental Analysis: UV-Vis is used in environmental monitoring, such as water quality analysis. 6. Advantages: It's a rapid and simple technique. It can be highly sensitive for many compounds. It is non-destructive to the sample. 7. Limitations: It does not provide structural information about the molecules. It may not be suitable for analyzing complex mixtures.

1. By- Prof. Ankita Raikwar
Department of Analysis
SOPS/JNU

2. Introduction
Principle- Beer Lambert Law
Absorbance ∝ Conc. & Path length
Instrumentation
Application

3.  Spectroscopy
 UV VIS Spectroscopy
 UV Visible Spectrum
 Electronic Transitions
 Types of electronic transitions
 Chromophores
 Auxochromes
 Spectral shifts
 Solvents used in UV VIS Spectroscopy
 Factors affecting the position of UV bands
 Principle
 Beer and Lambert’s law
 Instrumentation of UV visible Spectrophotometer
 Applications

4.  branch of science that deals with the study of
interaction of electromagnetic radiation with matter
 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)
 study of interaction of EMR with matter
 used for analysis of wide range of samples
 Spectrometer- instrument designed to measure the
spectrum of a compound.
 Spectroscopy is a necessary tool for structure
determination.

7.  absorption spectroscopy or reflectance
spectroscopy in the ultraviolet-visible spectral
region
 analytical method that can measure the
analyte quantity depending on the amount of
light received by the analyte
 ultraviolet-visible spectral field refers to
absorption spectroscopy or reflectance
spectroscopy. In the visible and neighbouring
(near-UV and near-infrared (NIR) ranges, this
means that it uses light.

8. Far/vaccum uv region (10-200nm)
near or quartz uv region (200-400nm)
visible region (400-780nm)

9.  based on the absorption of ultraviolet light or visible
light by chemical compounds when electrons are
promoted to higher energy levels, which results in the
production of distinct spectra.
 deals with the measurement of energy absorbed when
electrons are promoted to higher energy levels.
 involves electronic transitions, it is often called
electronic spectroscopy.
 plot of wavelength of light absorbed versus the
absorption intensity (absorbance or transmittance) and
is conveniently recorded by plotting molar absorptivity
(ε) against wavelength (nm).

10. electrons in organic molecules are
involved in bonding as strong bonds, or
weak bonds or present in the non-bonding
form (lone pair)
Hence, variety of energy absorptions for
electronic transitions within a molecule is
thus possible depending upon the nature
of bonding.

11. A) Transitions between bonding and antibonding orbitals : 2
Types-
 (i) σ to σ* Transition(120-200nm) : The excitation between
bonding sigma and antibonding sigma orbitals (σ to σ*
transition) requires large energies.
For example, methane, propane, cyclohexane etc., display σ
to σ* transitions λͅmax for each of these compounds is below
140 nm.
 (ii) π to π* Transition (170-190 nm): The transition or
promotion of an electron from a π bonding orbital to a π*
antibonding orbital. These type of transitions occur in
compounds containing one or more covalently unsaturated
groups like C=C, C≡C, C=O, N=N, aromatic rings, NO2, etc.
In unconjugated alkenes,; ethylene shows λͅmax at around
171 nm.

12. (ii) n to σ* Transition : The excitation of an
electron in an unshared pair (nonbonding
electrons) on nitrogen, oxygen, sulphur or
halogens to an antibonding σ orbital is called
n to σ* transition.
 transition is of moderate intensity located
around 180nm for alcohols, near 190 nm for
ethers or halogen derivatives and in the
region of 220 nm for amines.
(iii) σ to π* Transition and π to σ* Transition :
These electronic transition are forbidden
transitions and are theoretically possible.

13. B) Transitions between non-bonding atomic orbitals
and antibonding orbitals(200nm<) : 2 types
(i) n to π* Transition : Transitions between non-
bonding atomic orbitals holding unshared pair of
electrons and antibonding pi-orbitals are called n to
π* transitions. Non-bonding electrons are held
more loosely than σ bonding electrons and
consequently undergo transitions at comparatively
longer wavelengths.
For ex-compounds containing double bonds
involving hetero atoms bearing unshared pair(s) of
electrons (e.g., C=S, N=O etc.).

14. system responsible for imparting colour to
a compound.
functional group that absorbs
electromagnetic radiation, a 'color' is
thereby produced.
functional group containing multiple bonds
capable of absorbing radiations above
200nm due to n to π* and π to π*
examples are: C=O, C=C, C=N, N=N, C ≡
C, C ≡ N, C = S etc.

15.  functional groups attached to a chromophore
which modifies the ability of the chromophore to
absorb light, altering the wavelength or intensity of
absorption.
 auxiliary group which interacts with the
chromophore causing a bathochromic shift.
 enhances the colour imparting properties of a
chromophore without being itself a chromophore.
 examples are: methyl, hydroxyl, alkoxy, halogens,
and amino and substituted amino groups
(OR,NH2, NHR and NR2).

16.  Bathochromic shift (Red Shift): shift to lower
energy or longer wavelength.
 caused by change of medium (π to π*
transitions undergo bathochromic shift with an
increase in the polarity of the solvent) or
when auxochrome is attached to C=C double
bond
 for example, ethene absorbs at λͅmax = 175
nm while 1-butene absorbs at λͅmax = 190
nm.
 bathochromic shift is progressive as the
number of alkyl groups increases.

17.  Hypsochromic shift (Blue Shift): shift to higher
energy or shorter wavelength.
 caused by change of medium (n to π* transitions
undergo hypsochromic shift with an increase in the
polarity of the solvent)
 for example, acetone absorbs at 280 nm in hexane
and at 265 nm in water.
 shift results from hydrogen bonding which lowers
the energy of n orbital of oxygen atom. This can
also be produced when an auxochrome is attached
to double bonds e.g. C=O) where n electrons are
available.

18. Hypochromic effect: It is the effect leading
to decreased absorption intensity,
for example, benzoate ion has less
absorption intensity than benzoic acid.

19. Hyperchromic effect: effect leading to
increased absorption intensity.
If auxochrome is introduced to a
compound, the intensity of absorption
increases.
For example, phenolate ion has more
absorption intensity than phenol.

20. Solvent λ of absorption
Water 191 nm
Ether 215 nm
Methanol 203 nm
Ethanol 204 nm
Chloroform 237 nm
Carbon tetrachloride 265 nm
Benzene 280 nm
Tetrahydrofuran 220 nm

21. 1. Effect of solvent: choice of solvent can shift peaks to shorter
or longer wavelengths.
 depends on the nature of the interaction of the particular
solvent with the environment of the chromophore in the
molecule
 Water and alcohols can form hydrogen bonds which results
the shifting of the bands of polar substances.
 Since polarities of the ground and excited state of a
chromophore are different, hence a change in the solvent
polarity will stabilize the ground and excited states to different
extent causing change in the energy gap between these
electronic states.
 Highly pure, non-polar solvents such as saturated
hydrocarbons “do not “interact with solute molecules either
in the ground or excited state

22. (i) π to π* Transitions: excited states are more polar than the ground
state.
 If a polar solvent is used the dipole–dipole interaction reduces
the energy of the excited state more than the ground state.
 Thus a polar solvent decreases the energy of π to π* transition
and hence the absorption in a polar solvent such as ethanol will be
at a longer wavelength (red shift) than in a non-polar solvent such as
hexane.
(ii) n to π* transitions: polar solvents form hydrogen bonds with the
ground state of polar molecules more readily than with their
excited states.
 energy of ground state is decreased which further causing increase
in energy difference between the ground and excited energy levels
 Therefore, absorption maxima resulting from n to π* transitions are
shifted to shorter wavelengths (blue shift) with increasing
solvent polarity.

23. 2. Effect of Sample pH: The absorption spectra of certain
aromatic compounds such as phenols and anilines change on
changing the pH of the solution.
 Phenols and substituted phenols are acidic and display
sudden changes in their absorptions maxima upon the
addition of a base.
 After removal of the phenolic proton, we get phenoxide ion.
 In the phenoxide ion lone pairs on the oxygen is delocalized
over the π-system of the aromatic ring and increases the
conjugation.
 Extended conjugation leads to a decrease in the energy
difference between the HOMO and LUMO orbitals, which
results in red or bathochromic shift , along with an increase in
the intensity of the absorption.

24. aromatic amine gets protonated in an
acidic medium which disturb the
conjugation between the lone pair on
nitrogen atom and the aromatic π-system.
As a result, blue shift or hypsochromic shift
(to shorter wavelength) is observed along
with a decrease in intensity.

25. based on the fundamental law of
absorption called Beer-Lambert’s law.
absorption of radiation by an absorbing
medium (dilute solution)

26. states that the intensity of a beam
monochromatic light decreases
exponentially with the concentration of the
absorbing molecules.
absorbance ∝ concentration

27. states that the intensity of a beam
monochromatic light decreases
exponentially as the light travels through a
thickness of homogeneous medium
absorbance ∝ path length
But since dimension of cuvettee is fixed
hence Lambert Law is quite nullified.

28. A) Sources of radiation:
 Stable
 provide sufficient intensity of radiation
 supply continuous radiation over the entire wavelength.
i. Hydrogen/deuterium discharge lamp: consists of hydrogen
gas under relatively high pressure through which there is an
electrical discharge.
-hydrogen molecules are excited electrically and emit UV
radiation.
-which causes the hydrogen to emit a broad
band(continous) rather than a simple hydrogen line
spectrum.
-The lamps are stable, robust, and widely used.
-If deuterium (D,) is used instead of hydrogen, the emission
intensity is increased.

29. ii. Tungsten filament lamp: similar in its functioning to an electric
light bulb.
-heated electrically to white heat.
-It has 2 shortcomings. The intensity of radiation at short
wavelengths (350 nm<
) is small.
-to maintain a constant intensity, the electrical current to
the lamp must be carefully controlled.
-lamps are generally stable, robust, and easy to use.
iii. Mercury arc: mercury vapour is under high pressure, and the
excitation of mercury atoms is done by electric discharge.
-a standard source for much ultraviolet work, is generally
not suitable for continuous spectral studies because of the
presence of sharp lines or bands.

30. iv. Xenon discharge lamp: xenon gas is stored
under pressure in the range of 10-30
atmospheres.
-xenon lamp possesses two tungsten
electrodes separated by about 8 mm.
- when an intense arc is formed between
two tungsten electrodes by applying a low
voltage, the ultraviolet light is produced.
-intensity of ultraviolet radiation produced
by xenon discharge lamp is much greater
than that of hydrogen lamp.

31. B) Monochromator (wavelength selectors): used to disperse the radiation
according to the wavelength.
 essential elements of a monochromator are an entrance slit, a dispersing
element and an exit slit.
 entrance slit sharply defines the incoming beam of heterochromatic
radiation.
 dispersing element disperses the heterochromatic radiation into its
component wavelengths
 exit slit allows the nominal wavelength together with a band of wavelengths
on either side of it.
 position of the dispersing element is always adjusted by rotating it to vary
the nominal wavelength passing through the exit slit. The dispersing element
may be a prism or grating.
 The prisms are generally made of glass, quartz fused silica.
 Glass has the highest resolving power but it is not transparent to radiations
having the wavelength between 200 and 300 nm, because glass absorbs
strongly in this region.
 Quartz and fused silica prisms which are transparent throughout the entire
UV range are widely used in UV spectrophotometers.

32. C)Sample containers/sample cells/Cuvettes: The
cells that are to contain samples for analysis
should fulfill these main conditions:
 uniform in construction
 thickness must be constant
 surfaces facing the incident light must be optically
flat.
 material of construction should be inert to solvents.
 must transmit light of the wavelength used.
 made of quartz or fused silica.
 cells of pathlength 1cm are used.

34. D) Detectors
i) Photomultiplier tube: photomultiplier tube is a combination of a photodiode
and an electron multiplying amplifier.
 consists of an evacuated tube which contains one photo-cathode and 9-16
electrodes known as dynodes.
 When radiation falls on a metal surface of the photocathode, it emits
electrons.
 The electrons are attracted towards the first dynode which is kept at a
positive voltage.
 When the electrons strike the first dynode, more electrons are emitted by the
surface of dynode; these emitted electrons are then attracted by a second
dynode where similar type of electron emission takes place.
 The process is repeated over all the dynodes present in the photomultiplier
tube until a shower of electrons reaches the collector.
 number of electrons reaching the collector is a measure of the intensity of
light falling on the detector.
 The dynodes are operated at an optimum voltage that gives a steady signal.

35. ii) Photo voltaic cell (Barrier Layer Cell): consists of a semiconductor,
such as selenium, which is deposited on a strong metal base, such
as iron.
 Then a very thin layer of silver or gold is sputtered over the surface
of the semiconductor to act as a second collector electrode.
 radiation falling on the surface produces electrons at the selenium
silver interface.
 barrier exists between the selenium and iron which prevents the
electrons from flowing into iron.
 electrons are therefore accumulated on the silver surface.
 accumulation of electrons on the silver surface produces an
electrical voltage difference between the silver surface and the base
of cell.
 If external circuit has a low resistance, a photocurrent will flow which
is directly proportional to the intensity of incident radiation beam.

36. iii) Photo tube (Photocell): It consists of a high-sensitive cathode in the form of
a half-cylinder of metal which is contained in an evacuated tube.
 anode is also present in the tube which is fixed more or less along the axis
of the tube.
 inside surface of the photocell is coated with a light sensitive layer.
 light is incident upon a photocell, the surface coating emits electrons.
 These are attracted and collected by an anode.
 current, which is created between the cathode and anode, is regarded as a
measure of radiation falling on the detector.
iv) Silicon Photodiode: A silicon photodiode is a solid-state device which
converts incident light into an electric current. It consists of a shallow
diffused p-n junction, normally a p-on-n configuration. (p-n junction is an
interface or a boundary between two semiconductor material types, namely
the p-type and the n- type, inside a semiconductor. The p-side or the positive
side of the semiconductor has an excess of holes and the n-side or the
negative side has an excess of electrons.)

37. 1) Qualitative Analysis: presence or absence of certain functional
groups
2) Detection of Impurities: for example, benzene as impurity in
ethanol
3) Quantitative analysis: directly or by making calibration curve
4) Pharmaceutical analysis: Overlap of absorbance peaks in uv
spectra can be used to find out the pharmaceutical
compounds using mathematical derivatives. Chlortetracycline
(antibiotic) and benzocaine (anesthetic) are identified
simultaneously in veterinary powder formulation using first
mathematical derivative.
5) Determination of strength of H-bonding: It is based on shift
observed in polar solvents, for example, acetone absorbs at
279 (121kcal/mole) & 264.5 (126kcal/mole) nm resp. in
hexane and water

38. THANKYOU