1. UV-Visible Spectroscopy
By: Asst. Prof. Sanjaykumar Uchibagle

2. 1. Introduction
2. Absorption Law (Beers & Lamberts Law)
3. Theory of Electronic Spectroscopy
1. Types of Electronic Transitions (K, R, B, E Bands)
2. The Chromophore Concept
3. Auxochrome
4. Absorption and Intensity Shifts
a. Bathochromic effect. (Red Shift)
b. Hypsochromic shift (Blue Shift)
c. Hyperchromic effect (Abs Increase)
d. Hypochromic effect (Abs Decrease)

3. 5. Effect of Temperature and Solvent on the Fineness
of Absorption Band.
6. Woodward-fieser Rules for Calculating Absorption
Maximum in Dienes
7. Instrumentation.
A. Light Source
b. Collimating System
c. Monochromator
d. Sample Holder
e. Detector.

4. 1. Introduction
 light travels in the form of waves.
 These are characterized by their
wavelengths or frequencies or
wavenumbers.
 The emission or absorption of radiation is
quantised and each quantum of radiation
is called a photon.
 Wavelength. It is the distance between
the two adjacent crests (C—C) or troughs
(T—T) in a particular wave. It is denoted
by the letter (lambda). It can be
expressed in Angstrom units or in
millimicrons (mμ).
1 Å = 10–8 cm; 1 mμ = 10–7 cm.

6.  Spectroscopy is the branch of Science that deals with the study
of interaction of electromagnetic radiation with matter.
 Spectrometer is an instrument design to measure the spectrum
of a compound.
 Instrument used to measure the absorbance in UV (200- 400nm)
or Visible (400-800nm) region is called UV- Visible
Spectrophotometer.
 A Spectrophotometer records the degree of absorption by a
sample at different wavelengths and the resulting plot of
absorbance (A) versus wavelength (λ) is known as a Spectrum

7. 2. Absorption Law (Beers & Lamberts Law)
Lambert’s Law : When a beam of monochromatic radiation passes
through a homogeneous absorbing medium, the rate of decrease of
intensity of radiation with thickness of absorbing medium is proportional
to the intensity of the incident radiation.
Mathematically, the law is expressed as
Where, I = intensity of radiation after passing through a thickness x, of the
medium.
= rate of decrease of intensity of radiation with thickness of the absorbing
medium

8. 2. Absorption Law (Beers & Lamberts Law)
Beer’s Law : When a beam of monochromatic radiation is passed through
a solution of an absorbing substance, the rate of decrease of intensity of
radiation with thickness of the absorbing solution is proportional to the
intensity of incident radiation as well as the concentration of the solution.
Mathematically, this law is stated as
Where, c = conc. of the solution in moles litre–1.
k = molar absorption coefficient and its value depends upon the
nature of the absorbing substance

9. 3. Theory of Electronic Spectroscopy
1. Molecule absorbs – UV light- its electrons - Ground state to the higher
energy state
2. In the higher energy state, if the spins of the electrons are paired,
called an excited singlet state.
3. On the other hand, if the spins of the electrons in the excited state
are parallel, it is called an excited triplet state.
The triplet state is always lower in energy than the corresponding excited
singlet state.
Therefore, triplet state is more stable as compared to the excited singlet
state

10. σ→σ* transitions.
1. It is a high energy
process since, bonds are, in
general, very strong.
2. The organic ompounds
in which all the valence
shell electrons are involved
in the formation of sigma
bonds do not show
absorption in the normal
ultra-violet region.
n→σ* transition.
This type of transition takes
place in saturated compounds
containing one hetero atom
with unshared pair of electrons
(n electrons).
π→π* transitions.
This type of transition occurs
in the unsaturated centers of
the molecule; i.e., in
compounds containing double
or triple bonds and also in
aromatics.
n→π* transition.
In this type of
transition, an electron
of unshared electron
pair on hetero atom
gets excited to π*
antibonding orbital.

11. (a) K* Bands.
1. K-bands originate due to π→π*
transition from a compound
containing a conjugated system.
2. Such type of bands arise in
compounds like dienes, polyenes,
enones.
3. K-bands also appear in an
aromatic compound which is
substituted by a chromophore.
4. The intensity of K-band, is
usually more than 104 .
5. The K-band absorption due to
conjugated ‘enes’ and ‘enones’ are
effected differently by changing
the polarity of the solvent

12. (b) R* band.
1. Such type of bands
originate due to n→π*
transition of a single
chromophoric group
and having at least one
lone pair of electrons on
the hetero atom.
2. R-bands are also
called forbidden bands.
These are less intense.

13. (c) B-band.
1. Such type of bands
arise due π→π*
transition in aromatic or
hetero-aromatic
molecules.
2. Benzene shows
absorption peaks between
230–270mμ.
3. When a chromophoric
group is attached to the
benzene ring, the B-bands
are observed at longer
wave-lengths than the
more intense K-bands.

14. (d) E-bands.
1. Such bands originate due to the electronic transitions in the benzenoid
system of three ethylenic bonds which are in closed cyclic conjugation.
2. These are further characterized as E1 and E2-bands. E1 and E2 bands of
benzene appear at 184 and 204 mμ respectively.
3. E1 band which appears at lower wave-length is usually more intense
than the E2-band for the same compound which appears at longer
wavelength.

15. 3.2. The Chromophore Concept
1. It is defined as any isolated covalently
bonded group that shows a
characteristic absorption in the ultra-
violet or the visible region.
2. All those compounds which absorb
light of wavelength between 400-800
mμ appear coloured to the human eye.
3. Exact colour depends upon the
wavelength of light absorbed by the
compound
4. Nitro-compounds are generally yellow
in colour.
5. Similarly, aryl conjugated azo group is
a chromophore for providing colour to
azo dyes.

16. 3.3. The Auxochrome Concept
1. An auxochrome can be defined as any group-which does not itself act as a chromophore
but whose presence brings about a shift of the absorption band towards the red end of
the spectrum (longer wavelength).
2. The absorption at longer wavelength is due to the combination of a chromophore and an
auxochrome to give rise to another chromophore.
3. An auxochromic group is called colour enhancing group.
4. Some common auxochromic groups are —OH, —OR, —NH2, —NHR, —NR2, —SH etc.
5. For example, benzene shows an absorption maximum at 255 mμ [Abs. max 203] whereas
aniline absorbs at 280 mμ [Abs max 1430]. Hence, amino (—NH2) group is an
auxochrome.

17. 4. Absorption and Intensity Shifts
(a) Bathochromic effect/Red Shift
1. It is an effect by virtue of which the
absorption maximum is shifted towards
longer wavelength due to the presence of an
auxochrome or by the change of solvent.
2. Such an absorption shift towards longer
wavelength is called Red shift or
bathochromic shift.
3. For example, benzene shows an absorption
maximum at 255 mμ [Abs. max 203] whereas
aniline absorbs at 280 mμ [Abs max 1430].
Hence, amino (—NH2) group is an
auxochrome.

18. (b) Hypsochromic shift/Blue Shift
1. It is an effect by virtue of which the
absorption maximum is shifted towards
shorter wavelength..
2. The absorption shifted towards shorter
wavelength is called Blue shift or
hypsochromic shift
3. . In the case of aniline, absorption
maximum occurs at 280 mμ because the
pair of electrons on nitrogen atom is in
conjugation with the π bond system of the
benzene ring. In its acidic solutions, a blue
shift is caused and absorption occurs at
shorter wavelength (~203 mμ).

19. (c) Hyperchromic effect.
1. It is an effect due to which the
intensity of absorption maximum
increases.
2. The introduction of an auxochrome
usually increases intensity of absorption.
3.For example, benzene shows an
absorption maximum at 255 mμ [Abs.
max 203] whereas aniline absorbs at
280 mμ [Abs max 1430]. Hence, amino
(—NH2) group is an auxochrome.

20. (d) Hypochromic effect.
1 It is defined as an effect due to which the
intensity of absorption maximum
decreases.
2. For example, biphenyl absorbs at 250
mμ, Abs max 19000 whereas 2-methyl
biphenyl absorbs at 237 mμ, Abs max
10250

21. 5. Effect of Temperature
1. It is known that the vibrational
and the rotational states
depend on temperature.
2. As the temperature is
decreased, vibrational and the
rotational energy state of the
molecules are also lowered.
3. Thus, when the absorption of
light occurs at a lower
temperature, smaller
distribution of excited states
result. It produces finer
structure in the absorption
band than what is noticed at
higher temperature.
1.The solvent used also
effects the fineness of
absorption band in UV
spectrum.
2.If the dielectric constant
of the solvent is high, there
will be stronger solute-
solvent interactions.
3.Due to this, vibrational
and rotational energy states
of molecules increase and
thus, the fineness of the
absorption band falls.
Solvent on the Fineness of Absorption
Band.

22. Solvent Effects
1. A most suitable solvent is one
which does not itself absorb in the
region under investigation.
2. A dilute solution of the sample is
always prepared for the spectral
analysis. Most commonly used
solvent is 95% Ethanol.
3. Ethanol is a best solvent as it is
cheap and is transparent down to
210 mμ.
4.Some other solvents which are
transparent above 210 mμ are n-
hexane, methyl alcohol, cyclohexane,
acetonitrile, diethyl ether etc

23. 7. Instrumentation.
A. Light Source
b. Collimating System
c. Monochromator
d. Sample Holder
e. Detector.
Ideal Property of Source.
1. Stable
2. Not Fluctuating
3. Emit of light continuous &
Uniform Intensity for which its
Used.
4. It should not be fatigue on
continuous used.
For Visible Spectroscopy.
1. Tungsten Halogen Lamp
For UV Radiation.
1. Hydrogen Discharge Lamp.
2. Xenon Discharge Lamp.
3. Mercury Arc Lamp.

24. Light Source
For Visible Spectroscopy.
1. Tungsten Halogen Lamp
 Its Construction is similar to the house hold lamp.
 Bulb contains a filament of Tungsten fixed in evacuated condition &
then filled with inert gas.
 The filament can be heated up to 3000 k, beyond this this Tungsten
starts sublimating.
 It is used when polychromatic radiation is required. To prevent this
along with inert gas some amount of halogen is introduced. (usually
Iodine)
 Sublimated form of Tungsten is reacted with Iodine to form complex,
which migrate back to the hot filament.
 It provides a supply of radiation in the wavelength range of 320-
2500nm.
 Demerit- It emit the major portion of near IR region

26. Light Source
For UV Spectroscopy.
1. Hydrogen Discharge Lamp.
 In this pair of electrode is enclosed in a glass tube (provide with
silica or quartz for UV radiation to pass it) filled with hydrogen gas.
 When current is passed through the electrode maintained at high
voltage, discharge of electron occurs with excites hydrogen molecule
which in turn cause emission of UV radiation.
 They are stable and robust. Covers a range 160-375nm

27. Light Source
For UV Spectroscopy.
1. Xenon Discharge Lamp
 It possesses 2 tungsten electrode separated by some distance.
 This are enclosed in a glass tube with quartz / Fused Silica & Xenon
gas is filled under pressure.
 An intense arc is formed between electrode by applying high voltage.
 This is good source of continuous plus additional intense radiation.
Its intensity is higher than Hydrogen Discharge Lamp.
 Demerit: The lamp since operate at high voltage become very hot
during operation & hence need thermal insulation.

28. Light Source
For UV Spectroscopy.
1. Mercury Arc Lamp
 In Mercury arc lamp, mercury vapor is stored under high pressure &
excitation of mercury atom is done by electrical discharge.
 Demerit: Not suitable for continuous spectral studies because it
doesn’t give continuous radiations.

29. b. Collimating System
The radiation emitted by source is collimated (parallel) by lenses, mirror and slits.
Lenses:
 Material used for the lenses must be transparent to the radiation being used.
 Ordinary silicate glass transmits between 350 to 3000 nm & is suitable for visible
and near IR region.
 Quartz or fused silica is used as a material for lenses to work below 300 nm.
Mirrors:
 This are used to reflect, focus or collimating light beam in spectrophotometer.
 To minimize the light loss, mirror are aluminized on their surface
Slits:
 Important device in resolving polychromatic to monochromatic
radiation To achieve this entrance & Exit Slits are Used.
 The width of slit plays imp. role in resolution of polychromatic
radiation.

30. c. Monochromator
This device is used to isolate the radiation of desired wavelength from wavelength of
continuous spectra. Ex.
1. Filters
2. Prism
3. Grating.
1.Filters:
 Selection of filter is usually done on compromise between peak transaction &
band pass width; the former should be as high as possible & latter as narrow as
possible.
Absorption Filters: Works by selective absorption of unwanted radiation and
transmit the radiation which is required. Ex. Glass & Gelatin.

31. Selection of absorption filters is done according to the
following procedure.
 Draw a filter wheel.
 Write the color VIBGYOR in anticlockwise
 If solution to be analyzed is blue in color
filter having complimentary color
orange is to be used.
 Similarly we can select the required filters
in colorimeter, based upon the color of the solution
 An absorption glass filter is made of solid sheet of
glass that has been colored by pigment which is
dissolved or dispersed in the glass.
 The color in the glass filter is produce by incorporating metal oxide like
Cr, Mn, Fe, Ni, Co, Cu etc.

32. Gelatin filter is an example of absorption filter prepared by adding
organic pigments.
 Here instead of solid glass sheet thin gelatin sheet are used
 This are not used now days.
 It tends to be deteriorate to be time & glass affected by heat and
moisture The Color of dye gets bleached.
Merits:
1. Simple in construction.
2. Cheaper.
3. Selection of filter easy
Demerits:
1. Less Accurate.
2. Band pass (bandwidth) is ±30nm
If we have to measure the 400nm; we
get the radiation from 370-430 nm,
Less Accurate Results Obtained.

33. Interference filters:
 Work on the interference phenomenon cause rejection of unwanted
wavelength by selective reflection.
 It is constructed by using two parallel glass plate, which are silvered
internally & separated by thin film of dielectric material of different
(CaF2, SiO, MgF2) refractive index.
Merits:
1. These filters have band pass of 10-15 nm with peak transmittance of
40-60%.
2. Cheap
3. Additional filters can be used to cut off undesirable wavelength

34. 2. Prism.
 Prism is made up from glass, quartz or fused silica.
 Quartz or fused silica is the choice of material for UV.
 When the light is pass through the glass prism, dispersion of
polychromatic light in RAINBOW occurs.
 Now by rotation of Prism in different wavelength of the spectrum can be
made to pass through the exit slit on the sample.
 The effective wavelength depends on the dispersive power of Prism
material & Optical Angle of the Prism.

35. There are two types of mounting in
an instrument
 one is called ‘Cornu type’
(refractive), which has an optical
angle of 60o and its adjusted such
that on rotation the emerging
light is allowed to fall on exit slit.
 The other type is called “Littrow type”
(reflective), which has optical angle 30o
and its one surface is aluminized with
reflected light back to pass through prism
and to emerge on the same side of the light
source i.e. light doesn’t pass through the
prism on other side.

36. Grating:
 Are most effective one in converting a polychromatic light to monochromatic
light. As a resolution of +/- 0.1nm could be achieved by using gratings, they
are commonly used in spectrophotometers.
 Gratings are of two types.
1. Diffraction grating. 2. Transmission gratings.
1. Diffraction grating.
 More refined dispersion of light is obtained by means of diffraction gratings.
 These consist of large number of parallel lines ( grooves) about 15000-30000/
inch is ruled on highly polished surface of aluminium.
 These gratings are replica made from master gratings by coating the original
master grating with a epoxy resin and are removed after setting

37. 2. Transmission gratings.
 It is similar to diffraction grating but refraction takes place instead of
reflection.
 Refraction produces reinforcement. this occurs when radiation transmitted
through grating reinforces with the partially refracted radiation.

38. Advantages:
 Grating gives higher and linear dispersions compared to prism
monochromator.
 Can be used over wide wavelength ranges.
 Gratings can be constructed with materials like aluminium which is
resistant to atmospheric moisture.
 Provide light of narrow wavelength.
 No loss of energy due to absorption

39. d. Sample Holder
 The cells or cuvettes are used for handling liquid samples.
 The cell may either be rectangular or cylindrical in nature.
 For study in UV region; the cells are prepared from quartz or fused silica
whereas color corrected fused glass is used for visible region.
 The surfaces of absorption cells must be kept scrupulously clean. No
fingerprints or blotches should be present on cells.
 Cleaning is carried out washing with distilled water or with dilute alcohol,
acetone.

40. e. Detectors.
 Device which converts light energy into electrical signals,
that are displayed on readout devices.
 The transmitted radiation falls on the detector which
determines the intensity of radiation absorbed by sample The
following types of detectors are employed in instrumentation
of absorption spectrophotometer.
 1. Barrier layer cell/Photovoltaic cell
 2. Phototubes/ Photo emissive tube
 3. Photomultiplier tube

41. Ideal Properties of Detectors.
 It should give quantitative response.
 It should have high sensitivity and low noise level.
 It should have a short response time.
 It should provide signal or response quantitative to wide
spectrum of radiation received.

42. 1. Barrier layer cell/Photovoltaic cell
 The detector has a thin film metallic layer coated with silver or gold
and acts as an electrode.
 It also has a metal base plate which acts as another electrode.
 These two layers are separated by a semiconductor layer of
selenium.

43.  When light radiation falls on selenium layer, electrons become
mobile and are taken up by transparent metal layer.
 This creates a potential difference between two electrodes & causes
the flow of current.
 When it is connected to galvanometer, a flow of current observed
which is proportional to the intensity and wavelength of light falling
on it.

44. 2. Phototubes/ Photo emissive tube

45.  Consists of a evacuated glass tube with a photocathode and a
collector anode.
 The surface of photocathode is coated with a layer of elements like
cesium, silver oxide or mixture of them.
 When radiant energy falls on photosensitive cathode, electrons are
emitted which are attracted to anode causing current to flow.
 More sensitive compared to barrier layer cell and therefore widely
used.
 The principle employed in this detector is that, multiplication of
photoelectrons by secondary emission of electrons

46.  In a vacuum tube, a primary photo-cathode is fixed which receives
radiation from the sample.
 Some eight to ten dynodes are fixed each with increasing potential of
75-100V higher than preceding one.
 Near the last dynode is fixed an anode or electron collector electrode.
 Photo-multiplier is extremely sensitive to light and is best suited
where weaker or low radiation is received

47. 3. Photomultiplier tube
 The principle employed in this detector is that,multiplication of
photoelectrones by secondary emission of electrons.
 In a vaccum tube a primary photo-cathode is fixed which receives
radiation from the sample.
 Some eight to 10 dynodes are fixed each with increasing potential of
75-100V higher than preceeding one.
 Photomultiplier is extremely sensitive to light and is best suited
where weaker or low radiation is received.
 Near the last dynode is fixed an anode or electrone collector
electrode.

48. 3. Photomultiplier tube

49. DOUBLE BEAM SPECTROSCOPY

50. Thank You!