1. UV-VISIBLE
SPECTROSCOPY
By
E. Gireesh Kumar
Associate Professor
Annamacharya College of Pharmacy, Rajampet

2. INTRODUCTION
• The UV-VIS spectrophotometry is one of the oldest instrumental techniques of
analysis and is the basis for a number of ideal methods for the determination of
micro and semimicro quantities of analytes in a sample.
• It concerns with the measurement of the consequences of interaction of
Electromagnetic radiations in the UV and/or Visible region with the absorbing species
like, atoms, molecules or ions.
• UV-VIS spectrum results from the interaction of electromagnetic radiation in the UV-
Visible region with molecules, ions or complexes. It forms the basis of analysis of
different substances such as, inorganic, organic and biomolecules.
• These determinations find applications in research, industry, clinical laboratories and
in the chemical analysis of environmental samples.
• Ultraviolet and visible spectroscopy also known as electronic spectroscopy.

3. ● UV-Visible spectroscopy deals with the study of the
electronic transitions of molecules as they absorb light
in the UV (190-400 nm) and visible regions (400-800 nm)
of the electromagnetic spectrum.
● The absorption of ultraviolet or visible radiation lead
to transition among electronic energy levels, hence it
is also often called electronic spectroscopy.
● As a rule, the energetically favoured electron promotion
will be from the Highest Occupied Molecular Orbital
(HOMO) to the Lowest Unoccupied Molecular Orbital
(LUMO). The resulting species is said to be in an excited
state.
● The HOMO-LUMO gap decrease as the conjugation
increase.
INTRODUCTION

4. ● The wavelengths at which absorption occurs, together
with the degree of absorption at each wavelength is
recorded by Optical Spectrometer.
● A spectrum is obtained as a result.
● It commonly provides the knowledge about
○ π-electron systems,
○ conjugated systems,
○ aromatic compounds and
○ conjugated non-bonding electron systems etc.
INTRODUCTION

5. TYPES OF ELECTRONIC TRANSITIONS

6. DIFFERENT TYPES OF ELECTRONIC TRANSITIONS
n to π* represents the
excitation of an electron of a
lone pair (non-bonding
electron pair) to an π
antibonding orbital
σ to σ*
A transition involving
excitation of an electron
from s bonding orbital to σ
anti-bonding orbital is called
σ to σ* transition
n to σ* represents the
excitation of an electron of a
lone pair (non-bonding
electron pair) to an σ
antibonding orbital
n to σ* n to π*
A transition involving
excitation of an electron
from p bonding orbital to an
antibonding π orbital
π to π*
σ to σ* n to σ* π to π* n to π*

7. σ to σ* Transitions
• A transition of electrons from a bonding sigma orbital to the antibonding sigma orbital is
designated as σ to σ∗ transition.
• These are high energy transitions as σ bonds are generally very strong.
• Thus these transitions involve very short wavelength ultraviolet light (< 150 nm) and usually fall
outside the range of UV-visible spectrophotometers (200-800 nm).
• Alkanes can only undergo σ to σ* transitions.
• Methane and ethane undergo σ → σ* transitions with an absorbance maximum at 122 and 135 nm,
respectively.
• Study of such transitions is usually done in an evacuated spectrophotometer (< 200 nm) since
oxygen present in air absorbs strongly at 200 nm and below. Similarly nitrogen absorbs at ~150 nm
and below.

8. n to σ* Transitions
• The transition of an electron from a non-bonding
orbital to the antibonding sigma orbital is
designated as n to σ∗ transition.
• Saturated compounds containing atoms with lone
pairs (nonbonding electrons) like saturated
alcohols, amines, halides, ethers etc are capable
of showing n to σ∗ transitions.
• Energy required for these transitions is usually
less than σ to σ∗ transitions.
• Such compounds absorb light having wavelength
in the range 150-250 nm.
• For example the absorption maxima for
• Water - 167 nm,
• Methyl alcohol - 174 nm,
• Methyl chloride - 169 nm and
• Methyl iodide - 258 nm.

9. π to π* Transitions
• The transition of an electron from a π bonding orbital to
a π∗ antibonding orbital is designated as π to π∗
transition.
• These transitions require lesser energy than n to σ∗
transitions.
• These types of transitions take place in compounds
containing one or more unsaturated groups like simple
alkenes, carbonyl, aromatics, nitriles, nitro etc.
• In non-conjugated alkenes, this type of transition occurs
in the range 170-190 nm e.g. ethene shows absorption
maxima at 171 nm.
• Similarly, π to π∗ transition in the range of 180-190 nm
occurs in non-conjugated carbonyl compounds e.g.
acetone shows absorption maxima at 188 nm.

10. n to π* Transitions
• The transition of an electron from a non-bonding orbital
to a π∗ antibonding orbital is designated as n to π∗
transition.
• This transition involves the least amount of energy in
comparison to all other transitions and therefore gives
rise to an absorption band at longer wavelength.
• Saturated carbonyl compounds show two types of
transitions, low energy n to π∗ (270-300 nm) and high
energy π to π* (180-190 nm).
• The transition n to π∗ is of lowest energy but is of low
intensity as it is symmetry forbidden.
• Thus the most intense band for these compounds is
always due to π to π* transition.

11. ● Some electronic transitions, which are otherwise
theoretically possible, are generally not observed
in the UV/VIS spectroscopy. Therefore there are
some restrictions which govern the observable
transitions.
● Transitions, which involve change in the spin
quantum number of an electron during the
transitions do not occur, i.e. singlet-triplet
transitions are not allowed.
● Transitions between orbitals of different
symmetry do not occur. For example, transition
n to π∗ is forbidden because the symmetry of n
and π∗ do not match.
SELECTION RULE

13. Factors affecting
THE POSITION OF UV BANDS
• Effects of conjugation
• Effects of steric hindrance
• Effects of solvent
• Effect of pH
THE SHAPE OF UV-ABSORPTION BANDS
• Effects of temperature
• Effect of sample concentration
Factors affecting the Position & Shape of UV bands

14. IMPORTANT TERMINOLOGIES IN UV-VIS SPECTROSCOPY
Bathochromic
shift
or Red shift
Hypsochromic
shift
or blue Shift
Hyperchromic
effect
Hypochromic
effect
Chromophore Auxochrome
methyl, hydroxyl, alkoxy,
halogen, amino group
C=C, C=C, C=O, C=N, N=N,
C A

15. • If a compound absorbs light in the visible region
(400-800 nm), only then it appears coloured to our
eyes.
• Therefore a chromophore may or may not impart
colour to a compound depending on whether the
chromophore absorbs radiation in the visible or UV
region.
A chromophore is defined as an isolated
covalently bonded group that shows a
characteristic absorption in UV/Vis region.
For example
C=C, C=C, C=O, C=N, N=N, R-NO2 etc.
Chromophore
C
H
R
O
M
O
P
H
O
R
E

16. • These are also called colour enhancing groups.
• The actual effect of an auxochrome on a
chromophore depends on the polarity of the
auxochrome, e.g. groups like CH3, CH3CH2 and Cl
have very little effect, usually a small red shift of
5-10 nm. Other groups such as NH2 and NO2 show a
strong effect and completely alter the spectrum.
The substituents covalently attached to a
chromophore which themselves do not absorb
ultraviolet/ visible radiation, but their
presence changes both the intensity as well
as wavelength of the absorption maximum
are known as auxochromes.
Auxochrome
A
U
X
O
C
H
R
O
M
E
methyl, hydroxyl, alkoxy, halogen, amino group
etc. are some examples of auxochromes.

18. SHIFTS IN UV SPECTRUM
Bathochromic Shift or Red shift
• The shift of an absorption maximum towards longer wavelength or lower energy is
called as bathochromic shift.
• It may be produced due to presence of an auxochrome or change in solvent polarity.
• Because the red colour has a longer wavelength than the other colours in the visible
spectrum, therefore this effect is also known as red shift.
Hypsochromic Shift or Blue Shift
• The shift of an absorption maximum towards the shorter wavelength or higher energy is
called hypsochromic shift.
• It may be caused due to presence of an auxochrome or change in solvent polarity.
• Because the blue colour has a lower wavelength than the other colours in the visible
spectrum and hence this effect is also known as blue shift.

19. SHIFTS IN UV SPECTRUM
Hyperchromic Effect
• It is an effect that results in increased absorption intensity.
• The introduction of an auxochrome usually causes hyperchromic shift.
• For example benzene shows B band (the secondary band in UV-Vis spectra) at 256 nm and
Ɛmax 200, whereas aniline shows B-band at 280 nm and Ɛmax at 1430. The increase in
the value of Ɛmax is due to the hyperchromic effect of auxochrome NH2.
Hypochromic Effect
• An effect that results in decreased absorption intensity is called hypochromic effect.
• This is caused by a group which distorts the geometry of the molecule.
• For example, biphenyl shows λmax at 250 nm and Ɛmax at 19,000, whereas 2-methyl
biphenyl absorbs at λmax 237 nm, Ɛmax 10250. The decrease in the value of absorbance is
due to hypochromic effect of methyl group which distorts the chromophore by forcing
the rings out of coplanarity resulting in the loss of conjugation.

20. Fundamental law of absorption
The law that governs the absorption of UV-Visible light
Beer – Lambert’s Law
Where,
Io = intensity of incident light
I = intensity of transmitted light
ε = molar absorptivity M-1 cm-1 or mol-1 lit cm-1
b = path length of sample, cm
c = concentration of sample, M or mol/lit
A = Absorbance

21. Beer – Lambert’s Law

22. Beer – Lambert’s Law
b
P0 P
b, Cm
dx
dn
ds
Px
dPx
n
V, Cm3
S, Cm2
Consider a beam of parallel monochromatic radiation with Power P0 passing
through Cell having a path length of b, cm containing an absorbing
solution (n, number of absorbing species).
Let, a cross section of the block having an area ds and thickness dx
containing dn number of absorbing species on which photon capture occurs.
The power of the beam entering into the section Px, is proportional to the
number of the photons per unit area. Let dPx represents the radiant power
absorbed within the section.

23. Beer – Lambert’s Law
b
P0 P b, Cm
dx
dn
ds
Px
dPx
n
V, Cm3
S, Cm2
The fraction absorbed (-dPx/Px) must be equal to the ratio of the capture area
to that of the total area (ds/s).
Thus,
----- Equation (1)
The term dS represents the sum of the capture areas for the particles within
the section, which in turn is proportional to the number of absorbing particles
----- Equation (2)
Where a is proportionality constant which represents the capture cross section

24. Beer – Lambert’s Law
b
P0 P b, Cm
dx
dn
ds
Px
dPx
n
V, Cm3
S, Cm2
Combining and integrating the Equation (1) and (2) over the interval P0 to P & 0 to n
---------- Equation (3)
---------- Equation (4)
Rearranging, ---------- Equation (5)
Simplifying, ---------- Equation (6)

25. Beer – Lambert’s Law
b
P0 P b, Cm
dx
dn
ds
Px
dPx
n
V, Cm3
S, Cm2
The cross sectional area S, Cm2 can be expressed as the ratio of the Volume of
the block, V Cm3 to its lenght b, cm.
Thus,
---------- Equation (7)
Since, n/V is the number of Particles per Cm3, it is nothing but concentration.
It can be converted to moles/lit by dividing with Avogadro's number.

26. Beer – Lambert’s Law
b
P0 P b, Cm
dx
dn
ds
Px
dPx
n
V, Cm3
S, Cm2
Concentration C in mol/lit is given by
---------- Equation (8)
Combining this equation (8) with equation (7)
---------- Equation (9)

27. Beer – Lambert’s Law
b
P0 P b, Cm
dx
dn
ds
Px
dPx
n
V, Cm3
S, Cm2
All the constants in Equation (9) can be collected into a single term ε, to give
---------- Equation (10)
This equation represents Beer-Lambert’s Law.
Beer’s law relates Absorbance to Concentration A α C
Lambert’s law relates Absorbance to the Path length A α b

28. Application of Beer – Lambert’s Law
for Mixtures
• If multiple species that absorb light at a given wavelength are present in a sample, the
total absorbance at that wavelength is the sum due to all absorbers:
A = A1 + A2 +... + An
A = ε1 b c1 + ε2 b c2 + ... + εn b cn
where the subscripts refer to the molar absorptivity and concentration of the different
absorbing species that are present.

29. Beer – Lambert’s Law
Lambert’s Law
• When a beam of light is allowed to pass through a transparent medium, “the rate of
decrease of intensity with the thickness of medium is directly proportional to the
intensity of light.”.
• Mathematically,the Lambert’s law may be expressed as follows.
- dI / dt α I
- dI / dt = KI
Where, I = intensity of incident light
t = thickness of the medium
K= proportionality constant
Beer’s Law
• “Intensity of incident light decreases exponentially as the concentration of absorbing
medium increases arithmetically.”
• Mathematically,the Beer’s law may be expressed as follows.
- dI / dC α I
- dI / dC = KI

30. Beer – Lambert’s Law
Beer’s Law
• When a beam of light is allowed to pass
through a transparent medium, “the rate of
decrease of intensity with the concentration
of absorbing species is directly proportional
to the intensity of light.”.
• Mathematically, the Beer’s law may be
expressed as follows.
- dI / dc α I
- dI / dc = KI
Where, I = intensity of incident light
c = conc. of the absorbing species
K= proportionality constant
Beer’s Law
“Intensity of incident light decreases exponentially
as the concentration of absorbing medium
increases arithmetically.”
Lambert’s Law
• When a beam of light is allowed to pass
through a transparent medium, “the rate of
decrease of intensity with the thickness of
medium is directly proportional to the
intensity of light.”.
• Mathematically, the Lambert’s law may be
expressed as follows.
- dI / db α I
- dI / db = KI
Where, I = intensity of incident light
b = thickness of the medium
K= proportionality constant
Lambert’s Law
“Intensity of incident light decreases exponentially
as the path length of absorbing medium increases
arithmetically.”

31. Beer – Lambert’s Law
Derivation

32. Beer – Lambert’s Law
Derivation

33. Limitation
Beer – Lambert’s Law
• Following are the situations when Beer’s law is not obeyed:
• At higher concentration (>0.01M).
• When fluorescent compounds are used.
• When coagulated substances are used.
• When suspensions are used.
• When different types of molecules are in equilibrium with each other.
• An association complex is formed by the solute and the solvent.
• When thermal equilibrium is attained between the excited state and the ground
state.
• When polychromatic light is used
• When mismatched cells are used.
• When stray light enters.
• When scattering of light due to particulates in the sample

34. Deviations
Beer – Lambert’s Law
• It is often assumed that Beer’s Law is always a linear plot describing the relationship
between absorbance and concentration.
• Deviations do occur however that cause non-linearity.
• This can be attributed to a range of chemical and instrumental factors

35. Deviations
Beer – Lambert’s Law
• Generally, all the compounds/substances absorbing in UV-Visible range will follow
Beer’s law to only up to a particular concentration. At higher concentrations the curve
does not follow linearity. These deviations are grouped into THREE categories.
• Real deviations
• Instrumental deviations
• Chemical deviations

36. Real Deviations
• Beer’s law is limiting law (concentration < 0.01M).
• At higher concentrations (C > 0.01M),
• solute – solvent interactions and
• solute – solute interactions or
• hydrogen bonding
can affect the change in the density of the analyte environment and hence its
absorptivity.
• Up to concentration <0.01M, the refractive index remains relatively constant. But at
higher concentrations, as the refractive index increases, deviations from Beer’s law is
observed.

37. Instrumental deviations
• Several factors cause instrumental deviations.
• These include,
• Polychromatic radiation
• Stray radiation
• Mismatched Cells
• Instrumental noice

38. Chemical deviations
• Deviation from Beer-Lambert’s law often occurs due to chemical effects such as
• Dissociation
• Association
• Complex formation
• Polymerization
• Solvolysis
• Precipitation
• Temperature effects
• Photochemical reactions

40. GENERAL INSTRUMENT DESIGNS
Single beam
Requires a stabilized voltage supply

41. GENERAL INSTRUMENT DESIGNS
Double Beam: Space resolved (in-space)
Need two detectors

42. GENERAL INSTRUMENT DESIGNS
Double Beam: Time resolved (in-time)
Need two detectors

43. General Instrument Designs
Double Beam: Time resolved (in-time)

44. Source of Radiation
● Source of radiant energy for
○ UV region: Deuterium discharge lamp
○ Visible region: Tungsten Filament Lamp

45. Deuterium discharge lamp (D2 Lamp)
● Most common UV source
● Arc between Oxide coated filament and metal anode
● D2 at lo pressure
D2 + Ee → D2* → D’ + D’’ + h
Ee = ED2* = ED’ + ED’’ + Ehv
● Ee is the electrical energy absorbed by the molecule. ED2* is the fixed quantized energy of D2*, ED’ and ED’’ are kinetic energy
of the two deuterium atoms. ED’ & ED’’ continuously vary from 0 to ED2* Hence, Ehν varies continuously.
● Continuum from 160- 400 nm, emission lines >400nm
● As D2 lamp is operated at high temperature, glass housing can not be used for casing.
Instead, a fused quartz, UV Glass, or magnesium fluoride envelop is used.
Excited deuterium
molecule with fixed
quantized energy
Dissociated into two
deuterium atoms with
different kinetic energies
UV Photon

46. Tungsten Halogen Lamp
● Most common source for visible region
● This lamp consist of Tungsten filament enclosed in a glass envelop
● Operated at 2850K
● Produces continuum in the range 350 – 2200nm
● Tungsten – Halogen lamp: 240 – 2500 nm
○ Why add I2 in the lamps?
W + I2 → WI2
stikes the filament
decomposition occurs
redeposit of Tungsten
Life-time is increased

47. Mercury Vapour Lamp
● Give line spectra
● 254, 303, 313, 365, 436, 546, 578, 579, 691, 773nm

48. Xenon arc Lamp

49. Absorption Filters
● absorptive filters transmit desired wavelengths by absorbing unwanted ones.
● These filters typically consist of dyed glass or pigmented gelatin resins
● Band pass: ± 30 – 250nm

50. Interference filter
● Band pass: ±10 – 15nm

52. Monochromators
● Prism Type: Band Pass: ±1nm
● Gratings Type: Band Pass: ±0.1nm

53. Prism Type Monochromators
● Prism: Utilized from UV to IR
● Material is spectrum dependent
○ glass – from 350 nm up – VIS
○ quartz – UV
○ salts crystals (NaCl, KBr) - IR

54. Prism Monochromators
● 60° Prism - Bunsen Prism

55. Prism Monochromators
● 2x30° Prism --‐ Cornu prism

56. Prism Monochromators
● 30° Prism with mirrored back – Littrow prism

57. Grating Monochromators
● Replica Gratings manufactured from master gratings
● Optically flat, polished surface that has large no. of series of parallel and closely spaced
grooves made with diamond tool
● Space equivalent to wavelength:
○ for UV/VIS 500 to 5000 lines per mm (1200-1400 grooves/mm )
○ for IR 50 to 200 lines per mm (100 grooves/mm )

58. Echellette Gratings

59. Grating Monochromators

60. Sample Cells
● Used for sample handling
● Cells can be made of Plastic, Glass, or Quartz
● Only Quartz is transparent in full UV-Vis range;
● Plastic and Glass are only suitable for Visible region.
● Sample cell – commonly called as Cuvette
● All UV Spectra are recorded in Solution Phase
● Solvent must be transparent in the region to be observed.
(The wavelength where a solvent is no longer transparent is referred to as the Cutoff)
● Since spectra are only obtained up to 200nm, solvents typically only need to lack of
Conjugated p system or carbonyls.
● Common solvents and their cutoff wavelengths in nm:
Acetonitrile 190
Chloroform 240
Cyclohexane 195
1,4-dioxane 215
95% ethanol 205
n-hexane 201
Methanol 205
Isooctane 195
Water 190

61. Photovoltaic cell
● Also known as a self-generating barrier layer cell.
● A photoelectric detector that converts radiant flux directly into electrical current.
● Generally, it consists of a thin silver film on a semiconductor layer deposited on an iron
substrate.

62. Photo Tubes

63. Photo Tubes
● A caesium-antimony cathode
very sensitive in the violet to ultra-violet region with sensitivity falling off to blindness to
red light
● Caesium on oxidised silver
sensitive to infra-red to red light, falling off towards blue

64. Photomultiplier tubes

66. Photodiode
● A silicon photodiode is a semiconductor device that exploits the photoelectric effect to convert light into
an electrical current.
● When the incident photons’ energy is larger than the bandgap of silicon, the photons are absorbed and the
electrons in the valence band are excited to the conduction band, creating holes in the initial valence band.
● The photodiode is made up of a p- and a n- junction and a depletion region. An applied electric field in this
depletion region pushes the positive holes towards the n-junction while the negative electrons move
towards the p-junction, building up areas of highly positive and negative charges and thus producing a
photocurrent.
● Photodiodes have a quick response time, a slightly broader spectral range than a PMT, and low noise. While
silicon photodiodes are less sensitive than PMT detectors in the UV and visible regions, they are a cheaper
alternative for applications not requiring high sensitivity.

67. Photodiode

69. Applications

70. ● Detection of Impurities
● Structure elucidation of organic compounds
● Quantitative analysis
● Qualitative analysis
● Dissociation constants of acids and bases
● Chemical kinetics
● Quantitative analysis of Pharm. Substances
● Spectrophotometric titrations
● Molecular weight determination
● As HPLC detector
Applications

71. 1. Detection of Impurities
● 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.
● Benzene appears as a common impurity in cyclohexane. Its presence can be easily detected
by its absorption at 255 nm.

72. 2. Structure elucidation of organic compounds
● 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.

73. 3. Quantitative analysis
● 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 that is used in UV spectrophotometer.

74. Other methods for quantitative analysis are:
Calibration Curve method
Simultaneous Equation method
Absorbance Ratio Method
Difference Spectrophotometric method
Derivative Spectrophotometric method
Area Under Curve method

75. 4. Qualitative analysis
● UVabsorption 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.
● UV absorption spectroscopy is generally used for characterizing aromatic compounds and
aromatic olefins.

76. 5. Dissociation constants of acids & bases
● pH = pKa + log [A-] / [HA]
From the above equation, the PKa value can be calculated if the ratio of [A-] / [HA]
is known at a particular pH. and the ratio of [A-] / [HA] can be determined
spectrophotometrically from the graph plotted between absorbance and
wavelength at different pH values.

77. 6. Chemical kinetics
● Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is
passed through the reaction cell and the absorbance changes can be observed.

78. 7. Quantitative analysis of pharmaceutical substances
● Many drugs are either in the form of raw material or in the form of formulation.
They can be assayed by making a suitable solution of the drug in a solvent and
measuring the absorbance at specific wavelength.

79. 8. Spectrophotometric titrations
● Spectrophotometric titration curves:
(a) only the titrand absorbs;
(b) only the titrant absorbs;
(c) only the product of the titration reaction absorbs;
(d) both the titrand and the titrant absorb;
(e) both the titration reaction’s product and the titrant absorb;
(f) only the indicator absorbs.
● The red arrows indicate the end points for each titration curve.
where A indicates the analyte, P indicates the product
and T indicates the titration for the reaction A + T --> P

80. 9. Molecular weight determination
10. As HPLC detector

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