Spectroscopy is the study of interaction of electromagnetic radiation with matter. It involves measuring the spectrum (absorption or emission) of a sample when it interacts with electromagnetic radiation such as visible light, UV light, or infrared light. The main types of spectroscopy are absorption spectroscopy and emission spectroscopy. UV-visible spectroscopy measures absorption of ultraviolet and visible light by a substance in solution. It follows Beer-Lambert law where absorbance is directly proportional to concentration and path length of light through the sample. Electronic transitions that occur when absorbing UV-visible light include σ→σ*, n→π*, π→π*, etc. Factors like auxochromes, conjugation, and solvents can cause shifts in the absorption maximum

1. SPECTROSCOPY
OR
SPECTROPHOTOMETRY

2. Spectroscopy
• 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. Electromagnetic
Radiation

4. Electromagnetic Radiation
• Electromagnetic radiation is energy that is
propagated through free space or through a
material medium in the form of electromagnetic
waves, such as radio waves, visible light, and
gamma rays etc. Electromagnetic waves consist of
discrete packages of energy which are called as
photons.
• A photon consists of an oscillating electric field (E)
& an oscillating magnetic field (M) which are
perpendicular to each other.

6. Electromagnetic Radiation
• Wavelength (λ):
– It is the distance between any two consecutive
parts of the wave whose vibrations are in the same
phase i.e. distance between two nearest crest or
troughs.
– Can be expressed in angstrom (10-10), nanometre
(10-9),micrometre(10-6)
• Frequency (ν):
– It is defined as the number of waves passing form
a point in one second.
– The unit for frequency is Hertz (Hz).
1 Hz = 1 cycle per second

7. •Wave number: The reciprocal of the wavelength
expressed in cm, i.e. the number of waves per cm. Its
unit is cm-1
Wavelength(λ) = speed of light (c)/frequency(ν)
Wave number= 1/ wavelength(λ)
Wave number = frequency(ν)/ speed of light(c)
Energy of a photon(E)= h ν = h c / λ

8. Electromagnetic Radiation

9. Electromagnetic Radiation
Violet 400 - 420 nm Yellow 570 - 585 nm
Indigo 420 - 440 nm Orange 585 - 620 nm
Blue 440 - 490 nm Red 620 - 780 nm
Green 490 - 570 nm

10. Principles of
Spectroscopy

11. Principles of Spectroscopy
• The principle is based on the measurement of
spectrum of a sample containing atoms / molecules,
obtained as a result of interaction of matter with
electromagnetic radiations .
• 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.

12. 1. Absorption Spectroscopy:
• An analytical technique based on the measurement
of the absorption of monochromatic light by ground
state atoms.
• When the energy of radiation exactly matches to the
difference between energies of ground and excited
state, the excitation of electrons from ground state to
excited state takes place.
• The techniques based on absorption spectroscopy are
UV-visible spectroscopy, IR spectroscopy etc.
• For e.g. Atoms of sodium vapours absorb a radiation
of 589nm and get excited.

13. 2. Emission Spectroscopy:
• An analytical technique based on the
measurement of light emitted by thermally excited
atoms or molecules.
• When the sample is heated to high temperatures ,
it undergoes partial or complete dissociation into
free atoms and then volatilises into to free gaseous
atoms. The electrons in the outer orbitals of atoms
absorb thermal energy and get excited. When
these excited molecules get deactivated absorbed
energy is emitted in the form of radiations.
• The energy of emitted light is given by E= h c / λ

14. UV-visible
spectroscopy

15. UV- visible spectroscopy
• This technique involves the measurement of the
amount of ultraviolet (200-400 nm) or visible
radiation (400-800 nm) absorbed by a substance in
the solution.
• The absorption of light in both ultraviolet and visible
regions occurs when energy of light matches the
energy required to induce an electronic transition in
the molecule.

16. Principle of UV
Spectroscopy

17. Beer-Lambert law
• When a beam of light is passed through a
transparent cell containing a solution of absorbing
substance reduction in intensity of light may occur
due to:
Reflections at the surface of cell
Scattering by particles in solution
Absorption of light by molecules in the solution
• Reflections are compensated by a reference cell
containing solvent only
• Scatter is eliminated by filtration of solution

18. Thus intensity of light absorbed is given by
Iabs=Io-IT
where, Io is original intensity incident on cell
IT is reduced intensity transmitted from cell
Transmittance (T) is the ration of IT and Io

19. Lambert’s
Law

20. Lambert’s Law
• When a monochromatic radiation is passed through
a solution, the rate of decrease in the intensity of
radiation with thickness of the medium is directly
proportional to the intensity of the incident light.
or
• The intensity of a beam of parallel monochromatic
radiation decreases exponentially as it passes
through a medium of homogenous thickness.
• Simply,
Absorbance is proportional to the
thickness (Pathlenght) of solution.

21. Beer’s
Law

22. Beer’s Law
• When a monochromatic radiation is passed through
a solution, the decrease in the intensity of radiation
with concentration of the solution is directly
proportional to the intensity of the incident light.
Or
The intensity of a beam of parallel monochromatic
radiation decreases exponentially with the no of
absorbing molecules.
• Simply,
Absorbance is proportional to the
concentration of absorbing molecules.

23. A combination of two laws yields the Beer-Lambert law
The absorbance of light is directly proportional to the
thickness of the media through which the light is being
transmitted multiplied by the concentration of absorbing
chromophore
A = abc
Where,
A is the absorbance,
a is absorptivity , combination of K’/2.303 and K”/2.303
•When concentration in in moles per lit constant is called
Molar absorptivity or molar extinction coefficient
b is the thickness of the solution
c is the concentration.

24. Substances that have values less than 100 are weakly
absorbing and those with values above 10000 are
intensely absorbing.
Many absorbing drugs have an value of molar
extinction coefficient above 103 - 104

25. Electronic
Transitions

26. Energy absorbed in the ultra violet region by complex
molecules causes transitions
Of valence electrons in the molecules.
The possible electronic transitions can graphically
shown as:

27. • σ → σ* transition1
• π → π* transition2
• n → σ* transition3
• n → π* transition4
• σ → π* transition5
• π → σ* transition6
The possible electronic transitions are

28. • σ electron from orbital is excited to corresponding
anti-bonding orbital σ*.
• The energy required is large for this transition.
• These transitions can occur in compounds in which
all electrons are involved in formation of single
bonds.
• e.g. Methane at 121.9 nm and ethane at 135 nm
shows this type of transition
• σ → σ* transition1

29. • 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. Ethylene exhibits a intense band at 174nm
and weak band at 200 nm
• π → π* transition2

30. • Saturated compounds containing atoms with lone
pair of electrons like O, N, S and halogens undergo 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 (180 – 200 nm).
• n → σ* transition3

31. • An electron from non-bonding orbital is promoted
to anti-bonding π* orbital.
• Unsaturated Compounds containing 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.
• For eg: Aldehydes and ketones the band due to n →
π* trnsitions occur in the range 270-300nm
• n → π* transition4

32. •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.
• σ → π* transition5
• π → σ* transition 6&

33. Transition Probablity
• It is not necessary that exposure of a compound to
ultraviolet or visible radiation always give rise to
electronic transition. The probability of transition
depends on the value of molar extinction coefficient
and other factors. Accordingly transitions are of two
types
a. Allowed transitions: transitions having value of
molar extinction coefficient 104 or more.
E.g. π → π* transition in 1,3-butadiene etc.

34. a. Forbidden transitions: transitions for which value
of molar extinction coefficient is generally less
than 104
E.g. n → π* transitions of saturated aldehydes and
ketones etc.

35. Terms used
in
UV / Visible
Spectroscopy

36. Chromophore
Previously the chromophore was used to denote a
group presence of which gives a colour to a compound.
But now a days term chromophore is used to denote
any group which is capable of absorbing
electromagnetic radiations in the visible or ultraviolet
region.
It may or may not impart colour to the compound.

37. Two types of chromophores are known:
a. In which group is having π electrons undergo π →
π* transitions. Eg ethylene, acetylenes.
b. In which group is having both π and n electrons
undergo two types of transitions π → π* and n- π*
transitions

38. Chromophore
To interpretate UV – visible spectrum following
points should be noted:
1. Non-conjugated alkenes show an intense
absorption below 200 nm & are therefore
inaccessible to UV spectrophotometer.
2. Non-conjugated carbonyl group compound
give a weak absorption band in the 200 - 300
nm region.

39. e.g. Acetone which has λmax = 279 nm
and that cyclohexane has λmax = 291 nm.
When double bonds are conjugated in a
compound λmax is shifted to longer wavelength.
e.g. 1,5 - hexadiene has λmax = 178 nm
2,4 - hexadiene has λmax = 227 nm
CH3
C
CH3
O
O
CH2
CH2
CH3
CH3

40. Chromophore
3. Conjugation of C=C and carbonyl group shifts
the λmax of both groups to longer wavelength.
e.g. Ethylene has λmax = 171 nm
Acetone has λmax = 279 nm
Crotonaldehyde has λmax = 290 nm
CH3
C
CH3
O
CH2 CH2
C
CH3
O
CH2

41. Auxochrome
It is a group which itself does not act as a
chromophore but when attached to a chromophore it
shifts the absorption maximum towards longer
wavelength along with an increase in the intensity of
absorption.
Common auxochromic groups are -OH, -OR, -NHR, -
NH2 etc.
For eg when -NH2 group is attached to benzene ring
its absorption changes from λmax 255 to λmax 280 while
presence of –OH changes λmax of benzene from 255
to 270.

42. Auxochrome
e.g. Benzene λmax = 255 nm
Phenol λmax = 270 nm
Aniline λmax = 280 nm
OH
NH2

43. Absorption
& Intensity
Shifts

45. • When absorption maxima (λmax) of a
compound shifts to longer wavelength, it is
known as bathochromic shift or red shift.
• The effect is due to presence of an auxochrome
or by the change of solvent.
• e.g. An auxochrome group like –OH, -OCH3
causes absorption of compound at longer
wavelength.
• Bathochromic Shift (Red Shift)1

46. • In alkaline medium, p-nitrophenol shows red shift.
Because negatively charged oxygen delocalizes more
effectively than the unshared pair of electron.
λmax = 255 nm λmax = 265 nm
• Bathochromic shift is also caused when two or more
chromophores are present in conjugation
• Bathochromic Shift (Red Shift)1
OH
N
+ O
-
O
OH
-
Alkaline
medium
O
-
N
+ O
-
O

47. • When absorption maxima (λmax) of a
compound shifts to shorter wavelength, it is
known as hypsochromic shift or blue shift.
• The effect is due to presence of an group,
removal of conjugation or by the change of
solvent.
• Hypsochromic Shift (Blue Shift)2

48. • Aniline shows blue shift in acidic medium, it
loses conjugation.
Aniline
λmax = 280 nm λmax = 265 nm
• Hypsochromic Shift (Blue Shift)2
NH2
H
+
Acidic
medium
NH3
+
Cl
-

49. • When absorption intensity (ε) of a compound is
increased, it is known as hyperchromic shift.
• If auxochrome introduces to the compound,
the intensity of absorption increases.
Pyridine 2-methyl pyridine
λmax = 257 nm λmax = 260 nm
ε = 2750 ε = 3560
• Hyperchromic Effect3
N N CH3

50. • When absorption intensity (ε) of a compound is
decreased, it is known as hypochromic shift. Here
hypochromic effect if seen due to presence of methyl
group as it is able to distort he geometry of molecule
Naphthalene 2-methyl naphthalene
ε = 19000 ε = 10250
CH3
• Hypochromic Effect4

51. Wavelength ( λ )
Absorbance(A)
Shifts and Effects
Hyperchromic shift
Hypochromic shift
Red
shift
Blue
shift
λmax

52. INSTRUMENTATION

53. The various components of a UV Spectrometer are a
follows:
• Radiation source
• Monochromators
• Detectors
• Recording system
• Sample cell

54. Radiation source
• In UV spectrometers the most commonly used
radiation sources are:
Tungstan lamp
Hydrogen discharge lamp
Deuterium lamp
Xenon discharge lamp
Mercury arc
In all sources, excitation is done by passing electrons
through a gas and this causes collisions between gas
molecules and electrons and gas molecules get
excited. These excited molecules emit UV radiations

55. Monochromators
Monochromator is used to disperse the radiation
according to the wavelength.
The essential elements of a monochromators are:
An entrance slit: It sharply defines the incoming
beam of heterochromatic radiation
A dispersing element: It disperses the
heterochromatic radiation into its component
wavelengths
An exit slit: It allows the light of light of single
wavelength to pass through it.

56. Dispersing element may be a Prism or Grating
Prisms are generally made up of glass, quartz or fused silica.
Glass has the highest resolving power but not transparent to
radiation between 200 – 300 nm.
Quartz and fused silica are transparent to entire UV range and
hence are widely used.

57. Detectors
• There are three common types of detectors used in UV
spectroscopy. These are:
1. Barrier layer cell: This cell is also known as Photovoltaic
cell.
The radiation falling on the surface produces electrons at metal
semiconductor interface. A barrier exist between base(iron) and
semiconductor which prevent electrons from moving into iron.
This accumulation of electrons on metal surface produces a
voltage difference. If the resistance in external circuit is low
current flows which is directly proportional to intensity of
incident radiation

58. 2. Photocell: It consists of a high senstivity cathode in th form
of half cylinder of metal contained in a evacuated tube. Anode
is also present in the tube more or less fixed along the axis of
tube. The inside surface is coated with a light sensitive layer.
When light falls on photocell surface coating emits electrons
which are attracted and collected ny anode. The current flows
between cathode and anode dur to estabilsed potential
difference. Current produced is cosidered as a measure of
radiation falling on detector

59. 3. Photomultiplier tube: It is a combination of a photodiode
and electron multiplying amplifier. It consists of an evacuated
tube which contains one photocathode and 9-16 electrodes
known as dynodes. The surface of each dynode is of Be-Cu, Cs-
Sb or similar material.

60. UV Spectrophotometers
• Two types of spectrophotometers are available
1. Single beam system

61. Double beam system

62. APPLICATIONS OF
UV / VISIBLE
SPECTROSCOPY

63. Applications
• Qualitative & Quantitative Analysis:
– It is used for characterizing aromatic compounds
and conjugated olefins.
– It can be used to find out molar concentration of the
solute under study.
• Detection of impurities:
– It is one of the important method to detect
impurities in organic solvents.
• Detection of isomers are possible.
• Determination of molecular weight using Beer’s
law.