LAMBERT'S LAW : when monochromatic radiation is passed through a medium the rate of the decrease in the intensity of radiation with thickness of the medium is directly proportional to intensity of the incident radiation dI/dt α I  BEER'S LAW  According to this law, when a beam of monochromatic radiation is passed through a solution of absorbing species, the intensity of beam of monochromatic light decreases exponentially with increase in concentration of absorbing species dI/dc α I

1. UV-VISIBLE
SPECTROSCOPY
BEER-LAMBERT LAW
P R E S E N T E D BY
S . PA L A N I A N A N T H
P R E S E N T E D TO
D R . S . S I VA S A N K A R A N A R AY I N I

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.
• What is electromagnetic radiation ?
• Electromagnetic radiation is a type of energy that is transmitted through space at enormous
velocities.
• Radiant energy has wave nature and being associated with electric as well as magnetic field,
these radiations are called electromagnetic radiation.
• Electromagnetic radiation has it’s origin in atomic and molecular processes.
• The field may be represented as electric and magnetic vectors oscillating in mutually
perpendicular planes.

3. • The energy of an EMR can be given by the following equation:
• E=hµ
• Where , E= Energy of radiation h=Planck’s constant (6.62607015 × 10−34 joule second.)
• µ=Frequency of radiation
• Frequency (µ)= c/λ
• Where c=velocity of light in vacuum
• λ= wavelength
:-Hence, E=hµ
• Therefore , energy of a radiation depends upon frequency and wavelength of radiation.
E=hc/λ

4. ELECTROMAGNETIC WAVE HAS FOLLOWING CHARACTERISTICS
WAVELENGTH:
• It is the distance between two successive crest or trough of a wave. It is denoted by ‘λ’. It is
measured in meter(m), nano meter (nm), micro meter(µm) and Ao units.
• 1A0=10-8cm=10-10m
FREQUENCY:
• The number of complete wavelength units passing through a given point in unit time [number
of cycles per seconds].It is denoted by ‘ν’.
WAVE NUMBER:
• The number of waves per cm.

5. ELECTROMAGNETIC SPECTRUM
• The arrangement obtained by arranging various types of electromagnetic waves or radiations
in order of their increasing wavelength or decreasing frequencies is called electromagnetic
spectrum.
• The electromagnetic spectrum is divided into a number of regions; these are artificial divisions
in the sense that they have been defined solely as a result of differences in the
instrumentation required for producing and detecting radiation of a given frequency range

6. • Ultraviolet: 190~400nm
• Violet: 400 - 420 nm
• Indigo: 420 - 440 nm
• Blue: 440 - 490 nm
• Green: 490 - 570 nm
• Yellow: 570 - 585 nm
• Orange: 585 - 620 nm
• Red: 620 - 780 nm

7. SPECTROSCOPY
"The study of interaction of electromagnetic radiation with molecules/atoms
• Types:
• 1 )Absorption Spectroscopy: The study of absorbed radiation by molecule , in the of spectra.
• E.g.: UV, IR, NMR, colorimetry, atomic absorption spectroscopy
• 2) Emission Spectroscopy: The radiation emitted by molecules can also be studied to reveal
the structure of molecule. E.g. : flame photometry, fluorimeter.
Study of spectroscopy
• Atomic spectroscopy: interaction of EMR+ATOMS Changes in energy take place at atomic
level.
• Eg: atomic absorption spectroscopy, flame photometry
• Molecular spectroscopy: Interaction of EMR + molecules Changes in energy take place at
molecular level
• E.g.: UV, IR, colorimetry
• Results in transitions between vibrational,& rotational energy levels

8. THEORY INVOLVED
• When a beam of light falls on a solution or homogenous media ,a portion of light is reflected
,from the surface of the media, a portion is absorbed within the medium and remaining is
transmitted through the medium.
• Thus if 10 is the intensity of radiation falling on the media
• Ir is the amount of radiations reflected,
• la is the amount of radiation absorbed &
• It the amount of radiation transmitted
• then
• 10= Ir + la + It
• ABSORPTION LAWS
• Lambert's law
• Beer's law
• Beer-lambert's law

10. LAMBERT'S LAW :
• Lambert’s law states that the rate of decrease of intensity of monochromatic light with the
thickness of the medium is directly proportional to the intensity of incident light.
dI/dt α I
BEER'S LAW
• According to this law, when a beam of monochromatic radiation is passed through a
of absorbing species, the intensity of beam of monochromatic light decreases exponentially
with increase in concentration of absorbing species
dI/dc α I

11. Derivation of beer – lambert’s law

12. According to beer’s law
.

16. A= Kct Instead of K,
we can use ε A = εct K, AND K’ = ε
{ Mathematical eqn for beer lambert’s law}
Where ε is the Molar extinction coefficient, a constant dependent upon the wavelength of
incident radiation and the nature of absorbing material and the concentration is expressed in
gram mole/litre
Since absorbance A= log Io /It
we can infer that A= εbc (Equation of beer – Lambert’s law)
Where: A – Absorbance or optical density.
ε – Molar extinction coefficient
c – Concentration of the MEDIUM OR SOLUTION (mol/lit)
b – Path length (normally 10mm or 1cm)
Or A= abc
a –absorptivity,
if concentration is expressed in grams/litre

17. ABSORPTIVITY (a):
• It is characteristics of a particular sample for a given wavelength and it is defined as the ratio
of absorbance to the product of concentration and length of optical path. a= A/ bc
Absorbance (A):
• It is the logarithm to the base 10 of the reciprocal of the transmittance. A = log 10 (1/T)
Molar absorptivity ‘ɛ’:
• It is the absorptivity expressed in units of liter/moles cm. The concentration is in moles/liter
and the cell length is in centimeter. ɛ = AM/bc

18. LAMBERT- BEER'S LAW:
when a beam of monochromatic radiation passes through a
homogenous medium the rate of decrease in the intensity of
radiation with the thickness of medium and concentration of
solution is directly proportional.

20. REASONS FOR DEVIATION FROM BEER LAMBERT’S LAW:
. True deviation : True deviations are related to the concentration of the absorbing substance.
Beers law holds good only for dilute solutions.
• Chemical deviation : Chemical deviations arise if the absorbing species undergo chemical
changes such as association, complex formation, dissociation, hydrogen bonding, hydrolysis,
ionization or polymerization
• Instrumental Deviation : Only monochromatic light gives beers law use of polychromatic
gives negative deviation.
• Any fluctuations in intensity of light, change in the sensitivity of detector, improper slit width
can lead to deviation from beer lamberts law.

21. • A solution of Tryptophan has an absorbance at 280 nm of 0.54 in a 0.5 cm length cuvette.
Given the absorbance coefficient of trp is 6.4 × 10 3 LMol-1 cm-1 . What is the concentration
of solution? Solution:
• A = εct
• A= 0.54, ε = 6.4 × 10 3 LMol-1 cm-1 l= 0.5 cm C=?
• SO
• A/ε l = 0.54 / 6.4 × 10 3 × 0.5
• Answer = 0.000168

22. • A solution shows a transmittance of 20%, when taken in a cell of 2.5 cm thickness. Calculate
its concentration, if the molar absorption coefficient is 12000 dm3/mol/cm.
• Solution
A = 2 - log10 %T
= 2 - log10 20
= 2 – 1.301
= 0.698
A = ε l c
l= 2.5 cm ε = 12000 dm3/mol/cm A=0.698 c=?
SO
So c = A/ ε l
= 0.698/ 12000 × 2.5
Answer = 2.33 X 10 – 5 mol / dm3

23. UV SPECTROSCOPY
• UV spectroscopy involves absorption spectroscopy where molecules interact with UV radiation
and produce absorption spectra in the range of 200 nm to 400 nm.
• The UV range in electromagnetic spectra is subdivided into two
Far or vacuum UV region : 10 nm – 200 nm Near or
Quartz UV region : 200 nm – 400 nm
• UV spectroscopy is a molecular spectroscopic method arising due to transition of valence
electrons in a molecule from the ground state energy (E0) to the higher excited state (E1).
• Then the difference in change of energy is ΔE = E1- E0 =hν

24. UV SPECTROSCOPY

25. UV SPECTROSCOPY

26. SOME IMPORTANT TERMS
I. Chromophore:
A covalently unsaturated group responsible for electronic absorption is called
chromophore (Any functional group responsible for imparting color to the compound).
Eg: C=C, C=O, NO2 etc.
A compound containing chromophore is called a chromogen.
II. AUXOCHROME
It is a group of atoms attached to a chromophore which modify the ability of
chromophore to absorb light.
• They themselves fail to produce the color but when present along with chromophore in an
organic compound intensify the color of chromogen.
• E.g.: -OH group, -NH2 group, -CHO group and methyl mercaptan group

27. Absorption and intensity shifts

28. BATHOCHROMIC SHIFT OR RED SHIFT
• Bathochromic effect is a shift of an absorption maximum towards longer wavelength.
• It may be produced by a change of medium or by a presence of an auxochrome or by the
change of solvents.
• The n→ π* transition for carbonyl compounds experience bathochromic shift when the
polarity of the solvent is decreased.
• HYPOCHROMIC EFFECT OR BLUE SHIFT
• This shift towards shorter wavelength (blue shift).
• This is due to change in medium, solvents, substitution or removal of conjugation.
• E.g.: Aniline absorbs at 280 nm, but in acidic solutions the absorbance is at 203 nm due to
blue shift. 55

29. E.G.
Aniline shows blue shift in acidic medium since it loses conjugation. Aniline (280nm) &
Anilinium ion (203nm).

30. HYPERCHROMIC EFFECT
• This effect leads to increased absorbtion intensity that is ɛmax increases.
• E.g.: The B band for pyridine at 257 nm, ɛmax is 2750 is shifted to 262 nm with ɛ max 3560 for
2-methyl pyridine.
• The introduction of an auxochrome increase the intensity of absorption.
HYPOCHROMIC EFFECT
• This effect leads to decreased absorption intensity, ɛmax .
• The introduction of group which distorts the geometry of the molecule cause hypochromic
effect.
• Eg; Biphenyl absorbs at 250 nm, ɛmax 19000 where as 2- methyl biphenyl absorbs at 237 nm,
ɛmax 10250.
• It is due to distortion caused by the methyl group in 2-methyl biphenyl group.

32. TYPES OF ELECTRONS ARE INVOLVED IN UV VISIBLE
SPECTROSCOPY
1 . ’σ’ electrons:
• These electrons are involved in saturated bonds.
• The amount of energy required to excite σ electrons are high.
• σ electrons do not absorb near UV region, but absorb in far UV region .
2. ’π’electrons:
• These electrons are involved in unsaturated compounds.
• Typical compounds with π bonds are trienes and aromatic compounds.
3 . ’n’ electrons:
• These electrons are not involved in bonding between atoms in molecules.
• So it is called non bonded electrons. E.g.; Organic compounds containing nitrogen, oxygen or
halogens.

33. TYPES OF ELECTRONIC TRANSITIONS
• Energy absorbed in the UV region by valence electrons causes transition from ground state to
excited state.
• The valence electrons are exited from bonding to an antibonding orbitals.
• The energy required for various transitions are in the following order.
• σ → σ* > n → σ* c→ π* > n→ π* 19

34. ELECTRONIC TRANSITIONS

35. 1.σ → σ* Transition:
• σ → σ* Transition occurs in compounds in which all the electrons are involved in σ bonds.
• Eg: Saturated hydrocarbons.
• The energy required for this transitions is very high and the absorption band occurs in the far
UV region (125-135nm).
• Compounds which undergo σ → σ*
• Transition are methane (125nm), Ethane(135nm), cyclopropane (190nm).

36. 2) n → σ* Transition:
Saturated compounds containing atoms with unshared electron pairs (Oxygen, Nitrogen,
Sulphur, or Halogen) or non bonding electrons are capable of n → σ* Transition.
n → σ* Transition need less energy than the σ → σ* transition.
These transitions can be brought about by radiation in the range of 150- 250nm with most
absorption peaks appear below 200nm

37. 3) π→ π* Transition:
These transitions occurs in compounds with unsaturated compounds such as alkenes, carbonyl
compounds and aromatic compounds.
A selection rules determine whether a transition to a particular π* orbital is allowed or
forbidden.
Eg: The UV spectrum of ethylene exhibit an intense band at 174nm and a weak band at 200nm.
Both of these are due to π→ π* Transitions.
According to the selection rule, the band at 174 nm represents an allowed transition

38. Aromatic compound show a number of band.
Bbenzenoid (B) band:
• This band is due to π→ π* Transitions in aromatic or hetero aromatic molecules.
• The benzene shows a broad absorption band containing peaks between 230- 270nm.
• When a chromophoric group is attached to benzene ring, the B-bands are observed at longer
wavelength.
R-Band
• R-band transition originate due to n-π* transition of a single chromophoric group and having
at least one lone pair of electrons on the hetero atom
• These are less intense with εmax value below 100

39. Ethylenic (E) band:
• This band is due to the electronic transition in the benzenoid system of three ethylenic bonds
are in closed cyclic conjugation.
• These are further characterized as E1 and E2 bands.
• The E1 band of benzene, which appears at lower wavelength (184nm) is more intense than E2
band that occurs at longer wavelength (204nm)
K band:
• This band exhibited by aromatic compounds with the benzene ring directly attached to a
group containing multiple bond.
• E.g.: Styrene, Benzaldehyde etc.

40. 4) n→ π* Transition:
• n→ π* Transitions are shown by unsaturated molecules which contain atom such as oxygen,
nitrogen etc.
• These transition exhibit a weak band in their absorption spectrum.
• The peak due to this transition is called R-band.
• In aldehyde and ketones the band due to the n → σ* Transition generally occur in the range
of 270 to 300nm.
• In aldehydes and ketones n → σ* Transition arise from excitation of a lone pair of electrons in
the 2py orbital of the oxygen atom into the antibonding π* orbital of the carbonyl group.
• When hydrogen is replaced by methyl group in an aldehyde group, this results in a shift of
band due to the n→ π* to shorter wavelength.

41. Applications of UV Spectroscopy

42. 1. Detection of Impurities
• It 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.
2. Structure elucidation of organic compounds
• It is useful in the structure elucidation of organic molecules, such as in detecting the presence or
absence of unsaturation, the presence of hetero atoms.
3. 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, b- is the length of the cell that is used in UV spectrophotometer.
• UV absorption spectroscopy can characterize those types of compounds which absorbs UV
radiation thus used in qualitative determination of compounds. Identification is done by comparing
the absorption spectrum with the spectra of known compounds.

44. 4 . 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.
• Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength 284 nm.
5 . Molecular weight determination
Molecular weights of compounds can be measured spectrophotometrically by preparing the
suitable derivatives of these compounds.