UV Visible spectroscopy as per PCI syllabus: Electronic transitions, chromophores, auxochromes, spectral shifts, solvent effect on absorption spectra, Beer and Lambert’s law, Derivation and deviations.

1. by
Dr. MONIKA SINGH
(M.Pharm, PhD)
Identification
of Compounds

2. BP701T. INSTRUMENTAL METHODS OF
ANALYSIS (Theory)
Scope: This subject deals with the application of instrumental methods in
qualitative and quantitative analysis of drugs. This subject is designed to impart a
fundamental knowledge on the principles and instrumentation of spectroscopic
and chromatographic technique. This also emphasizes on theoretical and
practical knowledge on modern analytical instruments that are used for drug
testing. Objectives: Upon completion of the course the student shall be able to: •
Understand the interaction of matter with electromagnetic radiations and its
applications in drug analysis. • Understand the chromatographic separation and
analysis of drugs. • Perform quantitative & qualitative analysis of drugs using
various analytical instruments.
Unit -I 10 Hours
UV Visible spectroscopy: Electronic transitions, chromophores, auxochromes,
spectral shifts, solvent effect on absorption spectra, Beer and Lambert’s law,
Derivation and deviations.
Instrumentation - Sources of radiation, wavelength selectors, sample cells,
detectors-Photo tube, Photomultiplier tube, Photo voltaic cell, Silicon Photodiode.
Applications- Spectrophotometric titrations, Single component and multi component
analysis.

3. Identification of Compound
H2
CH3
CH3
CH3
H3C
H2
H
H
H
H
H
O

4. SPECTROMETRIC IDENTIFICATION
I. Introduction of Spectrometric Analyses
II. Ultra Violet Spectrometry
III. Infrared Spectrometry
IV. Nuclear Magnetic Resonance Spectrometry
V. Mass Spectrometry

5. Introduction of Spectrometric Analyses
The study how the sample interacts with different
wavelength in a given region of electromagnetic radiation
is called spectroscopy or spectrochemical analysis.
The collection of measurements signals (absorbance) as a
function of electromagnetic radiation is called a spectrum.

6. Spectroscopic Techniques
UV-vis UV-vis region bonding electrons
Atomic Absorption UV-vis region atomic transitions (val. e-)
FT-IR IR/Microwave vibrations, rotations
Raman IR/UV vibrations
FT-NMR Radio waves nuclear spin states
X-Ray Spectroscopy X-rays inner electrons, elemental
X-ray Crystallography X-rays 3-D structure

7. UVX-rays IRg-rays RadioMicrowave
Visible
Spectrum of Radiation

8. Electromagnetic Spectrum.
Wavelength, , cm
frequency, , (cycles/sec)
g-ray
-ray
ultraviolet
visible
infrared
microwave
radio
Power
violet
blue
green
yellow
orange
red
visible region
400 500 600 700 800
1020
1018
1016
1014
1012
1010
108
106
104
102
10-10
10-8
10-6
10-4
10-2
1 102
104
106
108
Wavelength, , nm

10. Energy Absorption
The mechanism of absorption energy is different in the
Ultraviolet, Infrared, and Nuclear Magnetic Resonance
regions. However, the fundamental process is the absorption
of certain amount of energy.
The energy required for the transition from a state of lower
energy to a state of higher energy is directly related to the
frequency of electromagnetic radiation that causes the
transition.

11. Spectral Distribution of Radiant Energy
ν = Wave number (cm -1
)
 = Wave length (nm)
C = Velocity of Radiation (constant) 3  1010
cm/sec
ν = Frequency of Radiation (cycles/sec)
ν = =
(The energy of photon) E = ν h(Planck's Constant 6.62  10-27
erg - sec)
E = ν h = h
C = ν 
ν =
C
ν

1

C

C
1 x 107
erg = I joule =0.239 calorie
Avogadro’s number = 6.02 x 10 23mol-1

12. Visible
Ultra
violet
Radio
Gamm
a ray
Energy
Wave
Number V
Wavelengt
h
λ
Frequen
cy
υ
Type
Radiatio
n
Type
spectrosco
py
Type
Quantum Transition
Kcal/mo
l
Electron
volts,
eV cm-1 cm Hz
9.4 x 107 4.9 x 106 3.3 x
1010
3 x 10-11 1021
9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017
9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015
9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013
9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011
9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107
X-ray
Infrared
Micro-
wave
Gamma ray
emission
X-ray
absorption,
emission
UV absorption
IR absorption
Microwave
absorption
Nuclear
magnetic
resonance
Nuclear
Electronic
(inner shell)
Molecular
vibration
Electronic
(outer shell)
Molecular
rotation
Magnetically
induced spin
states
Spectral Properties, Application and Interactions of Electromagnetic Radiation

13. Spectroscopy
Introduction
Absorption: electromagnetic (light) energy is transferred to atoms, ions, or molecules in
the sample. Results in a transition to a higher energy state.
- Transition can be change in electronic levels, vibrations, rotations, translation, etc.
- Concentrate on Molecular Spectrum in UV/Vis (electronic transition)
- Power (P): energy of a beam that reaches a given area per second
- Intensity (I): power per unit solid angle
- P and I related to amplitude2
Eo
E1
h Energy required of photon
to give this transition:
h = DE = E1 - Eo
(excited state)
(ground state)

14. Ultra Violet Spectrometry
The absorption of ultraviolet radiation by
molecules is dependent upon the electronic
structure of the molecule. So the ultraviolet
spectrum is called electronic spectrum.

15. Electronic Excitation
The absorption of light energy by organic compounds in the
visible and ultraviolet region involves the promotion of
electrons in , , and n-orbitals from the ground state to higher
energy states . This is also called Energy Transition. These
higher energy states are molecular orbitals called antibonding.

17. B.) Laws:
1.) Beer’s Law: ( August Beer) When a beam of monochromatic light passed
through a absorbing substance, the rate of decrease of Intensity of radiation is
(amount of light absorbed (A) by a sample) is dependent on Thickness of medium
{the path length (b or l)}, and a proportionality constant
Amount of light absorbed is dependent on frequency ()
2.) Lambert’s Law: () When a beam of monochromatic light passed through a absorbing
substance, the rate of decrease of Intensity of radiation with Thickness of medium of a
sample is dependent on concentration of medium (c) and a proportionality constant
c
Absorbance is directly proportional to concentration Fe+2
Increasing Fe+2 concentration 

18. 3.) Lambert-Beer’s Law: The amount of light absorbed (A) by a sample is
dependent on thickness of absorbing medium {the path length (b or l)}, concentration
of the sample (c) and a proportionality constant (e – molar absorptivity)
A = ebc or ecl
Transmittance (T) = I/Io %Transmittance = %T = 100T
Absorbance (A) = - log10T= - log10 I/Io = log10 Io/I
No light absorbed-
% transmittance is 100%  absorbance is 0
All light absorbed-
% transmittance is 0%  absorbance is infinite

19. T=I/Io
A= - log T = -log (I/Io)
Calibration curve

20. Relationship Described :
A = Absorbance = ebc or ecl = -log (%T/100)
e = molar absorptivity: constant for a compound at a given frequency ()
units of L mol-1 cm-1
b or l = path length: cell distance in cm
c = concentration: sample concentration in moles per liter.
Therefore, by measuring absorbance or percent transmittance at a given frequency can get
information related to the amount of sample (c) present with an identified e and .
Note: law does not hold at high
concentrations, when A > 1
The relationship between path
length absorbance and
concentration is known as Beer’s
law.

21. Why do we prefer to express the Beer-Lambert law using
absorbance as a measure of the absorption rather than %T ?
Now, suppose we have a solution of copper sulphate (which appears blue because it has
an absorption maximum at 600 nm). We look at the way in which the intensity of the light
(radiant power) changes as it passes through the solution in a 1 cm cuvette. We will look
at the reduction every 0.2 cm as shown in the diagram below. The Law says that the
fraction of the light absorbed by each layer of solution is the same. For our
illustration, we will suppose that this fraction is 0.5 for each 0.2 cm "layer" and calculate
the following data:

22. Deviation
Following are the situations when Beer’s law is
not obeyed:
• When different types of molecules are in
equilibrium with each other. eg. Keto-enol
tautomer
• An association complex is formed by the
solute and the solvent.
• When fluorescent compounds are present.
• When thermal equilibrium is attained
between the excited state and the ground
state.

24. Ultraviolet (UV) Spectroscopy
– The Output
Decreasing wavelength in nm
Increasingabsorbance*
*Absorbance has no
units – it is actually the
logarithm of the ratio of
light intensity incident on
the sample divided by
the light intensity leaving
the sample.
A = absorbance; c = concentration in moles l-1; l = pathlength in cm ; e = molar absorptivity (also
known as extinction coefficient) which has units of moles-1 L cm -1.
Beer Lambert Law
A = e.c.l

25. More Complex Electronic
Processes
• Fluorescence: absorption of radiation
to an excited state, followed by
emission of radiation to a lower state of
the same multiplicity
• Phosphorescence: absorption of
radiation to an excited state, followed
by emission of radiation to a lower
state of different multiplicity
• Singlet state: spins are paired, no net
angular momentum (and no net
magnetic field)
• Triplet state: spins are unpaired, net
angular momentum (and net magnetic
field)

26. Observed electronic transitions
1. The lowest energy transition (and most often obs. by UV) is
typically that of an electron in the Highest Occupied Molecular
Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital
(LUMO)
2. For any bond (pair of electrons) in a molecule, the molecular
orbitals are a mixture of the two contributing atomic orbitals; for
every bonding orbital “created” from this mixing (, ), there is
a corresponding anti-bonding orbital of symmetrically higher
energy (*, *)
3. The lowest energy occupied orbitals are typically the ; likewise,
the corresponding anti-bonding * orbital is of the highest
energy
4. -orbitals are of somewhat higher energy, and their
complementary anti-bonding orbital somewhat lower in energy
than *.
5. Unshared pairs lie at the energy of the original atomic orbital,
most often this energy is higher than  or  (since no bond is
formed, there is no benefit in energy)

27. Transitions
•  * transitions: high-energy, accessible in vacuum
UV (max <150 nm). Not usually observed in molecular UV-
Vis.
•n  * transitions: non-bonding electrons (lone pairs),
wavelength (max) in the 150-250 nm region.
•  * and n  * transitions: most common
transitions observed in organic molecular UV-Vis, observed in
compounds with lone pairs and multiple bonds with max =
200-600 nm.

28. 

*
*
n
Occupied Energy Levels
Unoccupied Energy Levels
UV
Increasingenergy
Energy required by different transitions:
* > n* > * > n*

29. Types of Bonds
Energy
 *
*
n


*
*
n*
n*
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Transitions * n* * n*
Examples  180 nm
C-C
Alkanes
C-O, C-S,C-
N
C=C,CC C=O,C=S,
C=N

30. Electronic Ground and Excitation
States
 *

* *

hv
*

hv
n
hv

*


hv
*
n
*

Energy
n *
*
hv
hv

31.   *
H-Bonding (Strengthen bonds) leads to Blue shift
(Hypsochromic shift)
*
Extending conjugation leads to Red shift
(Bathochromic shift)
max 253 239 256 248

32.   * -
Example:   * transitions responsible for ethylene UV absorption
at ~170 nm
HOMO u bonding molecular orbital LUMO g antibonding molecular orbital
h 170nm photon
-*; max=218
e=11,000
n-*; max=320
e=100

33. *

*

C C
C C

34. 
*
n
O
O
C O

35. Chromophore is a functional group which absorbs a
characteristic ultraviolet or visible region.
210 nm Double Bonds
233 nm Conjugated Diene
268 nm Conjugated Triene
315 nm Conjugated Tetraene
Carbonyls
Azo compounds
Nitro groups


 and * orbitals  and * orbitals
Chromophore

36. Auxochrome are the groups that enhances the colour
imparting property of a characteristic ultraviolet or visible
region.
Auxochrome
2 properties:
- Causes shift in absorption maxima
- Causes changes in intensity of Chromophore
Examples:
Hydroxyl groups
Amino groups
Halogen groups
- cause a red shift / Bathochromic Shift

37. 37
Shifts/Substituent Effects
(Substituents may have any of four effects on a chromophore)
i. Bathochromic shift (red shift) – a shift to longer ; lower energy
ii. Hypsochromic shift (blue shift) – shift to shorter ; higher energy
iii. Hyperchromic effect – an increase in intensity/ Energy
iv. Hypochromic effect – a decrease in intensity/ Energy
200 nm 700 nm
E
Hypochromic
Hypsochromic
Hyperchromic
Bathochromic

38. Solvents for UV
(showing high energy cutoffs)
Water 205
CH3CN 210
C6H12 210
Ether 210
EtOH 210
Hexane 210
MeOH 210
Dioxane 220
THF 220
CH2Cl2 235
CHCl3 245
CCl4 265
benzene 280
Acetone 300

39. Solvent Effect
•  * transitions: As Polarity of solvent Increases,
(G.S. Becomes less polar than E.S.)
 Increases,  decreases -RED shift
(Bathochromic shift)
•n  * and n  * transitions:
As Polarity of solvent Increases, due
to hetero atom H-bonding Increases,
(G.S. Becomes more polar than E.S.)
 decreases,  increases -Blue shift
(Hypsochromic shift)

40. Temperature Effect
In n  * and n  * transitions:
As Temperature of solvent Increases, due to
hetero atom H-bonding Decreases, (G.S. Becomes
less polar than E.S.),  Increases,  decreases -
RED shift (Bathochromic shift)

41. Derivation
Derivative UV-spectrophotometry is an analytical technique of
enormous implication commonly in obtaining mutually
qualitative and quantitative in order from spectra that are of
unresolved bands, with respect to qualitative and quantitative
analysis, it uses first or higher derivatives of absorbance in
accordance with wavelength.
In quantitative analysis, derivative spectra enlarge difference
between spectra to resolve overlapping bands.
Derivative spectra can obtain by variety of experimental techniques; the
differentiation can be done numerically even if the spectrum has been
recorded digitally or in computerized readable form.
When spectrum is scanned at a constant rate, real time derivative
spectra can be recorded either by achieving the time derivative of the
spectrum or by wavelength modulation.

42. The first derivative spectrum (D1) is a plot of the rate of change of absorbance
with wavelength against wavelength, i.e. plot of ΔA/Δλ vs. λ.
The second derivative spectrum(D2) is a plot of Δ2A/ Δλ2vs. λ. Not only can the
first and second derivative of the absorbance spectrum be obtained, but up to the fourth
derivative is possible. However, as the differentiation order increases, the noise increases
as well, and if a lower derivative is fine, going to higher derivatives is a waste of time
and effort.

43. Zero
order
First
order
Second
order
Zero, first and Second-order UV derivative spectrum

44. 1st order Derivative spectra

45.  Second derivative spectrum is characterised by two satelite maxima and an
of theinverted band of which the minimum corresponds to the λmax
fundamental band.
Satelite maxima's
Second derivative spectrum eliminates the broad band absorption.

46. Advantages of First derivative spectroscopy:
(1) Precise determination of the λmax can be obtained from the zero crossing
of the first derivative.
(2)Improved spectral resolution
(3)Discrimination of broad bands
Resolution enhancement in derivative spectroscopy

48. Applications of derivative
spectroscopy
 Multicomponent analysis Derivative spectrophotometry (DS) has been
mainly used in pharmaceutical analysis for assaying of a main ingredient
in a presence of others components or its degradation product.
 Calculation of some physico-chemical constants, e.g. reaction,
complexation or binding constants.
Disadvantage
The main disadvantage of derivative spectrophotometry is
its poor reproducibility.

49. THANKS