1. JSPM’s
Charak College of Pharmacy & Research,
GAT No.720/1&2,WAGHOLI, Pune-NAGAR ROAD,PUNE-412 207
Theory of U.V Spectrophotometry
Presented By, Guided By,
Miss. Pranita Kore Dr. Rajesh J Oswal
Prof.Sandip Kshirsagar
Department Of Pharmaceutical Chemistry
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2.  Introdution
 Electronic Transition’s
 Chromophores
 Auxochromes
 Beer’s-Lambert Law
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3. I.Introduction
A.UV radiation and Electronic Excitations:
1.The difference in energy between molecular bonding, non-bonding and
anti-bonding orbitals ranges from 125-650 kJ/mole
2.This energy corresponds to EM radiation in the ultraviolet (UV) region,
100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum
3.For comparison, recall the EM spectrum:
-rays X-rays UV IR Microwave Radio
Visible
4.Using IR we observed vibrational transitions with energies of 8-40
kJ/mol at wavelengths of 2500-15,000 nm
5.For purposes of our discussion, we will refer to UV and VIS spectroscopy
as UV
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4. Introduction
B. Band Structure
6. When these energy levels are superimposed, the effect can be
readily explained – any transition has the possibility of being
observed
Disassociation
R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
V2
E1 Vo
V1 R1 - Rn
R1 - Rn
Energy
Disassociation
R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
V2
V1 R1 - Rn
E0 Vo R1 - Rn
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5. II. Electronic transitions
The absorption of UV or visible radiation corresponds to the excitation of outer
electrons.
There are two types of electronic transition which can be considered;
1.Transitions involving p, s, and n electrons
2.Transitions involving charge-transfer electrons
When an atom or molecule absorbs energy, electrons are promoted from
their ground state to an excited state.
In a molecule, the atoms can rotate and vibrate with respect to each other.
These vibrations and rotations also have discrete energy levels, which can be
considered as being packed on top of each electronic level.
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6. Absorbing species containing p, s, and n electrons
Absorption of ultraviolet and visible radiation in organic molecules is
restricted to certain functional groups (chromophores) that contain
valence electrons of low excitation energy.
The spectrum of a molecule containing these chromophores is
complex.
This is because the superposition of rotational and vibrational
transitions on the electronic transitions gives a combination of
overlapping lines.
This appears as a continuous absorption band.
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7. Possible electronic transitios of , , and n electrons are;
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8. s -s* Transitions
An electron in a bonding s orbital is excited to the corresponding
antibonding orbital. The energy required is large.
For example, methane (which has only C-H bonds, and can only
undergo s -s* transitions) shows an absorbance maximum at 125 nm.
Absorption maxima due to s - s* transitions are not seen in typical UV-
Vis. spectra (200 - 700 nm)
n -s* Transitions
Saturated compounds containing atoms with lone pairs (non-bonding
electrons) are capable of n - s* transitions.
These transitions usually need less energy than s - s * transitions. They
can be initiated by light whose wavelength is in the range 150 - 250
nm. The number of organic functional groups with n - s* peaks in the
UV region is small.
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9. n - p* and p - p* Transitions
Most absorption spectroscopy of organic compounds is based on
transitions of n or p electrons to the p* excited state.
This is because the absorption peaks for these transitions fall in an
experimentally convenient region of the spectrum (200 - 700 nm).
These transitions need an unsaturated group in the molecule to
provide the p electrons.
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10. III.Chromophores
A. Definition
1. Remember the electrons present in organic molecules are
involved in covalent bonds or lone pairs of electrons on atoms
such as O or N
2. Since similar functional groups will have electrons capable of
discrete classes of transitions, the characteristic energy of these
energies is more representative of the functional group than the
electrons themselves
3. A functional group capable of having characteristic electronic
transitions is called a chromophore (color loving)
4. Structural or electronic changes in the chromophore can be
quantified and used to predict shifts in the observed electronic
transitions
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11. III.Chromophores
B. Substituent Effects:
General – from our brief study of these general chromophores, only
the weak n  * transition occurs in the routinely observed UV
The attachment of substituent groups (other than H) can shift the
energy of the transition
Substituents that increase the intensity and often wavelength of
an absorption are called auxochromes
Common auxochromes include alkyl, hydroxyl, alkoxy and amino
groups and the halogens
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12. III.Chromophores
C. Substituent Effects
General – 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
iv. Hypochromic effect – a decrease in intensity
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13. Hyperchromic
Hypsochromic Bathochromic
Hypochromic
200 nm 700 nm
Fig: Absorption & intensity shifts
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14. IV. Auxochromes
The color of a molecule may be intensified by groups called auxochromes which generally do
not absorb significantly in the 200-800nm region, but will affect the spectrum of the
chromophore to which it is attached.
The most important auxochromic groups are OH, NH2, CH3 and NO2 and their properties are
acidic (phenolic) or basic.
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 5-10nm. Other groups such as -NH2 and -NO2
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15. V. Beer-Lambert law
cuvette
slit
source
detector
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16. The method is most often used in a quantitative way to determine
concentrations of an absorbing species in solution, using the Beer-Lambert
law:
A=log10 (I0/I)=ε.C.L
Where,
A is the measured absorbance,
I0 is the intensity of the incident light at a given wavelength,
I is the transmitted intensity, L the pathlength through the sample, and
c the concentration of the absorbing species.
For each species and wavelength, ε is a constant known as the molar
absorptivity or extinction coefficient.
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17. 11
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