1

UV/VIS Spectroscopy
Humera Naz
PHC 403
By
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Introduction to UV-Visible Spectroscopy
It deals with the measurement of energy absorbed when
electrons are promoted to higher energy levels.
It provides information about the length and structure of
the conjugated part of molecule.
The excitation process
3
A UV spectrum is a plot of wavelength (or frequency) of
light absorbed versus the absorption intensity (absorbance
or transmittance).
The range of this absorption spectroscopy is 190 800
nm.
The UV spectrum may be divided into three regions.
Far (or vacuum) ultraviolet (10-200 nm)
Near or Quartz ultraviolet (200-380 nm)
Visible region (380-780 nm)
Types of electronic transition
The total energy of a molecule is the sum of E
elec
, E
vib
, and E
rot
.
Energy absorbed in the UV region produces changes in the
electronic energy of the molecule resulting from transitions of
valence electrons in the molecule.
Excitation of an electron from bonding or nonbonding
molecular orbital to antibonding orbital.
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5
Electronic energy levels and transitions
Unoccupied
levels
Occupied
levels
6
Some possible transitions in different compounds
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Relative energies of the t MOs of ethene and
1,3-butadiene
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o o* and o t* transitions:
High-energy
Accessible in vacuum UV (
max
<150 nm)
Not usually observed in UV-Vis spectroscopy
n o* transition:
Non-bonding electrons (lone pairs)
wavelength (
max
) is in the 150-250 nm region
n t* and t t* transitions:
most common transitions in UV-Vis spectroscopy
observed in compounds with lone pairs and multiple bonds
with
max
= 200-600 nm.
The UV Absorption process
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Principles of Absorption Spectroscopy
The measurement of light absorption by a solution of molecules is
governed by the Beer-Lambert Law, which is written as follows:
A = log (I

/I ) = c b c
A = absorbance
I

= intensity of light incident upon sample cell

I = intensity of light leaving sample cell
c = molar concentration of solute in moles per liter
b = length of sample cell in cm
c = molar absorptivity (0 10
6
)
or molar extinction coefficient
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The Molar absorptivity
of a compound is a constant that is
characteristic of the compound at a particular wavelength. The size
of the absorbing system and the probability that the electronic
transition will take place control the absorptivity. It ranges from 0 to
10
6
. (value above 10
4
are termed high intensity absorption,
while value below 10
3
are low intensity absorption)
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UV-visible Spectrum
Plot of absorbance versus wavelength.
Spectra often displayed (after simple calculation) as a
plot of either c or log c versus wavelength.

max
= 230 nm log c = 4.2
= 272 nm = 3.1
= 282 nm = 2.9
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Choice of solvents
It should be transparent over the desired range of
wavelength.
It should not contain conjugated system.
Water, 95% ethanol and hexane are most commonly used
solvent.
Polarity of solvent
Non polar solvent clear spectrum (fine structure)
Polar solvent fine structure
disappear
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Important Terms in UV spectroscopy
Chromophores
It is a group of atoms (part of molecule) that gives rise
an electronic absorption.
OR
The colored substances have their color due to the
presence of one
or more unsaturated groups responsible for electronic
absorption.
(C C, C C, C N, C N, C O, N N)
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Auxochrome
Substituents that increase the intensity of the absorption,
and possibly the wavelength, are called auxochrome.
For e.g. CH
3
, OH, OR, X, and NH
2
groups)
Auxochrome Unsubstituted
chromophore

max
(nm)
Substituted chromophore
max
(nm)
CH
3
OR
Cl
H
2
C=CH CH =CH
2
H
3
CCH =CHCOOH
H
2
C=CH
2
217
204
175
H
2
C=CH CH =CHCH
3
H
3
CC(OCH
3
) =CHCOOH
H
2
C=CH
2
Cl
224
234
185
Auxochromic effects
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Bathochromic shift (red Shift)
Shift of an absorption maximum to longer wavelength due to
substitution or solvent effect.
t t- transitions undergo bathochromic shift with a increase
in the polarity of the solvent or
When an auxochrome is attached to a C=C.
Benzene,
max
= 256 nm Aniline,
max
= 280 nm
The bathochromic shift is progessive as the number of
alkyl group increases.
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Hypsochromic shift (blue shift)
A shift of absorption maximum to higher energy or shorter
wavelength.
This is caused by the removal of conjugation or change in the
polarity of the solvent.
n t- transitions undergo this shift with an increase in the
polarity of solvent.
Aniline,
max
= 280 nm
Anilinium ion,
max
= 254 nm
CH
3
H
3
C
O

max
= 279 nm in hexane,

max
= 264.5 nm in water
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OR
When an auxochrome is attached to C=O where n electrons are
available.
H
C
H
O
NH
2
C
H
O
OH
C
H
O
Cl
C
H
O

max
(nm) 295 205 205 235
Hyperchromic effect
An increase in absorption intensity c
max
The introduction of an auxochrome usually
causes hyperchromic effect. For example
benzene shows B-band at 256 nm, c
max
200,
whereas aniline shows B-band at 280 nm,
c
max
1430.
E = 180 nm B = 250 nm
K = 200 nm
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Hypochromic effect
A decrease in absorption intensity c
max
.
This is caused by the introduction of a group which
distorts the chromophore.
H
3
C
H
3
C

max
= 252 nm
c
max
= 19,000

max
= 270 nm
c
max
= 800
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The Effect of Conjugation
In the presence of conjugated double bonds, The electronic energy
levels of a chromophore move closer together. As a result, the energy
required to produce a transition from an occupied electronic energy
level to an unoccupied level decreases, and the wavelength of the light
absorbed becomes longer.
CH
3
-(CH=CH)
n
-CH
3
of dimethylpolyenes
(A) n = 3; (B) n = 4; (C) n = 5
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The Effect of Conjugation on Alkenes
Increase in conjugation decreases the energy required for
electronic excitation.
A comparison of the t t- energy gap in a
series of polyenes of increasing chain length
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Auxochromes increase wavelength of absorption by
extension of the length of conjugating system. Resonance
interaction of lone pair with double bond(s) increase the
length of conjugated system.
As a result of this interaction, the nonbonded electrons
become part of the t system of molecular orbitals.
-OH, -OR, -X, or NH
2
are the examples of auxochromic
group.
C C B
C C
B
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Aromatic Compounds
Primary bands
184 nm
Primary bands
202 nm
Secondary bands
255 nm
Substitution on benzene ring can cause bathochromic and hyperchromic shifts
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A. Substituents with Unshared Electrons:
Substituents that carry nonbonding electrons (n electrons)
can cause shifts in the primary and secondary absorption
bands to longer wavelength (extended conjugation). The
nonbonding electrons can increase the length of the t
system through resonance.
The more available these n electrons for interaction with
the t e system of the aromatic ring, the greater will be the
shift.
Presence of n electrons in these compounds gives the
possibility of n t
-
transitions.
Y Y
+
Y
+
Y
+
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In compounds that are acids or bases, pH changes can
have very significant effects on the position of the primary
and secondary bands.
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B. Substituents Capable of t-Conjugation:
Substituents that are themselves chromophores usually
contain t electrons, interaction of the benzene-ring electrons
and the t electrons of the substituents can produce a new
electrons transfer band.
C
O
R
C O
R
C O
R
C O
R
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C. Electron-releasing and Electron-Withdrawing
Effects:
e-Releasing or e-withdrawing substituents have differing
effects on the position of absorption maxima.
Any substituent, regardless of its influence on the electron
distribution elsewhere in the aromatic molecule, shift the
primary absorption band to longer wavelength.
e-Withdrawing gps have essentially no effect on the
position of the secondary absorption band unless, they are
capable of acting as chromophore.
e-Releasing gps increase both the wavelength and intensity
of the secondary absorption band.
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D. Disubstituted Benzene Derivatives:
The effect of each two substituents to be considered.
For para-disubstituted benzene, two possibilities exist:
Both groups are e-releasing or e-withdrawing
(They exert effects similar to those observes with monosubstituted
benzenes)
If one of the gps is e-releasing while the other is e-
withdrawing
(The magnitude of the shift of the primary band is greater than the
shifts due to the individual gp, the enhanced shifting is due to
resonance interactions)
H
2
N N
O
O
H
2
N N
O
O
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If the two groups of a disubstituted benzene derivative
are either ortho or meta to each other, the magnitude of
the observed shift is approximately equal to the sum of the
shifts caused by the individual group.
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Added conjugation of benzene
The added conjugation in naphthalene, anthracene
and tetracene -> bathochromic shifts of absorption
bands.
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UV spectra of some representative drug molecules
Steroid enones:
Steroids usually have absorbance maxima at around 240 nm.
The extra double bond in betamethasone as compared with
hydrocortisone does not make a great difference to the
max
since
it does extend the original chromophore linearly.
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Ephedrine: The benzoid chromophore
Ephedrine has the simplest type of benzene ring chromophore.
Its spectrum is similar to benzene spectrum.
There are no polar groups involved in the chromophore so that its
fine structure is preserved.
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Ketoprofen: extended benzene chromophore
The benzoid chromophore has been extended by four double bonds
and thus the symmetry of the benzene ring has been altered.
The strong absorbance band present in benzene at 204 nm has
undergo bathochromic shift and giving
max
for ketoprofen at 262
nm.
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Procaine: amino group auxochrome
The benzene chromophore has been extended by addition of a C=O
group.
The molecule also contains an auxochrome in the form of amino
group.
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Phenylephrine: hydroxyl group auxochrome
The chromophore of phenylephrine is not extended but its structure
includes a phenolic hydroxyl group.
The phenolic group function as an auxochrome under acidic and
alkaline condition.
UV Spectrum of phenylephrine under acidic and basic condition
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Application of UV / visible spectroscopy in
pharmaceutical quantitative analysis
Pharmacopoeial methods rely heavily on simple analysis by
UV/visible spectrophotometry to determine active
ingredients in formulations. These methods are usually
based on the use of a standard A (1%, 1 cm) value for the
active ingredient being assayed. Such method also
presume that there is no interference from excipients
(preservatives, colorants, etc.) present in formulation and
that the sample is free of suspended matter, which will
cause light scattering.
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The ultraviolet spectrum of benzonitrile shows a primary absorption
band at 224 nm and a secondary band at 271 nm.
a. If a solution of benzonitrile in water, with a concentration of 1 x 10
-4
M,
is examined at a wavelength of 224 nm, the absorbance is determined
to be 1.30. The cell length is 1 cm. What is the molar absorptivity of
this absorption band?
b. If the same solution is examined at 271 nm, what will be the
absorbance reading (c = 1000)? What will be the intensity ratio, I
0
/I ?
Ans. c = 13000
I
0
/I = 1.26
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Recommended Book:
Introduction to Spectroscopy, 4
th
Edition 2001, Pavia, D. L.,
Lampman, G. M., Kriz, G. S. & Vyvyan, J. R., Brooks/Cole
Cengage Learning.