Ultraviolet and Visible (UV-Vis)

Spectroscopy
Belay Haile, MSc (Analytical Chemistry), PhD Scholar (Postharvest Technology), Arba Minch
University, CNS, Chemistry Department, Arba Minch Ethiopia
February 2021

SPECTROSCOPY
“SEEING THE UNSEEABLE”

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 Introduction
• Ultraviolet and visible (UV-Vis) The human eyes sees the complementary to
Spectrophotometry is based on which that is absorbed
the ability of atoms, molecules
and ions to absorb light at
wavelengths in the ultraviolet
(180-400 nm) and visible (400-
800 nm) range.

• The region of UV radiation is

classified into: far UV (10nm-
200nm) and near UV (200 nm-380
nm).

• Visible region (380-780nm) consist

of colored radiations.

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 Principle of UV-Vis Spectrophotometry
• Absorption of light in the UV-Visible
region of EMR result in transitions
between electronic energy levels, and this
technique is also known as electronic
spectroscopy.

– The absorption of UV-VIS radiations by an

organic molecule is associated with the
excitation of valence electrons from
ground state to excited state

• The absorption of radiation in UV-VIS

region depends on the electronic
configuration of the absorbing species like
atoms, molecules, ions, or complexes.
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 Cont. ….
The analyte absorbs the light of specific wavelength (UV and VIS only) and the
amount of radiation absorbed by the analyte is measured.

UV–vis spectroscopy is used to determine elemental concentrations quantitatively

in a solution according to Beer–Lambert law:

A=Log10(I0/I)=εcL,

where A is the measured absorbance, I0 is the intensity of the incident light at a

given wavelength, I is the transmitted intensity, L is the path length through the
sample, c is the concentration of the absorbing species, and ε is a constant known as
the molar absorptivity or extinction coefficient for each species and wavelength.

The molar absorptivity is a measure of the ability of an analyte to absorb light at a

specified wavelength.

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 Electronic Transitions
When UV-Vis radiation excites the molecule,
the electrons are temporarily moved from
one orbital (bonding orbital) to another
orbital (Anti-bonding orbital) according to
MOT.

These electrons may be σ, π or n (non-

bonding) electrons.

Thus possible transitions are: σ→σ*, π→π*,

n→σ*, n→π*
Schematic representation of various
levels of electronic transitions
 There is no n* orbital as n electrons don’t
form bonds

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 Cont. …
σ→σ* transition
o Large amount of energy is required for this transition.
o It is corresponding to the far UV region with the range of 120 –
200 nm.
o This type of transition is present in saturated hydrocarbons such
as CH3–CH3.
π→π* transition
 Present in the compounds that contain double and/or triple
bonds or aromatic rings and the excitation of electrons requires
less energy as compared to the σ →σ*.
 transition occurs at larger wavelength (160 – 190 nm).
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 Cont. …
n→σ* transition
 The excitation of electrons in an unshared pair form on an atom to an
antibonding sigma orbital.
 Present in saturated molecules such as halides, ethers, alcohols, etc
 results in the absorption of longer wavelength of the UV-VIS region
(between 150 and 250 nm)
n→π* transition
 the unshared pair electrons on heteroatoms are excited to antibonding
pi star (π*) orbital and in this orbital, the non-bonding electrons are
held loosely; therefore, the transition requires longer wavelengths.
 Occurs in compounds that contain double bonds involving heteroatoms
having unshared pair of electrons such as C=O, C=S, N=O, etc.

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Typical examples

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 Question
What types of electronic transitions are possible
for each of the following compounds?
A. Ethane 
B. Methyl vinyl ether
C. Triethylamine
D. Cyclopentene

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(a) Ethane (CH3-CH3)
Solution

(b) Methyl vinyl ether (CH₃OCH=CH₂)

(c) Triethylamine (N(CH₂CH₃)₃ )

(d) Cyclopentene (C5H8)

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 The Absorption Spectrum
• When sample molecules are exposed to light having an energy that matches
a possible electronic transition within the molecule, some of the light energy
will be absorbed as the electron is promoted to a higher energy orbital.

• An optical spectrometer records the wavelengths at which absorption

occurs, together with the degree of absorption at each wavelength.

• The resulting spectrum is presented as a graph of absorbance (A) versus

wavelength (λ) is known as a spectrum. The significant features:

λmax (wavelength at which there is a maximum absorption)

єmax (The intensity of maximum absorption)

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 Cont. …

UV-visible spectrum of isoprene showing maximum absorption at 222 nm

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 Absorption and Intensity Shifts in UV-VIS Spectroscopy
a)Bathochromic shift: It is also known
as Red shift, in this case absorption
shift towards longer wavelength(𝜆max).

b)Hypsochromic shift: It is also known

as Blue shift, in this case absorption
shift towards shorter wavelength
(𝜆max).

c)Hyperchromic shift: Intensity of

absorption maximum (𝜀max) increases.
Schematic representation of absorption
d)Hypochromic shift: Intensity of and intensity shifts
absorption maximum (𝜀max) decrease. 13
 Chromophores and Auxochromes
a)Chromophores: Chromophore is a
covalently unsaturated group absorbed
electromagnetic radiation in UV-visible
region and impart color to the
compound. For example, C=C, C≡C,
benzene, NO2etc.

b)Auxochromes: Auxochrome is a
saturated group (containing lone pair
of electron).

When auxochrome attach to the

Chromophore absorption shift towards
longer wavelength. For example, -OH,
-SH, -NH etc.

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 Factor affecting absorbance & intensity
a) Conjugation
• Absorption shift towards longer
wavelength, if double bonds (chromophore)
present in the molecule are in conjugation.

• In compound ‘A’, double bonds are in

conjugation therefore ‘A’ possessing higher
wavelength (𝜆max) as compare to compound
‘B’ (non-conjugated derivative).

• As the conjugation increase transition

energy (E) between the orbitals is decrease
and therefore wavelength (𝜆max) increase
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 Cont. …
b) Effect of solvent

• In polar solvent, 𝜋−𝜋* transitions shift

towards longer wavelength (Red
shift),because dipole interactions with polar
solvent molecules lower the energy of excited
state (𝜋*) than that of the ground state.

• Whereas in polar solvent, 𝑛−𝜋* transitions

shift towards lower wavelength (Blue shift),
because dipole interactions with polar
solvent molecules lower the energy of ground
state (n) than that of the excited state.

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Woodward's rules for prediction of λmax of
organic molecules
• In the middle of the last century, R. B.
Woodward studied the UV spectra of
conjugated dienes and developed a
set of rules for predicting the
wavelength of maximum UV
absorption based on the structure of
the diene.
• Later L. M. Fieser extended the
rules to conjugated aldehydes and
ketones.
Conjugated Dienes:-organic
compound containing two or
more double bonds each
separated from other by a
single bond
 The fundamental of
conjugated dienes are
the s-cis and the s-trans
conformation.
Conformations of 1,3-butadiene;
s =single bond that connects
the two double bonds
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 Conjugated diene correlations
a) Homoannular Diene:- Cyclic diene having
conjugated double bonds in same ring. 

b) Heteroannular Diene:- Cyclic diene having

conjugated double bonds in different rings.

c) Endocyclic double bond:- Double bond present in a

ring.

d) Exocyclic double bond: - Double bond in which one

of the doubly bonded atoms is a part of a ring system. 
Here Ring A has one exocyclic and endocyclic double bond.
Ring B has only one endocyclic double bond. 18
 Woodward's rules
implementation for
conjugated dienes
Important Note:
1. While calculating λmax only those alkyl
substituents or ring residues are
counted which are directly attached to
the carbon atoms making conjugated
system within the compound.
2. If a molecule has both homoannular
diene and heteroannular diene then
always use the homoannular diene as
the core chromophore.
CONJUGATED DIENE CORRELATIONS
a) Base value for homoannular
diene=253nm
b) Base value for heteroannular
diene=214nm
c) Alkylsubstituent to ring
residue attached to the parent
diene =5nm
d) Double bond extending
conjugation=30nm
e) Exocyclic double bonds=5nm
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 Application Examples for Woodward’s Rules

1)
Calc. λmax= 217 (acyclic base) + 5 (alkyl auxochrome
at C3 ) + 5 (alkyl auxochrome at C4 )
= 227 nm

2)
Calc. λ max = 214 (heteroannular since two pi
bonds are not in the same ring) + 20
(5 + 5 + 5 + 5 = 20, for each of the
alkyl or ring auxochromes attached
to C1 , C2 , C4 , and C4 ) + 5 (pi
bond of C1 -C2 is exocyclic to ring B)
= 239 nm
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 Cont. …
Calc. λmax= 253 (choose diene with highest base
value, pi bonds C1-2 and C3-4 are
within same ring, so homoannular base
3)
should be selected) + 30 (C5-6 pi bond
is conjugated to diene and is therefore an
extending diene) + 5 (C5-6 is exocyclic
to ring B) + 30 (5 + 5 + 5 + 5 + 5 + 5 = 30,
for the alkyl or ring auxochromes at C1 , C1
, C2 , C3 , C5 , and C6 ) = 318 nm

4)

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Components of UV-Visible Spectrophotometer

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 Types of UV-VIS Spectrophotometer
Based on the number of cuvettes and beams used, UV-VIS
spectrophotometer is classified into Single- and Double-Beam

Instrumental designs for UV/visible photometers in a single-beam

 Radiation from the filter or monochromator passes through either the

reference cell or the sample cell before striking the photodetector.

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 Cont. …

Instrumental designs for UV/visible photometers in a double-beam

 In a double-beam instrument, radiation from the filter or monochromator is

split into two beams that simultaneously pass through the reference and
sample cells before striking two matched photodetectors.

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 Instrumentation
1. A Stable and cheap radiant energy source.

2. A monochromator, to break the polychromatic radiation into

component wavelength (or) bands of wavelengths.

3. Transport vessels (cuvettes), to hold the sample.

4. A Photosensitive detector and

5. Readout system
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Cont. …
1) Light Sources
It provides a sufficient light in the form
of polychromatic light that is appropriate
for marking the measurements.

In UV-VIS spectrophotometer, two types

of light sources are used.
Schematic representation of light source
1.For UV sources, deuterium, hydrogen,
tungsten, mercury, and xenon lamps
are used.

2.for VIS sources, tungsten, mercury

vapor, and carbon lamps are used.

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Cont. …

2) Monochromator
• It is a device that receives
the polychromatic light as
input from a lamp and
provides output in the form
of monochromatic light.

• This device is used to

disperse the radiations of
polychromatic light Schematic representation of components of
according to the wavelength. monochromator

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Cont. …
Entrance Slit
• defines the incoming polychromatic beam of light and imparts it on dispersing
element.
Dispersing Device
• It disperses the polychromatic light that is coming from the entrance slit into its
component wavelengths with the help of focusing lens.
• Dispersing element, such as prism or grating is usually made up of quartz, fused
silica, or glass.
Exit Slit
• allows the minimal wavelength together with the band of wavelengths on either
side.
• The position of exit slit is not fixed and it is mostly needed to adjust it by
rotation to vary the minimal wavelength passing through the exit slit.

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Cont. …

3) Sample Device/Cuvette
• specifically designed to hold the
sample for spectrophotometric
analysis.
• They vary with respect to shape, size,
path length, and transmission
characteristics of required wavelength.
• Cuvettes are made up of plastic, glass,
or optical grade quartz that does not
absorb the λ range of interest. Schematic representation of
sample device or cuvette

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Cont. …
4) Detector
• It is a device that can be used for the measurement of the amount of light passing through
the sample and convert the light signals (coming from the sample) into the electrical
signals.
• Three kinds of detectors are used in UV-VIS spectroscopy:
i. Photovoltaic Cells or Barrier Layer Cell
ii. Phototubes or Photoemissive Tubes
iii. Photomultiplier Tubes

5) Recorder
 Spectrometer provides the signals to the recorder and intensity of signals depends upon
light absorption by the analyte at a particular wavelength.
 These signals are amplified by amplifier and elaborated by the personal computer.
 Digital read-out devices like light emitting diode or liquid crystal display (LCD) are used for
clarity purpose that removes the ambiguity.

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 Applications of UV-VIS spectroscopy
UV-VIS spectroscopy is regularly used in different fields of science for the
qualitative and/or quantitative analysis of different types of analytes. Followings
are the important applications of UV-VIS spectroscopy:

 Determination of Molecular Weight

 Detection of Impurities
 Quantitative Analysis
 Qualitative Analysis of Pharmaceuticals
 Detection of Functional Group
 Chemical Kinetics
 Determination of Extent of Conjugation
 Structural Elucidation of Organic Compounds
 Determination of Unknown Concentration

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 Determination of Unknown Concentration

• Unknown concentration of any compound can also be measured

using UV-VIS spectroscopy.

• It involves the following steps:

1. First of all, prepare the samples.
2. Then prepare series of standard solutions having known concentrations.
3. Set spectrophotometer to the wavelength at which sample shows
maximum absorption of light.
4. Draw the standard plot.
5. Finally, measure the absorption of unknown analyte and determine its
concentration with the help of standard plot.

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EXAMPLE: The determination of Fe in an industrial waste
stream was carried out by using o-phenanthroline. Iron in the
+2 oxidation state reacts with o-phenanthroline to form the
orange-red Fe(o-phen)32+ complex. The absorbance of samples
and standards is measured at a wavelength of 510 nm using a
1-cm cell. If Beer’s law is obeyed for concentrations of iron
within the range of 0.2 - 4.0 ppm, what is the concentration of
Fe in the waste stream using the data shown in the table?

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 SOLUTION
• Linear regression of 0.8
absorbance versus the
f(x) = 0.18 x + 0
concentration of Fe in the 0.6 R² = 1

Absorbance
standards gives:
0.4

Y=0.1817x (ppm Fe) + 0.0006 0.2

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
• Substituting the unknown’s
absorbance into the Concentration
calibration expression gives
the concentration of Fe in the
waste stream as 1.48 ppm.
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 Beware of impurities

"one man's signal is another's impurity"

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Surprise test (5%)
 Surprise test (5%)
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× 10−4 molar, 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 (e = 1000)? What will be the intensity
ratio, I0 /I?
Given (a) •  
Solution for (a)

 C= 1× 10−4 mol/L A= εbc

 A= 1.30
ε=a/bc=
 b=1 cm
 ε=? =13,000 Lmol-1cm-1
Given (b) Solution for (b)
 ε=1000 A= Log10(I0/I)=εbc
 C= 1× 10−4 mol/L =1000 ×1 cm× 10−4 mol/L
 b=1 cm =0.1
 A= ? I0/I=antilog A
 I0/I =10(0.1)
=1.26
 Solution for (b)
•  
Solution for (a)
A= Log10(I0/I)=εbc
A= εbc =1000 ×1 cm× 10−4 mol/L
=0.1
ε=a/bc= I0/I=antilog A
=10(0.1)
=13,000 Lmol-1cm-1
=1.26

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