UV-Ultraviolet Visible Spectroscopy MANIK

PHARM 3235
Md. Imran Nur Manik
Lecturer
Department of Pharmacy
Northern University Bangladesh

Visible and Ultraviolet Spectroscopy
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 1
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Ultraviolet-Visible Spectroscopy
Introduction:
Spectroscopy:
Spectroscopy is the study of interaction between matter (mass) and radiated energy.
Such interaction includes –
1. Absorption of the incident radiant energy.
2. Emission of radiant energy.
3. Scattering or reflection of incident radiant energy.
4. Impedance of radiant energy transmission.
5. Changing the frequency/wavelength of the transmitted radiant energy.
6. Causing interaction between molecules in a non-stationary state and sustaining it.
Absorption spectroscopy/spectrometry:
Absorption spectroscopy may be defined as the analysis of chemical substances by
measurement of the amount of radiant energy absorbed by the substance.
UV-Visible spectroscopy:
UV-visible spectroscopy is a type of absorption spectroscopy which uses the UV and
visible part of the EM spectrum.
Electromagnetic (EM) spectrum:
Spectrum:
Spectrum is the condition where a characteristic is not limited to a fixed set of values rather it
varies infinitely within a continuum.
By that definition, radiation arranged by wavelength is a spectrum; drug activity in terms of
action against bacteria is a spectrum.
Electromagnetic spectrum:
It is the arrangement of all types of electromagnetic radiation in order of their increasing or
decreasing wavelength (or frequency).
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The above diagram shows the electromagnetic spectrum where radiations are arranged in the
decreasing order of wavelength from left to right.
Visible and UV spectrum:
Color Wavelength
Violet 400-420
Indigo 420-440
Blue 440-490
Green 490-570
Yellow 570-585
Orange 585-620
Red 620-780
The visible spectrum starts at 400 nm and ends at around 700 nm. The electromagnetic radiation
adjacent to the violet visible radiation is called ultraviolet radiation. The electromagnetic
radiation adjacent to the red visible radiation is called infrared radiation.
The UV spectrum ranges from 10 nm to 380 nm. The UV radiation has been classified as UV A
(400-315 nm), UV B (315-280 nm) and UV C (280-10 nm) based on their wavelength.
Relationship between wavelength & color
Wavelength (nm) Spectrum region Colour absorbed Colour transmitted
400-420 Visible Violet Green-yellow
420-500 Visible Blue yellow
500-570 Visible Green Red
570-600 Visible yellow Blue
600-630 Visible orange Green-blue
630-700 Visible Red Green
Electromagnetic radiation:
Electromagnetic radiation is a form of energy that shows wave-like characteristics and –
1. It can travel without any medium
2. At vacuum it moves at speed of light
3. Contains both electric and magnetic field components. These two components oscillate
(moving back and forth) in a phase perpendicular to each other. When an electric field
oscillates it causes the oscillation of the corresponding magnetic field which in turn
oscillates the next electric field. Thus the EM radiation moves forward without any
medium.
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EM radiation is described by wavelength () or frequency (n). The relationship between
wavelength and frequency is given in following equation.
nλc 
Where c is the speed/velocity of the wave. At vacuum c is equal to the speed of light i.e.
11018
103103 
 cmsms . Pharmacists rarely use frequency value for analytical purposes and
wavelength value is of more importance.
Wavelength:
The wavelength of an wave is the linear distance between two consecutive corresponding
points of the same phase.
Since wave repeats a cycle over and over again, the lineardistance between two identical
points of two adjacent cycle is the wavelength. Thus wavelength is the linear distance
between the two corresponding peaks of a wave.
Frequency:
It is the number of cycles occuring per second

 
One cycle completed
Effect of electromagnetic radiation on a substance:
When a beam of electromagnetic radiation passes through a transparent medium of the
substance (the substance under analysis must be in solution for proper analysis), the radiation is
absorbed and/or transmitted by the substance. The absorption of the radiation depends on the –
- Chemical nature of the substance
- Wavelength of the radiation
The radiation not absorbed is transmitted by the substance and it is called transmitted light
which can be measured by an instrument called the spectrophotometer. The record is called the
spectrum of that substance.
Transmittance “T”
The amount of light that passes through a particular thickness of solution is known as
transmittance T.
Transmittance can be expressed as the ratio of the intensity of the transmitted light I to the
intensity of the incident light beam Io
I
I
T
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Absorbance “A”
The negative logarithm of the base ten of transmittance is called absorbance.
logTA 
I
I
logA 
I
I
logA


It is also called optical density.
Absorbance is directly proportional to the concentration of the solution when the thickness of
the medium is constant.
Laws governing spectrophotometry
As described before, when a substance is placed in the path of light (EM radiation), a number of
events e.g. absorption, transmission, elastic and nonelastic scattering of light may take place.
The absorption of EM radiation/light by molecules is governed by two laws – Lambert’s law
and Beer’s law.
LAMBERT'S LAW
Lambert described how intensity changes with distance in an absorbing medium.
Lambert’s Law: When a beam of monochromatic radiation is passed through a solution of an
absorbing medium, the decrease in the intensity of radiation with thickness of the solution is
directly proportional to the intensity of the incident light.
Incident
light
Transmitted
light
1/6 1/6 1/61/6 1/6 1/6
Intensity decreases
as light pass through
each layer
The solution has been hypothetically divided
into 6 layers of equal thicknes. According to
the Lambert's law, each layer absorbs same
fraction of the passing light even though the
intensity decreases.
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Let,
I be the intensity of incident radiation.
b be the thickness of the solution.
Then
I
db
dI

KI
db
dI

K'=Absorbing co efficient or Proportionality constant
I
db
dI
K
Now integrating the equation between the limit I=Iₒ at b=0
And I=I at b=b
We get,
 
I
I
b

dbK
I
dI
0
   b
0
I
I bKlnI 
b.K
I
I
ln 

b.K
I
I
log303.2 

b.
303.2
K
I
I
log 

Eb
I
I
log 

Where 2.303
K
=E= Extinction co efficient
We know that absorbance I
I
log

A
Thus A= Eb
A∞b
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The amount of light absorbed by the medium (solution/ sample) at a given wavelength is
proportional to thickness of the absorbing layer i.e. path length of the light
Basically it states that the absorbance is proportional to the path length i.e.
A∞b
Beer’s law
Relates the absorption of light to the properties of the material through which the light is
travelling.
Beer’s Law: When a beam of monochromatic radiation is passed through a solution of an
absorbing medium, the decrease in the intensity of radiation with thickness of the solution is
directly proportional to the intensity of the incident light as well as concentration of the solution.
Let I be the intensity of incident radiation.
x be the thickness of the solution.
C be the concentration of the solution.
C.I
dx
dI

C.I
dx
dI
K
K'=Absorbing co efficient or Proportionality constant
C.dx
I
dI
K
Now integrating the equation between the limit I=Iₒ at x=0
And I=I at x=l (l=Path length of the sample which is usually 1 cm)
we get
 
I
I
l
K

.dxC
I
dI
0
   l
0
I
I xCKlnI 
.C.lK
I
I
ln 

.C.lK
I
I
2.303log 

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C.l
2.303
K
I
I
log



lCE ..
I
I
log 

Where 303.2
K
=Extinction co efficient
We know that absorbance I
I
log

A
Thus A=E.C.l
From the equation it is seen that the absorbance which is also called as optical density (OD) of a
solution in a container of fixed path length is directly proportional to the concentration of a
solution.
Accordingly The amount of light absorbed by the a medium (solution/ sample) is proportional to
the concentration of the absorbing material or solute present.
This law states that absorption of the incident light is proportional to the number of absorbing
molecules. So according to this law –
A∞c
Where, c is concentration of the substance in solution and A is absorbance.
Thus the concentration of a coloured solute in a solution may be determined in the lab by
measuring the ABSORBANCY OF LIGHT AT A GIVEN WAVELENGTH
Combining Beer’s law and Lambert’s law – Beer-Lambert law/Equation of absorbance:
According to the Lambert’s law –
bA 
According to the Beer’s Law –
cA 
Combining the two laws we have –
A∞bc
abc
I
I
abcA
KbcA



0
log
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Where,
ty"absorptivi"astermedandconstantalityproportionaisIta
moleculesabsorbingtheofionConcentratc
solutionabsorbingorsampleoflengthPathb
lightdtransmittetheofIntensityI
lightincidentofIntensityI
AbsorbanceA
0






Absorptivity:
Absorptivity is a measure of how easily the absorbing molecules can absorb the
electromagnetic radiation. It is specific for a given substance. It is defined as the absorbance by
a sample of unit concentration and where the path is of unit length.
It is mathematically quotient of the absorbance (A) divided by the concentration of the solution
(gm/L) and the path length (cm).
bc
A
a 
When c is expressed in moles/litre in the Beer-Lambert law, the absorptivity is called molar
absorptivity or molar extinction coefficient and it is expressed by 
bc
A

When, c is expressed as %w/v, the absorptivity is expressed as %1
1cmA .
Deviations from Beer-Lambert law
The accuracy of the Beer Lambert law is dependant upon some chemical and instrumental
factors –
1. This law specifically the part concerning concentration is not maintained in high
concentrated solutions. This is because –
a. In highly concentrated solutions, degree of interaction between molecules is higher
at higher concentration. Thus the molecules will not absorb radiation in same
manner as when they are in dilute solution.
b. The absorptivity of solution doesn’t remain constant as the concentration changes.
c. The refractive index of the solution may change in high concentration.
2. If the solution contains particulate matter then scattering of light occurs which interfere
with the absorption process.
3. If the sample is fluorescent or phosphorescent then this law is not obeyed.
4. If the light is non-monochromatic (in practice, monochromatic light is difficult to produce)
i.e. not completely monochromatic.
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Absorption of radiant energy
Effect on molecules upon absorption of radiation:
Electromagnetic radiation is energy. When a molecule absorbs radiation it gains energy.
The higher the frequency the greater will be the gain in energy. This energy can bring about one
or more of the following changes –
1. The absorbed energy may break bonds within molecule. e.g. conversion of ergosterol to
calciferol.
2. The absorbed energy may increase vibration or rotation of atoms within the molecule.
This principle is used in IR spectroscopy.
3. It may change nuclear or electronic spin. This property is used in NMR spectroscopy.
4. It may cause electrons to rise to higher energy level.
The UV and visible radiation absorption will cause the last effect i.e. transition of electrons
within the molecule.
Energy of a molecule
Every molecule has a definite energy state. Energy possessed by a molecule can be classified in
several categories. These are
1. Transitional energy (Etrans)
2. Vibrational Energy (Evib)
3. Rotational Energy (Erota)
4. Electronic energy (Eelec)
Transitional energy
Molecule as a whole can move from one place to another and the energy associated with this
motion (Velocity) is called transitional energy as well as the movement is called transitional
movement.
Vibrational Energy (Evib)
The movement of a part of a molecule or a group of molecules that move within themself is
called vibrational movement and the energy associated with this vibration is called vibrational
energy.
Rotational Energy (Erota)
Molecule can rotate along its axis and the energy associated with this rotation is called
rotational energy.
Electronic Energy (Eelec)
Electrons of each atom moves around the central nuclei and the energy associated with this
movement is called electronic energy
Translational < Rotational < Vibrational < Electronic
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For example, a simple molecule, such as H2, may have the following energy levels:
Theory or principle of Electronic spectroscopy or molecular Spectroscopy
When a molecule absorbs electro magnetic radiation like UV or Visible light its electron gets
promoted from the ground state to the higher energy state or excited state.
(Ground State: The molecules when attained in a state, having lower energy level i.e. normal
unexcited condition or state is called ground state. Excited state: Absorbsion of EMR by the
molecule results in the shipment of the molecule in the higher energy state called excited state)
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In the ground state the spin of electrons in each molecular orbital is essentially paired. In the
higher energy state, the spins of electrons are either paired i.e. excited singlet state or parallel
excited triplet state. Normally the absorption of the UV or visible light results in the transition of
the singlet ground state to become singlet excited state.
To get the molecule in the higher energy level the energy difference between the two energy
level must be equal to the energy of photon absorbed, which can be expressed as
Energy required to get in the excited state, ΔE=E2-E1=hν
Here, E1= Lower energy level
E2=Higher Energy Level
h= Planks Constant
ν=Frequency
Generally the energy of the triplet excited state is lower than the singlet excited state. Thus to
be stable the molecule quickly returns from one of its higher energy level to lower one by the
emission of radiation.
Therefore an electron that has been raised to the higher energy level by the absorption of
radiation, quickly returns to the ground energy level within 10-8 seconds either directly or by the
way of second excited state and will give molecular absorption spectrum.
(Molecular absorption spectrum: A set of bands or lines generated, due to the absorption of EMR by the
molecules is called MAS. It is actually a graph obtained by plotting the absorption against wavelength)
As the transmission rates are different for different molecule or structure, so the absorption
spectra will be different also. Greater the number of molecules capable of absorbing the light of
different given wavelength the greater will be the extent of light absorption. MAS is
characteristics for a molecule and there is no similarity in MAS of one molecule with other, Thus
it is used to in the quantitative analysis of the molecules.
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Generally the absorption spectra is graphically represented by plotting the absorbance (A) on
the Y axis and the wavelength on the x axis.; This is the principle or theory of UV or visible
molecular absorption spectroscopy.
Electronic transition
When UV and visible radiation excites the molecule the electrons are temporally moved from one
orbital (bonding orbital according to MOT) to another orbital (Anti-bonding orbital according to
MOT). These electrons may be σ, π or n (non-bonding) electrons. (Time requires 10-8
sec)
Thus possible transitions are –
1. σ → σ*
2. n → σ*
3. π → π*
4. n → π*
(There is no n*
orbital as n electrons don’t form bonds).
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σ → σ*
transition:
C C C C

1. This type of transition takes place in the saturated centre of the molecules.
2. Here, an electron from a stable σ orbital goes to an unstable or anti-bonding σ orbital
(represented by σ*
).
3. High energy is required to excite electrons present in the σ orbital as they are held tightly.
Therefore saturated compounds do not absorb radiation from normal UV-Vis
spectrophotometer which generates radiation of 190-780 nm region.
n → σ*
transition:
C X C
n
O
N
X
O
n
n
n
N
SS
1. This type of transition occurs in saturated compounds containing heteroatom which has
an unshared electron pair.
2. Electron transits from unshared pair to anti-bonding σ orbital.
3. This type of transition may take place in halogen, sulfur, nitrogen and oxygen containing
compounds.
4. This transition requires high energy and therefore doesn’t occur in presence of the EM
radiation produced by normal UV-Vis spectrophotometer.
π → π*
transition (K-band):
C O C

O
1. Takes place in unsaturated compounds containing double or triple bonds.
2. Electron transits from stable π orbital to an unstable/anti-bonding π orbital (represented
by π*
).
3. It requires less energy as π bonds are sufficiently weaker than σ bonds. Conjugated
double bonds further lowers required energy. For example β-carotene containing 11
conjugated double bonds absorbs energy at 451 nm (visible region).
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n → π*
transition (R-band):
C OC O
n *
1. This type of transition takes place in compounds having heteroatom which contains
unshared electron pair.
2. Electron transits from unshared pair to anti-bonding π orbital.
3. Requires lower energy and therefore respond to the radiation produced by normal UV-Vis
spectrophotometer.
4. Carbonyl compounds, cyanides etc. show this type of transition.
Effect of solvents on electronic transition
The polarity of the solvent affect the energy required for electronic transition. This is described
below. We need to remember that polarity results from unequal distribution of electrons – the
greater is the difference the greater is the polarity.
n → π*
transition:
More energy required
Shift to Shorter wavelength
Ground state
Excitated state
C OC O
More polar Less polar
n *
x
y
n

The diagram above illustrates the polarity of the molecule at excited state and ground state. If
the solvent is polar, then dipole-dipole interaction between the solvent molecule and sample
molecule will be greatest at ground state. Hence, energy of the grounds state is lowered
significantly but that of excited state is raised (or not lowered significantly). The result is that
more energy i.e. radiation of shorter wavelength is required to excite the electrons.
π → π*
transition:
Ground state
Excitated state
C OC O
Less polar More polar
 *
x
y


Less energy required
Shift to longer wavelength
Here polarity increases after transition.
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Thus the polar solvent molecule will show greater interaction with the excited state than with
ground state. Hence, energy required for excitation is decreased.
n → σ*
transition:
Ground state
Excitated state
C OC O
More polar Less polar
n *
x
y
n

More energy required
Shift to shorter wavelength
The polarity decreases after transition to excited state. So the polar solvent prefers the ground
state. Hence more energy is required for excitation.
σ → σ*
transition:
Polarity unchanged
C C C C

In saturated compounds (no atom with lone pair of electrons though) transition doesn’t change
polarity much. Thus solvent effect is not seen.
Impurities
Impurities in the solvent affect the result in UV-Vis spectroscopy. The impurity will absorb light
hence the absorbance is exaggerated. This is why commercial absolute ethanol should not be
used to prepare sample solution as it contains benzene as impurity which strongly absorbs light
in the UV region.
Nature of the absorption band
The solvents used in sample preparation will absorb radiation at some specific
wavelength. So, radiation of that wavelength becomes unavailable for spectral studies. For
example, chloroform will strongly absorb radiation of below 245 nm. Thus this solvent can’t be
used in a sample that will be run at a wavelength lower than 245 nm. The table below shows
the minimum wavelength at which the solvent can be used to make sample solution. For
example, methanol should not be used if the sample is to be run at wavelength lower than 203
nm.
Solvents Minimum wavelength for 1 cm cell,
nm
Solvents Minimum wavelength for 1 cm cell,
nm
Acetonitrile 190 Ethanol 204
Water 191 Ether 215
Hexane 201 Chloroform 237
Methanol 203
Carbon
tetrachloride
257
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Absorbing groups
There are two types of groups present in a molecule which is responsible for characteristic
electromagnetic radiation absorption by the molecule. These are described below –
1. Chromophore
A chromophore is a chemical group that absorbs light at a specific frequency and so imparts
color to a molecule.
It is defined as any isolated covalently bonded group that shows a characteristic absorption in
the ultraviolet or visible region.
When a chromophore is attached to a saturated hydrocarbon, produces a molecule that absorbs
a maximum of UV or visible energy at some specific wavelength. Compounds containing a
chropmophoric group called a chromogen.
Some chromophores are listed below –
Chromophore Structure Example of molecule max
Acetylene (carbon-carbon triple bond) C C Acetylene 173
Amide CONH2 Acetamide <208
Azo N N Azomethane 347
Carbonyl (ketone)
C
O Acetone 271
Carbonyl (Aldehyde)
C
O
H
Acetaldehyde 293
Ethylene (carbon-carbon double bond)
C C
Ethylene 234
Nitrile C N Acetonitrile <160
A compound may contain two or more chromophores and then the relative position of the
chromophores will determine the absorption of radiation.
Chemical structure of beta-carotene. The eleven conjugated
double bonds that form the chromophore of the molecule
are highlighted in red.
2. Auxochrome:
An auxochrome is a group of atoms attached to a chromophore which modifies the ability of
that chromophore to absorb light. They themselves fail to produce the colour; but when present
along with the chromophores in an organic compound intensifies the colour of the chromogen.
Auxochrome group contains at least one atom with lone pair of electrons. Examples include
the hydroxyl group (-OH), the amino group (-NH2), the aldehyde group (-CHO), ─NHR, ─NR2.
Benzene shows absorption maximum at 255 nm whereas aniline at 280 nm.
=255 nm = 280 nm
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M/A: They have lone pair electrons that interferes with the chromophores electron transition system i.e.
with the electronic localization. This affects the conjugation system of the compound and causes the
shipment of the max as well as ϵmax to the higher value. Higher the conjugation higher the shipment.
Terminologies describing effects of solvent, chromophores and auxochromes
Absorption and intensity shifts
1. Bathochromic shift or Red shift: It is the displacement/shift of max (absorption maximum)
towards longer wavelength. This may occur due to solvent effect, conjugation or
auxochrome addition.
Absorbance

0 X
Y
max
max
Shift


Hexane
Aqueous acid
max
max
= 230
=203
It is known as red shift. Following is an example of this effect.
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2. Hypsochromic shift or Blue shift: it is the displacement of max towards the shorter
wavelength. This is usually due to solvent effect.
Absorbance

0 X
Y
max
max
Shift


Hexane
Aqueous acid
max
max
= 230
=203
3. Hyperchromic effect: It is the effect of increased intensity of radiation absorption by the
molecule.
Absorbance

Pyridine 2-methyl pyridine
λmax = 257 nm λmax = 260 nm
ε = 2750 ε = 3560
When absorption intensity (ε) of a compound is increased, it is known as hyperchromic shift.
If auxochrome introduces to the compound, the intensity of absorption increases.
4. Hypochromic effect: It is the effect of decreased intensity of radiation absorption by the
molecule.
When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift.
Absorbance

Naphthalene 2-methyl naphthalene
ε = 19000 ε = 10250
CH3
N N CH3
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Vacuum ultraviolet region
Oxygen of atmosphere absorbs UV light of below 200 nm. So it is difficult to analyze a
sample by running it at wavelength below 200 nm. If we want to measure absorption of UV
radiation below 200 nm we must remove all the air from the instrument.
Thus vacuum is used. So the UV radiation below 200 nm is known as vacuum ultraviolet region.
This region is relatively uninformative as it excites the σ electrons. Above this region, π electrons
are excited. The double and triple bonds are comparatively more informative in the analysis of a
compound.
Explanation of electronic transitions in polyenes
Compound max (nm)
H2C CH2 175
217
256
290
334
364
As we can see that, ethylene with only one double bond show most absorption at 175nm,
whereas 1,3-butadiene show most absorption at 217 nm. Generally the longer the conjugated
system the longer is the wavelength of maximum absorption. For example, β-carotene contains
11 double bonds and show maximum absorption at 451 nm (visible range).
When two atomic orbitals are combined, two molecular orbitals are produced. One is of high
energy. This is the unstable molecular orbital called the anti-bonding orbital. Another is of lower
energy and this is the stable orbital. Electrons are present in the stable orbital.
Two atomic p orbitals
High energy: Antibonding orbital. Electron not present in this
molecular orbital i.e it is the Lowest unoccupied molecular
orbital (LUMO).
Low energy: Bonding orbital. Electrons are present in this
molecular orbital. It is the Highest occupied molecular
orbital (HOMO)
Substraction of the wave functions of the atomic orbitals
Addition of the wave functions of the atomic orbitals
Figure: Molecular orbitals in ethylene
Energy
Eenrgy required for
excitation in ethylene.
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Below is the molecular orbitals of 1,3-butadiene. There are two double bonds in this
molecule. Each double bond forms two molecular orbital – one bonding orbital and one anti-
bonding orbital.
The two bonding orbitals of the two double bonds combine to form two molecular orbitals.
Among them one is higher in energy than ethylene molecule and another is lower. The higher
energy molecular orbital is the HOMO.
Similarly, the two anti-bonding orbitals of two double bonds will produce two new
molecular orbitals. One of them will be lower in energy than the excited ethylene molecule and
another will be higher in energy than excited ethylene molecule (excited is the keyword – in
HOMO determination we took the molecular orbital that was higher in energy than the ground
state ethylene molecule, not excited ethylene molecule). Among the two the molecular orbital
with lower energy than the excited ethylene molecule is the LUMO. One should remember that
anti-bonding molecular orbitals are normally unoccupied and becomes occupied when electrons
are excited.
As we can see that the energy difference between LUMO and HOMO of 1,3-butadiene
will lower than that of ethylene. (Ethylene has only one anti-bonding orbital which is the LUMO
for ethylene, 1,3-butadiene has two anti-bonding orbitals where the one which has lower energy
than LUMO of ethylene is the LUMO of 1,3-butadiene).
This is the HOMO
This is the LUMO
Anti-bonding
molecular orbitals
Bonding
molecular orbitals
Eenrgy for
excitation
Energy
Figure: Molecular orbitals of 1,3-butadiene
Each of these two MO is equivalent
to the LUMO of
ethylene
Each of these two MO is equivalent
to the HOMO of
ethylene
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As the double bonds/conjugation increases, so the energy difference between LUMO and HOMO
decreases (e.g. in ethylene the difference is 176 kcal/mol and in 1,3-butadiene the difference is
131 kcal/mol). Thus increase of double bonds decrease energy required for absorption.
Antibonding
orbitals
Bonding
orbitals
H2C CH2
Ethylene Butadiene Hexatriene Octetraene
Energy
Figure: Energy gap for excitation in polyenes
Instrumentation
Spectrometer
Spectrometer is an instrument that is used to measure a physical property over a specific
region of the spectrum of that property.
The physical property is usually the intensity of light but other properties can also be
used. For example in mass spectrometer, relative abundance of particles is measured over mass-
to-charge ratio.
Simply spectrometer is any instrument
used to measure spectrum.
Spectrophotometer
A spectrophotometer is an instrument
used to measure the intensity of light over a
specific region of the electromagnetic spectrum.
In another words, a spectrophotometer
is a spectrometer with a photomultiplier that is
used to detect light and determine its intensity
as a function of wavelength.
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Colorimeter
A colorimeter is a spectrophotometer which is designed to detect only the absorption at
the visible region i.e. it will measure the intensity of light at the visible region of the
electromagnetic spectrum.
Fig: Colorimeter
Parts of the UV-Visible spectrophotometer:
Basic Parts
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 an associated readout system.
Fig: Basic Parts of spectrophotometer
The UV-visible spectrometer is a spectrophotometer designed to work in the UV and visible
range of the electromagnetic spectrum.
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It consists of following parts –
1. Radiation source: A source for UV and visible light is perquisite. A UV-visible
spectrophotometer works at around 190-800 nm range. Usually two sources are used –
a. Hydrogen or deuterium discharge lamp: It emits electromagnetic radiation of
185-365 nm wavelengths.
b. Tungsten lamp (6V or 12V): It emits electromagnetic radiation of 350-800 nm.
Since, none of them produces the complete range of the target spectrum. They are used
simultaneously.
Requirements for radiation source
1. The radiation should be continuous
2. Its spectrum should include all the wavelengths required for the analysis.
3. The power of the beam should be such that, the solution can transmit some or all of the
radiation energy at all wavelengths.
4. The power of the beam must remain constant throughout the measurement.
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
RADIANT ENERGY SOURCES
Materials which can be excited to high energy states by a high voltage electric discharge (or) by electrical
heating serve as excellent radiant energy sources.
Sources of Ultraviolet radiation: Most commonly used sources of UV radiation are the hydrogen lamp and the
deuterium lamp. Xenon lamp may also be used for UV radiation, but the radiation produced is not as stable
as the hydrogen lamp.
Sources of Visible radiation: “Tungsten filament” lamp is the most commonly used source for visible
radiation. It is inexpensive and emails continuous radiation in the range between 350 and 2500nm. “Carbon
arc” which provides more intense visible radiation is used in a small number of commercially available
instruments.
Sources of IR radiation: “Nernst Glower” and “Global” are the most satisfactory sources of IR radiation.
Global is more stable than the nearest flower.
For Visible region
Tungsten filament lamp
• Use for region 350nm to 2000nm.
• These measure most effectively in the visible
region from 320 - 1100 nm
• Instruments that only use Tungsten halogen
lamps as the light source will only measure in the
visible region.
For ultra violet region
Hydrogen discharge lamp Fig. Tungsten filament lamp
• Consist of two electrode contain in Hydrogen filled silica envelop.
• Gives continuous spectrum in region 185-380nm. above 380nm emission is not continuous
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Fig. Hydrogen discharge lamp
Deuterium lamps:
• Deuterium arc lamps measure in the UV region 190 -
370 nm
• As Deuterium lamps operate at high temperatures,
normal glass housings cannot be used for the casing.
Instead, a fused quartz, UV glass, or magnesium fluoride
envelope is used.
• When run continuously typical lamp life for a Deuterium
lamp is approximately 1000 hours, however this can be
extended by up to a factor of three using PTR technology.
• Deuterium lamps are always used with a Tungsten halogen lamp to allow measurements to be performed
in both the UV and visible regions.
2. Monochromator: The monochromator disperses the polychromatic light by means of a
prism or grating (in grating there is a flat surface having alternating reflective and
non-reflective portions). The desired monochromatic light is obtained by changing the
angular position of prism or grating and this monochromatic light is allowed to pass to
sample.
Monochromatic Light: The light having a single wavelength and frequency i.e. the light that vibrates at
a single wavelength.
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Polychromatic Light: The light having several colours i.e. light having electromagnetic radiation of
several wavelengths.
The light must be monochromatic as extinction coefficient is variable with wavelength.
2. WAVELENGTH SELECTORS
Wavelength selectors are of two types.
1. Filters: “Gelatin” filters are made of a layer of gelatin, colored with organic dyes and sealed between glass
plates.
2. Monochromators: A monochromator resolves polychromatic radiation into its individual wavelengths and
isolates these wavelengths into very narrow bands. The essential components of a monochromator are.
a. Entrance slip-admits polychromatic light from the source
b. Collimating device – Collimates the polychromatic light onto the dispersion device.
c. Wavelength resolving device like a PRISM (or) a GRATING
d. A focusing lens (or) a mirror
e. An exit slip – allows the monochromatic beam to escape.
The kinds of the resolving element are of primary importance
PRISMS: A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its
ability to reflect different wavelengths to a different extent;
The degree of dispersion by the prism depends on upon
a. The optical angle of the Prism (usually 600)
b. The material of which it is made
Two types of Prisms are usually employed in commercial instruments. Namely, 600cornu quartz prism and
300 Littrow Prism.
GRATINGS: Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible
and infrared regions.
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3. Sample compartment: The sample is kept in a small tube of square cross section called
“cuvette” i.e. the tube has four sides.
There are two opposite sides through which the light is supposed to pass (i.e. these two sides
form the optical window). The other two sides are usually unclear (in some cases, all four sides
may be clear) and used for handling. The cuvette (at least the optical window of the cuvette) is
made of glass, plastic or fused quartz. The glass and plastic cuvettes are used to measure at
visible spectrum while quartz cuvettes are used to measure at ultraviolet spectrum. The length
of the optical window is usually 1cm.
Fig. cuvette
4. Detector: The detector converts the radiant energy into electrical energy.
Most detectors depend on the photoelectric effect. The current is then proportional to the light
intensity and therefore a measure of it.
Important requirements for a detector include
i. High sensitivity to allow the detection of low levels of radiant energy
ii. Short response time
iii. Long term stability
iv. An electric signal which easily amplified for typical readout apparatus.
In spectrophotometer the detector is a photomultiplier tube. It consists of following parts –
a. One cathode (electron emitter): It emits electrons when the photon of radiation strikes it.
b. Several electrodes called dynodes: After the cathode there are a number of electrodes called
the dynodes. The dynode next to cathode is more positive than cathode and produces several
electrons for each electron striking it. The dynode next to the 1st
dynode is even more positive
and it also produces more electrons. By the time electrons are collected at anode, for each
photon about 106
-107
electrons are generated.
c. One anode: It receives the electrons generated on the way. The resulting current is then
measured.
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Anode
Dynode I
Dynode II
Dynode III
Dynode IV
Cathode
Light
Fig. Photomultiplier tube
5. Recorder: From the measurement of current obtained from transmitted light, it plots a
graph of absorbance versus wavelength.
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The chart recorder
Chart recorders usually plot absorbance against wavelength. This particular substance
has what are known as absorbance peaks at 255 and 395 nm. How these arise and how
they are interpreted are discussed on another page.
Fig. The output of chart recorder.
Working principle
Firstly, the machine is started. The machine will run and take a reading of the intensity of
the radiation source. This is the incident light intensity.
Entrance slit
Exit slit
Detector
Cuvette
Red
Violet
Prism
MonochromatorLight source
I0
I
Readout
device
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The sample is placed inside the instrument in cuvette. Light is generated by the radiation source
and then passed through the monochromator. The monochromatic light is passed through the
sample and then the intensity of the transmitted light is measured in the detector. From the
intensity of the incident light and intensity of the transmitted light the computer will calculate
and show the absorbance.
Then the blank/reference (sample solution minus the sample molecules) is placed in the
instrument.
The intensity of the transmitted light is measured again. From these two values the relative
intensities light before passing the sample molecules and after passing the sample molecules
can be determined.
So basically there are two measurements involved.
In some single beam spectrophotometer a “zeroing” method is used where the
absorbance by the reference is set as baseline value and then the absorption by the sample is
measured relative to it.
Double beam spectrophotometer:
In single beam spectrophotometer there is a necessity to make two separate
measurements. This has been eliminated with the double beam spectrophotometer.
In this instrument the blank (the
reference cell) and the sample solution
(sample cell) is placed simultaneously
in the spectrophotometer. The light
generated by the radiation source is
passed through the monochromator and
the monochromatic light is split into
two beams (this is done by a rotating
disc).
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One beam passes through the reference cell and other passes through the sample cell. Then the
transmitted light (whether from reference cell or sample cell) is sent to the detector. The detector
will measure the intensities of the transmitted light. A computer will analyze the intensities and
subtract the intensity of transmitted light from sample cell from the intensity of the transmitted
light from reference cell.
Thus the absorption by the rest of the part of solution is cancelled.
a) Single-beam spectrophotometer
b) Double-beam spectrophotometer
a) Single beam spectrophotometer
A single-beam instrument uses only single beam of radiation through a single cell. The reference
cell is used to set the absorbance scale at zero for the wavelength to be studied. It is then
replaced by sample cell to determine the absorbance of the sample at that wavelength.
Fig: Single-beam spectrophotometer
b) Double-beam spectrophotometer
A double beam instrument divides the radiation into two beams of equal intensity which are
passed through two separate cells. One of the two cells contains the sample solution, while
other, called the reference cell, contains either the pure solvent or a blank solution. Since the
absorption by the sample is automatically corrected for absorption occurring in the solvent, the
readout from the instrument is the difference between amounts of the radiations absorbed in the
two cells.
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Fig: Double-beam spectrophotometer
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Applications of UV spectroscopy
1. Structure elucidation of organic compounds: UV spectroscopy is useful in the structure
elucidation of organic molecules,( the presence or absence of unsaturation, the presence of
hetero atoms). From the location of peaks and combination of peaks, it can be concluded that
whether the compound is saturated or unsaturated, hetero atoms are present or not etc.
2. Identification of an unknown compound- An unknown compound can be identified with the
help of UV spectroscopy. The spectrum of unknown compound is compared with the spectrum
of a reference compound and if both the spectrums coincide then it confirms the identification of
the unknown substance.
3. Quantitative analysis: UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation. This determination is based on Beer’s
law. A = abc
4. Detection of Impurities: Additional peaks can be observed due to impurities in the sample
and it can be compared with that of standard raw material. Benzene appears as a common
impurity in cyclohexane. Its presence can be easily detected by its absorption at 255 nm.
5. Qualitative analysis: In UV absorption spectroscopy identification is done by comparing the
absorption spectrum with the spectra of known compounds. UV absorption spectroscopy is
generally used for characterizing aromatic compounds and aromatic olefins.
6. Chemical kinetics: Kinetics of reaction can also be studied using UV spectroscopy. The UV
radiation is passed through the reaction cell and the absorbance changes can be observed.
7. Molecular weight determination: Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable derivatives of these compounds.
8. As HPLC detector: A UV/Vis spectrophotometer may be used as a detector for HPLC.
9. Detection of extent of conjugation- The extent of conjugation in the polyenes can be
detected with the help of UV spectroscopy.
10. Determination of configurations of geometrical isomers- It is observed that cis-alkenes
absorb at different wavelength than the trans-alkenes. The cis-isomer suffers distortion and
absorbs at lower wavelength as compared to trans-isomer.
Theoretical determination of max of compounds:
In 1941, Robert Burns Woodward first put forward a set of rules to predict the λmax of a
given compound in UV-Vis spectroscopy. These rules were applicable to open chain and 6-
membered ring dienes.
Later the rules were modified by Fischer Scott and Louis Fieser. This is known as the
Woodward-Fieser rules (This rule works well when the conjugated system is only 4 double
bonds long. When more than four double bonds are present, one must use the Fieser-Kuhn rules
instead).
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Various types of double bonds in conjugation are:
1. Homoannular dienes: It is a cyclic diene having conjugated doubles bonds in
the same ring. e.g. Toluene, Naphthalene.
2. Acyclic dienes: Diene containedvin an open chain syatem. Where the basic
unit is butadiene.
3. Heteroannular dienes: It is a cyclic diene in which double bonds in conjugation
are present in different ring.
4. Endocyclic conjugated double bond: It is the double bond present in the ring.
5. Exocyclic conjugated double bond: It is a double bond is a double bond, part of the
conjugated system and formed by any carbon atom of any ring but present outside of the ring.
According to the rules each type of diene or triene system have a fixed value at which
absorption takes place.
This constitutes the basic value or parent value.
The contributions made by various alkyl substances or ring residue, double bonded extending
conjugation and polar group such as –Cl, -Br, -OR are added to the basic value to obtain λmax for
a particular compound.
Ring residue is a C-C bond, not a part of the conjugated system; but attached to any one of the
carbon atoms of the conjugated poyene system.
The parent values and contribution of different substituents or groups are given below
(Conjugated trienes and dienes/solvent ethanol/pi-pi* transition)
1. Parent Values:
Cyclic conjugated dien or butadiene 217nm
Acyclic triene 245 nm
Heteroannular conjugated diene 215 nm
Homoannular conjugated diene 253 nm
2. Increment for each substituent:
Alkyl substituent or ring residue 5 nm
Exocyclic double bond 5 nm
Double bond extending conjugation 30 nm
3. Auxochromes
–OR +6 nm
–SR +30 nm
–BR, -Cl +5 nm
-NR2 +60 nm
-OCOCH3 0
Modified rules for open chain dienes:
According to the modified rules, calculation can be made as follows –
  onscontributiOtheroncontributitsSubstituenvalueParentmax
Md.
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Such values are listed below –
Class Structure Wavelength
value (nm)
Parent/base/core
C C
C
H
H C
H
H
H
H
217
A double bond extension of
the conjugation C C
Thus, following structure has the
λmax of 24430214 
C C
C
H
H C
H
C
H
H C
H
HH
30
Alkyl group substituent ─R 5
Auxochromic (attached to the
chromophore) OX
─OH, ─OR 6
Auxochrome – Acyloxy
Auxochrome – Ester C
O
R O C
O
R
0
Axochrome – Amine
N
R
R
60
Auxochrome – Halogen (X) Cl, Br 5
Sulfide SR 30
Phenyl 60
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Example explaining the modified rules for open chain dienes:
H3C C
C C
C C
C C
H
C
H
H
H
H NH2
Cl H
OH
C
CH3
NH2
Here,
Parent value 217
Double bond extension of the conjugation 30
One Cl group 5
One NR2 group 60
two alkyl group (shown in sky blue and
orange)
52
One OH group 6
total =328 nm
Modified rule for benzoyl derivatives:
Class Structure Wavelength value (nm)
Parent/base/core
C
O
X
1. When X = Alkyl group
or ring residue, 246.
2. When X = H, 250.
3. When X = OH or OR,
230.
Alkyl or ring residue on ortho or meta
position.
3
Alkyl or ring residue on para position. 10
OH, OR at ortho or meta position. 7
OH, OR at para position. 25
NH2 at ortho or meta position. 13
NH2 at para position. 58
NHAc at ortho or meta position. 20
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NHAc at para position. 45
Cl at ortho or meta position. 0
Cl at para position 10
Br at ortho or meta position. 2
Br at para position. 15
Explanation of the modified rules for benzoyl derivatives:
O
H3CO
H2N
OCH3
OH
Here,
Parent value 246 (Since X = R)
Ring residue at ortho position 3
Ring residue at meta position 3
Ring residue at para position 10
NH2 group at ortho position 13
OR group at meta position 7
Total = 282 nm
[(A more elaborate description of the rules that can be used to determine the λmax of
compounds can be found on www.pharmaxchange.info) – for conjugated diene systems,
transoid and cisoid concept is important).
Md.
Imran
Nur
Manik

Md.
Imran
Nur
Manik

UV-Ultraviolet Visible Spectroscopy MANIK

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